The Pennsylvania State University
The Graduate School
College of Medicine
AN INVESTIGATION OF MU-OPIOID RECEPTOR INTERACTING PROTEINS
WITHIN THE MU-OPIOID RECEPTOR SIGNALING COMPLEX
A Dissertation in
Genetics
by
Stephanie Justice-Bitner
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
December 2012
The dissertation of Stephanie Justice-Bitner was reviewed and approved* by the following:
Robert Levenson Distinguished Professor of Pharmacology Co-Director of MD/PhD Program Dissertation Advisor Chair of Committee
Laura Carrel Associate Professor of Biochemistry and Molecular Biology
Vincent Chau Professor of Cellular and Molecular Physiology
Victor Ruiz-Velasco Associate Professor of Anesthesiology
David Spector Professor and Distinguished Educator of Microbiology and Immunology Co-chair, Intercollege Graduate Program in Genetics
*Signatures are on file in the Graduate School
iii
ABSTRACT
Opioids are powerful analgesic drugs that lead to the development of tolerance, physical dependence, and addiction with prolonged use or abuse. Chronic exposure appears to cause alterations in the neuronal morphology of brain regions implicated in opioid dependence and addiction. Many of these changes reflect the inhibitory effects of these drugs and other opioid receptor agonists on neuron size, neurite outgrowth, dendritic arborization, and neurogenesis that occur in brain regions important for reward processing, learning, and memory. The mu-opioid receptor (MOR) is the G-protein coupled receptor (GPCR) primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. However, the precise role of the MOR in the development of these neuronal alterations remains elusive. A growing body of evidence suggests that G-protein-coupled receptor (GPCR) signaling is modulated by proteins that bind GPCRs and form multiprotein signaling complexes.
A number of proteins that interact with the MOR have recently been identified and have been shown to affect MOR biogenesis, trafficking, and signaling. Therefore, identifying and characterizing MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms of opioid addiction.
Using a combination of conventional and modified membrane yeast two-hybrid (MYTH) screening methods, a cohort of novel MOR interacting proteins (MORIPs) was identified. The interaction between the MOR and a subset of MORIPs was validated in pull-down, co- immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta (DOR) and kappa (KOR) opioid receptors. Finally, three of these proteins showed altered expression in the brains of morphine treated mice.
The ubiquitin pathway has been shown to be altered in the addiction process, therefore
iv two novel MORIPs, seven in absentia 1(SIAH1) and 2 (SIAH2), were of particular interest due to their known function as E3 ligases in the ubiquitin pathway. The effects of altering SIAH1 and/or SIAH2 protein expression on opioid receptor ubiquitination and protein levels was investigated using human embryonic kidney (HEK) stably transfected with MOR, delta opioid receptor (DOR), or kappa opioid receptor (KOR). These experiments suggest that SIAH2 is an
E3 ligase for the MOR, while both SIAH1 and SIAH2 may conjugate ubiquitin to DOR and
KOR. The ubiquitination by SIAH2 appears to signal for degradation of the MOR, whereas
SIAH1 seems to act as a shuttle protein or chaperone assisting in degradation. In contrast, the ubiquitination of DOR and KOR may be a signal for degradation or trafficking of receptors.
Additionally, opioid agonist treatment of neuroblastoma cells promoted the formation of
MOR/SIAH1 complexes. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.
v
TABLE OF CONTENTS
LIST OF FIGURES ...... viii
LIST OF TABLES ...... x
LIST OF COMMON ABBREVIATIONS ...... xi
ACKNOWLEDGEMENTS ...... xiii
Chapter 1 Literature Review ...... 1
1.1 Opioid Drugs ...... 1 1.1.1 Analgesia and Addiction ...... 1 1.1.2 Opioid Pharmacology ...... 3 1.2 The Opioid System ...... 5 1.2.1 The Opioid Receptors ...... 6 1.2.3 Mu-Opioid Receptor Genetic Variations ...... 8 1.2.4 Dimerization of Opioid Receptors ...... 17 1.2.5 Endogenous Opioid Peptides ...... 19 1.3 Neurobiology of Opioid Exposure ...... 21 1.3.1 Mechanisms of Opioid Addiction ...... 21 1.3.2 Animal Models of Addiction-Like Behavior ...... 22 1.3.3 Opioid Receptor Knockout Studies ...... 23 1.3.4 Physiological and Psychological Adaptations to Opioid Exposure ...... 26 1.3.5 Regulation of MOR ...... 29 1.4 GPCR Interacting Proteins ...... 31 1.4.1 MOR Interacting Proteins ...... 32 1.4.2 Identifying Candidate MORIPs ...... 35 1.5 Rationale and Hypothesis ...... 36
Chapter 2 Identification of MOR Interacting Proteins ...... 38
2.1 Introduction ...... 38 2.2 Experimental Procedures ...... 40 2.2.1 Membrane Yeast Two-Hybrid Screen ...... 40 2.2.2 Conventional Yeast Two-Hybrid Screen ...... 41 2.2.3 Mapping of Binding Regions ...... 42 2.2.4 Glutathione S-transferase Pulldown Assay ...... 42 2.2.5 Cell Culture ...... 44 2.2.6 Co-immunoprecipitation and Western blot Analysis ...... 45 2.2.7 Immunofluorescence Microscopy ...... 46 2.3 Results ...... 46 2.3.1 Identification of Novel MOR Interacting Proteins ...... 46 2.3.2 Mapping of Binding Regions ...... 51 2.3.3 Validation of MOR/MORIP interaction by GST Pulldown ...... 53
vi
2.3.4 Interaction of MORIPs with MOR, DOR, and KOR in Cultured Mammalian Cells ...... 55 2.3.5 Immunofluorescence Microscopy ...... 57 2.4 Discussion ...... 59
Chapter 3 Functional Analysis of the MOR/SIAH Interaction ...... 64
3.1 Introduction ...... 64 3.2 Experimental Procedures ...... 68 3.2.1 Mapping of binding sites ...... 68 3.2.2 Cell culture and transfections ...... 69 3.2.3 Treatment of cells with inhibitors of the proteasome and lysosome ...... 70 3.2.4 Detection of ubiquitinated MOR using TUBEs ...... 70 3.2.5 siRNA knockdown of SIAH proteins ...... 71 3.2.6 Co-immunoprecipitation and western blot analysis ...... 71 3.2.7 Quantitation of Western blots ...... 73 3.3 Results ...... 73 3.3.1 Mapping of the MOR binding site on SIAH1 ...... 73 3.3.2 SIAH1 regulates the amount of MOR modified with ubiquitin ...... 75 3.3.3 siRNA knockdown of SIAH1 increases the ubiquitination of the opioid receptors ...... 78 3.3.4 Knockdown of SIAH2 results in decreased ubiquitination of MOR, but increased ubiquitination of DOR and KOR...... 81 3.3.5 The MOR undergoes degradation via the proteasome and lysosome ...... 83 3.3.6 Overexpression of SIAH1 causes a decrease in MOR protein levels...... 85 3.3.7 siRNA knockdown of SIAH1 increases MOR protein levels, but decreases DOR and KOR protein ...... 86 3.3.8 siRNA knockdown of SIAH2 decreases MOR protein levels, but increases DOR and KOR protein ...... 89 3.3.9 SIAH1 and SIAH2 interact in HEK-MOR cells...... 91 3.3.10 SIAH1 regulates the levels of SIAH2 in HEK cells ...... 92 3.3.11 Knockdown of both SIAH1 and SIAH2 results in a decrease in the ubiquitination of the MOR and an increase in MOR protein levels ...... 94 3.4 Discussion ...... 96
Chapter 4 Effect of Opioid Exposure on MORIPs ...... 104
4.1 Introduction ...... 104 4.2 Experimental Procedures ...... 107 4.2.1 Cell culture and drug treatment ...... 107 4.2.2 Co-immunoprecipitation and Western blot analysis ...... 108 4.2.3 Animal Care and Drug Treatment ...... 109 4.2.4 Tissue Preparation and Protein Analysis ...... 110 4.3 Results ...... 111 4.3.1 Drug exposure alters strength of interaction between SIAH1 and opioid receptors ...... 111 4.3.2 Interaction of MOR and MORIPs in Rodent Brain ...... 114 4.3.3 The Effect of Morphine on MORIP Expression ...... 117 4.4 Discussion ...... 119
vii
Chapter 5 Overall Discussion ...... 123
REFERENCES ...... 140
viii
LIST OF FIGURES
Figure 1.1: Non-selective agonists and antagonists at opioid receptors ...... 5
Figure 1.2: Structural similarity between opioid receptors ...... 7
Figure 1.3: Mouse strains show varied responses to morphine and other opioid drugs ...... 10
Figure 1.4: Schematic of the human OMPR1 gene structure and splice variants ...... 13
Figure 2.1: Mapping of MORIP binding regions ...... 52
Figure 2.2: GST pull down of MORIPs and MOR ...... 54
Figure 2.3: Co-immunoprecipitation of MORIPs and MOR, KOR, and DOR ...... 56
Figure 2.4: Co-localization of MOR and MORIPs ...... 59
Figure 3.1: Mapping of SIAH1 binding site ...... 74
Figure 3.2: SIAH1 decreases the ubiquitination of the MOR ...... 77
Figure 3.3: Knockdown of SIAH1 increases ubiquitination of MOR, DOR, and KOR ...... 80
Figure 3.4: Knockdown of SIAH2 decreases ubiquitination of MOR, but increases ubiquitination of DOR and KOR ...... 82
Figure 3.5: Inhibitor treatment of HEK-MOR cells ...... 85
Figure 3.6: SIAH1 induces MOR degradation...... 86
Figure 3.7: Knockdown of SIAH1 changes MOR, DOR, and KOR protein levels ...... 88
Figure 3.8: Knockdown of SIAH2 changes MOR, DOR, and KOR protein levels ...... 90
Figure 3.9: Co-immunoprecipitation of SIAH1 with SIAH2...... 92
Figure 3.10: SIAH1 knockdown causes an increase in SIAH2 protein levels ...... 93
Figure 3.11: Knockdown of SIAH2 does not alter SIAH1 protein levels...... 94
Figure 3.12: Knockdown of both SIAH1 and SIAH2 decreases MOR ubiquitnation and increases MOR protein levels ...... 96
Figure 4.1: DAMGO treatment of SH-SY5Y cells promotes MOR/SIAH1 complex formation ...... 112
Figure 4.2: Morphine treatment promotes DOR/SIAH1 complex formation ...... 114
ix
Figure 4.3: MORIP expression and interaction with MOR in mouse brain ...... 116
Figure 4.4: MORIP expression in brain regions of morphine treated mice ...... 118
Figure 5.1: MOR interaction map ...... 124
Figure 5.2: Models for SIAH1 and SIAH2 function in HEK-DOR and HEK-MOR cells ...... 130
Figure 5.3: Simplified model of Siah protein-mediated MOR degradation ...... 132
Figure 5.4: Model of the role of SIAH1 and SIAH2 in ER-associated degradation of the MOR ...... 137
x
LIST OF TABLES
Table 1.1: Legal Definitions of Schedule I and Schedule II Controlled Substances ...... 2
Table 1.2: Association studies investigating A118G SNP in opioid dependence in humans ...... 16
Table 1.3: Opioid receptor dimers with related G protein-coupled receptors ...... 18
Table 1.4: Endogenous opioid peptides ...... 20
Table 1.5: Summary of opioid receptor knockout mice studies...... 26
Table 1.6: Previously identified MORIPs ...... 33
Table 2.1: PCR primers used for cloning MORIPs ...... 44
Table 2.2: Putative MORIPs identified in MYTH screen ...... 48
Table 2.3: Putative MORIPs identified in conventional yeast two-hybrid screens ...... 50
Table 3.1: PCR primers used for cloning of SIAH1 deletion mutants ...... 69
Table 3.2: Summary of SIAH overexpression and knockdown experiments ...... 99
xi
LIST OF COMMON ABBREVIATIONS
AUP1 ancient ubiquitous protein 1 bp base pairs coIP co-immunoprecipitation
CSN5 COP9 signalosome subunit 5
DOK4 docking protein 4
DOK5 docking protein 5
DOR δ-opioid receptor
ECL enhanced chemiluminescence
FBS fetal bovine serum
GPCR G protein-coupled receptor
GST glutathione S-transferase
HEK human embryonic kidney
HRP horseradish peroxidase
IL intracellular loop
IP immunoprecipitation kDa kilodalton
KO knockout
KOR κ-opioid receptor
MOR μ-opioid receptor
MORIP μ-opioid receptor interacting protein
MYTH membrane yeast two-hybrid
PAGE polyacrylamide gel electrophoresis
xii
PBS phosphate buffered saline
PVDF polyvinylidine difluoride
SIAH1 seven in absentia homolog 1
SIAH2 seven in absentia homolog 2
SDS sodium dodecyl sulfate
SNP single nucleotide polymorphism
VAPA vesicle-associated membrane protein-A
VTA ventral tegmental area
WLS Wntless
Y2H yeast two-hybrid
xiii
ACKNOWLEDGEMENTS
I am forever grateful to all of those who have contributed to the completion of my graduate work. First, I would like to thank Dr. Robert Levenson, my thesis advisor, for his support, encouragement, and patience. I am grateful to the members of my thesis committee, Drs.
Vincent Chau, Victor Ruiz-Velasco, and Laura Carrel for their advice and support throughout my graduate school career. I especially thank Dr. Vincent Chau for his technical advice and support.
I would also like to thank the past and present members of the Levenson Lab, who have made the lab an enjoyable place to work, especially Dr. Jessica Petko for her unending encouragement, advice, and friendship.
I extend gratitude to my collaborators, including Drs. Igor Staljar, Tom Ferraro, and
Jessica Petko for their assistance and advice. I am also indebted to the faculty and staff of the
Pharmacology Department and the Genetics Program.
Finally, I want to acknowledge my family members. I thank my parents for their continued love and support throughout my schooling. I thank my husband’s aunt for providing me a place to live, while working on my research. Lastly, I thank my husband for having the patience to care for our dogs and home miles away while I worked on my graduate work.
Chapter 1
Literature Review
1.1 Opioid Drugs
1.1.1 Analgesia and Addiction
Opioid analgesics are invaluable in the treatment of severe, chronic, and/or unremitting pain symptoms. The effectiveness of these drugs in relieving pain has been known for hundreds of years (Dhawan et al., 1996). Unfortunately, these pain-relieving benefits come with a number of negative side effects, which limits their use for only the most severe pain conditions. The major side effects of opioid analgesics include respiratory depression, alterations in heart rate and blood pressure, severe constipation, and mood changes. The most significant of these effects are respiratory depression and mood changes. The most common cause of death in cases of opioid agonist overdose is respiratory depression. The reported feelings of euphoria and relaxation that typically occur shortly after administration of opioid agonist drugs have been implicated in the abuse liability of these drugs. Psychologically, this euphoric state is considered to be the motivating or rewarding factor that drives an individual to use these drugs inappropriately (Bailey and Connor, 2005; Corbett et al., 2006). Due to their potential for abuse, opioid agonist drugs fall under the auspices of the Controlled Substances Act, and are considered either Schedule I or Schedule II controlled substances depending on whether or not the drug is clinically useful (Table 1.1). For example, heroin (diacetylmorphine) has been deemed to have no medical use in the United States, and is therefore listed as a Schedule I controlled substance. On the other hand, morphine is considered to have an important medical use and is therefore listed as a Schedule II controlled substance. The existence of the Controlled Substances Act affirms
2 the larger problem that these drugs pose from a biological, societal, and legislative perspective.
Table 1.1: Legal definitions of Schedule I and Schedule II controlled substances These definitions were reproduced verbatim from Section 812 of the Controlled Substances Act that was enacted into law by the Congress of the United States as Title II of the Comprehensive Drug Abuse and Prevention and Control Act of 1970. It should be noted that three more schedules exist, but only one opioid mixed agonist/antagonist, nalorphrine, is schedule III. Schedule III drugs have less potential for abuse than schedule I and II, but can still cause substance dependence.
Schedule I (A) The drug or other substance has a high potential for abuse. (B) The drug or other substance has no currently accepted medical use in treatment in the United States. (C) There is a lack of accepted safety for use of the drug or other substance under medical supervision.
Schedule II (A) The drug or other substance has a high potential for abuse. (B) The drug or other substance has a currently accepted medical use in treatment in the United States or a currently accepted medical use with severe restrictions. (C) Abuse of the drug or other substances may lead to severe psychological of physical dependence.
In the 2010 National Survey on Drug Use and Health, it was estimated that 22.6 million Americans 12 years or older had used an illicit drug or used a prescription drug inappropriately in the past year. Of these 22.6 million, an estimated 9.0 million people
3 were current users of drugs other than marijuana and 5.3 million of these current drug users were using prescription pain killers (opioid drugs) or heroin. Opioid drugs are the third most abused drug behind alcohol and marijuana. The use of prescription opioid drugs among first-time initiators of drug abuse was found to be almost equal to marijuana. However, the ability of many users to obtain opioid drugs from a friend or relative for free could lead to an increase of opioid drug users. The simplest way to resolve these issues would be the development of an analgesic drug that is as efficacious as known opioid analgesics, but does not have potential for abuse. Some researchers have called this endeavor “the exciting but vain quest for the Holy Grail” (Corbett et al., 2006). When considering the efforts made in the diverse field of opioid-related research, the sum total of work can all be traced back to this goal of finding or creating a drug that can alleviate severe pain without being addictive.
1.1.2 Opioid Pharmacology
Opioid compounds have been used since antiquity; long before the opioid receptors and their peptide ligands were discovered. It wasn’t until 1805 that the first and prototypical opioid agonist, morphine, was isolated from opium. Morphine is invaluable as an analgesic agent, but it can induce a sense of euphoria, which contributes to non- medical use and abuse (Dhawan et al., 1996). Receptor knockout studies in mice have shown that both the analgesic and rewarding properties of morphine are mediated by the mu-opioid receptor (MOR) (Matthes et al., 1996). Since these drugs are such powerful analgesics, much effort has gone into synthesizing new drugs that retain the pain- relieving qualities of morphine, but are less rewarding.
4 The quest to find a better opioid drug has led to the development of a large library of compounds. This library consists of substances that act as agonists, antagonists, and mixed agonist-antagonists. Morphine is the archetypal opioid agonist that mediates its effects by binding to and activating opioid receptors. Antagonists, such as naloxone and naltrexone, block the effects of agonists by competing for receptor binding sites and preventing activation of receptors by agonists. Antagonists are useful in clinical settings to rapidly reverse the effects of opioid agonist overdoses. Mixed agonist-antagonists are compounds, such as nalorphine (n-allylnormorphine), which can act as an agonist at one opioid receptor subtype, but also act as an antagonist at another opioid receptor subtype.
The development of these mixed action drugs initially held the promise of promoting one effect (analgesia) while controlling another (addiction), but these drugs have still had enough abuse liability to remain problematic (Snyder, 2004).
Most of these opioid drugs were created by making small chemical modifications to morphine. Morphine is a naturally occurring plant alkaloid isolated from opium, which is harvested from the unripe seed capsule of the poppy plant, Papaver somniferum
(Dhawan et al., 1996). When the structure of morphine and naloxone is compared, all that is required to convert a potent agonist into an antagonist is an ethylene side-chain
(Figure 1.1). Although this effect is not fully understood, the fact that small differences in chemical structure can have large impacts on pharmacological activity is clear from studies that show that the opioid binding sites in the brain are stereospecific (Simon et al.,
1973; Terenius, 1973).
5
Figure 1.1: Non-selective agonists and antagonists at opioid receptors Each of the compounds above contains the pentacyclic structure of morphine. This basic structure contains a benzene ring with a phenolic hydroxyl group at position 3 and an alcohol hydroxyl group at position 6 and at the nitrogen atom. The addition of relatively simple side chains to this basic structure results in the altered activity of the resulting compound. Reproduced from (Dhawan et al., 1996).
1.2 The Opioid System
Given the centuries-old knowledge and use of opioid drugs, it is surprising that the existence of an endogenous opioid receptor was not postulated until the 1950s (Lasagna, 1954). Since that time, pharmacological studies (Martin et al., 1976) and the cloning of specific genes have led to the identification of the components of the endogenous opioid system (Kieffer and Gaveriaux-Ruff, 2002).
6 The endogenous opioid system is composed of a subfamily of G-protein coupled receptors (GPCRs) and their peptide ligands, most of which are derived from precursor proteins. Many important physiological functions are regulated by this system, including respiration, gastrointestinal motility, hormone secretion, thermoregulation, immune function, mood, and pain modulation (Dhawan et al., 1996). The anatomical expression profile of opioid receptors and ligands throughout the central and peripheral nervous system and in other parts of the body, such as the gastrointestinal tract, explains why this system is involved in so many physiological functions (Snyder, 2004; Snyder and Childers, 1979). Our knowledge of the endogenous opioid system is largely derived from the major effects and side effects that are seen when exogenous opioid drugs act on the endogenous receptors (Dhawan et al., 1996; Snyder, 2004).
1.2.1 The Opioid Receptors
There are four main subtypes of endogenous opioid receptors, which are designated delta (DORkappa (KOR), mu (MOR), and nociceptin (NOR). All four receptors belong to the Class A (rhodopsin-like) GPCRs, and share similar structural features, including an extracellular amino-terminus, seven transmembrane spanning domains linked by three extracellular and intracellular loops, and an intracellular carboxy-terminus. The opioid receptors are often posttranslationally modified through glycosylation and palmitoylation (Minami and Satoh, 1995), and exert their major downstream effects through pertussis toxin-sensitive heterotrimeric G-proteins (Gαo) (Johnson et al., 1994; Ueda et al., 1988; Wong et al., 1988). Each receptor is encoded by a single gene in humans and presumably forms a single gene product (Knapp et al., 1994; Wang et al., 1994; Zhu et al., 1995), although a number of splice variants have been reported for the MOR (Pasternak, 2004). Amino acid sequence alignments show that the receptors are homologous to each other (MORand KOR 68% identical, MOR and DOR 66% identical, KOR and DOR 58% identical; NOR shares 50-60% identity with DOR, , and MOR) with most of the variation between receptors occurring in the
7 extracellular loops and the carboxy-terminus (Dhawan et al., 1996). Figure 1.2 summarizes the structural similarities and differences between the opioid receptor types. Recombinant chimeric receptor studies have found that the extracellular regions are responsible for the specificity of ligand binding (Kong et al., 1994; Onogi et al., 1995; Xue et al., 1994). Additionally, single nucleotide polymorphisms can alter ligand binding. For example, an amino acid substitution at residue 40 (Asn to Asp) of the MOR has been shown to increase -endorphin binding affinity (Bond et al., 1998) and alters the functional properties of the receptor (Kroslak et al., 2007).
Figure 1.2: Structural similarity between opioid receptors Model for the mu-opioid receptor in the rat. Amino acid residues that are conserved between the mu-opioid receptor and both delta- and kappa- opioid receptors are shown in black. Gray indicates an amino acid conserved with two opioid receptors and white indicates non-conserved residues. Branched structures indicate potential N-linked glycosylation sites. Reproduced from Minami and Satoh, 1995.
8 The distribution of opioid receptors has been reviewed by Le Merrer et al (2009) and will not be addressed in detail in this chapter. Opioid receptors are primarily expressed in the cortex, limbic system, and the brain stem. Ligand binding studies have shown that the three opioid receptors exhibit an overlapping expression pattern in most brain structures; however, some structures exhibit a higher expression of one receptor over the others. For example, the MOR is more highly expressed in the thalamus when compared to either the DOR or the KOR. Interestingly, MORs and KORs are often found together in most brain regions, whereas the distribution of DORs is more restricted. Importantly, all three receptors have been shown to have expression in areas of the brain believed to be involved with addiction, such as the hippocampus, ventral tegmental area, and the nucleus accumbens (Le Merrer et al., 2009). The distribution of opioid receptors also correlates with the reported functions of the opioid system. For example, activation of MORs located in the periaqueductal grey area (PAG) leads to analgesia, while activation of NORs in the same area reduces the analgesic effect of MOR agonists. This difference is due to the location of these receptors within the PAG. MORs are located on presynaptic inhibitory interneurons, which leads to disinhibition of the neural output from the PAG, whereas NORs are located on postsynaptic neurons, which leads to inhibition of the neural output from the PAG. This anatomical difference explains the opposing effects of these two opioid receptors, despite the fact that both receptors are coupled to the same downstream effectors (Corbett et al., 2006). Knockout studies in mice have further confirmed the role of these receptors in pain modulation, reward, and behavior (Kieffer and Gaveriaux-Ruff, 2002).
1.2.3 Mu-Opioid Receptor Genetic Variations
Opioid agonists used in the treatment of pain have a narrow therapeutic index and large interpatient variability in response. Patients may respond better to one opioid than another with respect to both analgesia and side effects (Pasternak, 2004). Even in relatively homogeneous patient cohorts, dosage requirements can vary substantially. For example, in a study of postoperative hip replacement therapy encompassing over 3,000
9 patients, morphine dosage requirements varied almost 40-fold (Aubrun et al., 2003). There also is great variability in the responses to these drugs, which may lead to the development of addiction (Trigo et al., 2010; Volkow and Li, 2005). Similar observations have also been seen in animal models (Figure 1.3). The analgesic sensitivity of various strains of mice to a fixed morphine dose was observed to vary substantially, with BALB/c mice the most sensitive and CXBK mice being the least sensitive (Picker et al., 1991). When similar doses of opioids were given to CD-1 and CXBK mice, there were more varied differences in analgesia depending on the opioid drug administered (Figure 1.3) (Chang et al., 1998; Rossi et al., 1996). In addition to inbred mouse strains, inbred rat strains have also shown variation in response to opioid drugs. Among these inbred strains, Lewis and Fischer rats are the most often compared because of their behavioral and neuronal molecular differences in responding to drugs. Lewis rats tend to be more responsive in behavioral tests because they more easily self- administer morphine and other drugs, such as cocaine (Ambrosio et al., 1995). In a more recent study, Lewis rats were found to escalate both the dose and total amount of heroin administered, whereas Fischer rats did not escalate either (Picetti et al., 2012). These observed varied responses in animal models and in human subjects are believed to be mediated by splice variants and polymorphisms in the MOR (Kasai and Ikeda, 2011).
10 A
B
Figure 1.3: Mouse strains show varied responses to morphine and other opioid drugs These two graphs represent some of the mouse strain studies that have been reported in the literature. (A) The strains of mice received a fixed dose of morphine and were assessed for analgesia using the radiant heat tailflck assay (Pick et al., 1991). (B) Doses of opioids were chosen to give similar responses between CD-1 mice. These same doses were then administered to CXBK mice (Rossi et al., 1996; Chang et al., 1998). Images reproduced from Paskernak, 2004.
There are at least three different categories of factors that contribute to the vulnerability of developing addiction: environmental factors, such as cues and conditioning; drug-induced factors, which lead to molecular and neurobiological changes; and genetic factors, which represent 40-60% of the risk for developing addiction (Kreek et al., 2005). Included in these genetic factors are opioid receptor splice variants and
11 single nucleotide polymorphisms (SNPs), both of which have been studied in animal models and in humans. Data from animal models has provided important information regarding the role of genetic factors in addiction. In mu-opioid receptor knockout mice, homozygous mice have lower nociceptive thresholds than heterozygous knockout mice that have 50% less wild-type receptor density. In turn, these heterozygous mice have lower nociceptive thresholds than wild-type mice with normal mu-opioid receptor densities (Sora et al., 1997). Mouse-strain comparison studies in recombinant inbred mouse lines have provided additional evidence to implicate genetic factors in addiction. The CXBK mouse strain exhibited reduced responses to some opioid agonists. Like the heterozygous MOR knockout mice, CXBK mice express approximately 50% less MOR compared with progenitor strains (BALB/c and C57BL/6) and have a similar phenotype (Ikeda et al., 1999; Ikeda et al., 2001). The human MOR gene (OPRM1) spans over 200 kilobases and consists of 11 exons that combine to yield splice variants (Xu et al., 2009). The most abundant transcript, MOR-1, which consists of exons 1, 2, 3, and 4, is approximately 15 kb in length, and encodes 400 amino acids (Ide et al., 2005). Mouse MOR-1 shares 94% amino acid identity to human MOR-1. The first splice variant to be discovered was human MOR-1A. This variant was shown to encode for only 392 amino acids and differs from MOR-1 only at its 3’end (Bare et al., 1994). Since this initial discovery, over 28 splice variants from the mouse OPRM1 gene (Doyle et al., 2007a; Doyle et al., 2007b; Pan, 2005) have been isolated with similar splicing patterns observed in rats (Pasternak et al., 2004) and humans (Pan, 2005; Pan et al., 2003). The majority of these splice variants yield receptors that differ from each other at the C-terminus. Splicing typically occurs between exon 3 and more than 10 downstream exons, yielding at least 16 carboxyl terminal variants (Figure 1.3). Although these full-length variants share a common binding pocket (as seven transmembrane domains are conserved), they display significant differences in mu opioid receptor agonist-induced G protein activation (Bolan et al., 2004; Oldfield et al., 2008; Pan et al., 2005; Pasternak et al., 2004). These studies used [35S] GTPγS binding assays (measures G-protein activation) to show that the potency and
12 efficacy of opioid agonists, such as DAMGO and morphine, differed among splice variants. Additionally, these variants have different distributions in the types of cells and regions of the central nervous system (Abbadie et al., 2000a; Abbadie et al., 2000b; Abbadie et al., 2000c; Abbadie et al., 2001; Schnell and Wessendorf, 2004). For example, in the dorsal horn of the spinal cord, cells express either MOR-1 or MOR-1C, but not both together (Pasternak, 2005). In addition to the 3’end splice variants described above, there are also 5’ end splice variants that contain exon 11. This exon is located 28-30 kb upstream from exon 1(species dependent). To date, nine exon 11-associated splice variants have been identified (Xu et al., 2009). Interestingly, three of the transcripts generate the same protein as MOR-1, while others predict a protein with six transmembrane domains, and/or a protein with one transmembrane domain (Pan et al., 2001). These variants also show differing expression patterns in the brain (Abbadie et al., 2004). Recently, a naltrexone derivative, iodobenzoylnaltrexamide (IBNtxA), was synthesized in the Pasternak laboratory (Majumdar et al., 2011). This new opioid drug is a potent analgesic, but lacks the common opioid side effects, such as respiratory depression, constipation, or physical dependence. In studying this new drug, Pasternak and colleagues found that IBNtxA analgesia persisted in triple knockout mice (no MOR-1, DOR, or KOR). In contrast, there was a loss of both IBNtxA analgesia and receptor binding in exon 11 knockout mice (Majumdar et al., 2011). These results demonstrate that exon splice variants may be of interest in designing opioid drugs with analgesic power and little abuse liability.
13
Figure 1.4: Schematic of the human OMPR1 gene structure and splice variants Exons are represented by boxes, while introns are represented by horizontal lines. Translational start sites are indicated by downward lines on exon boxes. Translation stop sites are indicated by upward lines on exon boxes. Exons are numbered by the order of their isolation and/or similarity to exons identified in the mouse. C-terminal splice variants would include MOR-1A, MOR-1B-1B5, MOR-1O, MOR-1S, MOR-1X, MOR- 1Y, Mu3, SV1, and SV2. Exon 11 splice variants would include MOR-1G1, MOR-1G2, MOR-1H, and MOR-1i. Reproduced from Xu et al., 2009.
14 According to the dbSNP database and the NCBI database of genetic variation, the OPRM1 gene contains over 700 single nucleotide polymorphisms (SNPs). This genetic variation differs greatly between different races and ethnicities. For example, the minor allele frequency (MAF) of A118G, which is a well-studied nonsynonymous SNP in exon 1 leading to an Asn40Asp substitution, are 0.047 in the African population, 0.154 in the European population, 0.485 in the Japanese population, 0.14 in the Hispanic population, and 0.08 in the Bedouin population (Gelernter et al., 1999). Many SNPs have been studied to determine their association with pain sensitivity. Four SNPs (G-172T, A118G, IVS1-C2994T, and IVS2 +G31A) were analyzed in association studies with pain. Among these SNPs, two (IVS1-C2994T and IVS2 +G31A) were found to be significantly associated with pain sensitivity scores and pressure pain thresholds, respectively (Fillingim et al., 2005; Huang et al., 2008; Shabalina et al., 2009). However, there have been no other reports showing an association between these two SNPs and pain-related traits. Significant associations have also been observed in studies of the A118G SNP. The G-allele carriers of the A118G SNP showed higher pressure pain thresholds and pain tolerance thresholds (Fillingim et al., 2008). Other than the highly studied A118G SNP, few other polymorphisms have been investigated for functional significance. Among those that have been studied are two promoter SNPs, G-554A and A-1320G. G-554A decreases transcription of MOR, but is rare with a MAF of less than 0.001. In contrast, the A-1320G variant increases transcription of MOR (Bayerer et al., 2007). Three others, G779A, G794A, and T802C, are located in exon 3 and cause a decrease in receptor coupling and signaling (Befort et al., 2001; Koch et al., 2000; Wang et al., 2001). Other OPRM1 polymorphisms have been identified and associated with either pain or opioid dependence, but have no in vitro evidence for function. These include C17T (A6V), which is found primarily in African Americans (Crystal et al., 2012; Hoehe et al., 2000) and C440G (S147C), which is rare with an MAF of less than 0.006 (Glatt et al., 2007). The MOR mediates analgesia and side effects, such as nausea and respiratory depression, of opioid drugs. The analgesic and side effects of opioids were abolished or reduced in homozygous or heterozygous MOR-deficient mice, indicating that MOR gene
15 dosage is related to the clinical efficacy of these drugs. Although many SNPs in the OPRM1 gene have been investigated in regard to opioid sensitivity, the one most often studied is A118G. The A118G SNP has been shown to be associated with the analgesic and side effects of opioids. In these studies, opioid dose (Ginosar et al., 2009; Reyes- Gibby et al., 2007) and consumption (Lotsch et al., 2002; Sia et al., 2008) were greater in G-allele carriers when compared to AA individuals. G-allele carriers exhibited a lower analgesic efficacy and a lower incidence of side effects than AA individuals (Kasai and Ikeda, 2011; Mague and Blendy, 2010; Somogyi et al., 2007). However, other studies have found no such associations (Kasai and Ikeda, 2011; Mague and Blendy, 2010). The A118G SNP has been studied in a variety of substance abuse studies, including those for alcohol, nicotine, heroin, and alcohol. Findings differ greatly from study to study (Kasai and Ikeda, 2011; Somogyi et al., 2007). As opioid dependence is the focus of this work, a summary of the studies on the A118G allele and opioid dependence are shown in Table 1.2. Many OPRM1 gene variations have been identified and analyzed for their associations with pain sensitivity, opioid sensitivity, and susceptibility to drug dependence. These studies have revealed significant associations between genetic variations with regard to opioid sensitivity and vulnerability to substance dependence. However, not every analysis finds a significant association; therefore, the significance of variations in the OPRM1 gene is still debatable. More research into the mechanisms underlying MOR expression and function are required to settle this debate.
16
Table 1.2: Association studies investigating A118G SNP in opioid dependence in humans This table summarizes association studies done with the A118G SNP and heroin dependence. Risk or protection is associated with having at least one copy of the SNP. These studies indicate that A118G SNP can have varied outcomes depending on the race or ethnicity studied and the sample size. Adapted from (Mague and Blendy, 2010).
Effect Finding Population Reference (number of subjects) Risk G118 associated with Swedish (139 heroin- (Bart et al., heroin dependence dependent, 149 control) 2004)
Risk 90% of G118 were European Caucasian (118) (Drakenberg et heroin users al., 2006)
Risk Higher frequency of Indian (126 opioid- (Kapur et al., G118 in opioid dependent and 156 control) 2007) dependent subjects
Risk G118 associated with Chinese men (200 heroin (Szeto et al., heroin dependence dependent and 97 control) 2001)
Protective A118 associated with Indian (20 dependent, 117 (Tan et al., heroin in Indian, but control), Malaysian (25 2003) not East Asian dependent, 131 control), populations Chinese (52 dependent, 156 control)
Protective A118 associated with African American (46 (Bond et al., heroin dependence in dependent, 16 controls), 1998) Hispanics Caucasian (60 dependent, 44 controls), Hispanic (116 dependent, 78 controls)
None No association found Chinese (48 heroin (Shi et al., dependent, 48 controls) 2002)
17 1.2.4 Dimerization of Opioid Receptors
Opioid receptors belong to the G protein-coupled receptor (GPCR) Class A superfamily. GPCRs can form homo- and heterodimers. Importantly, these dimers can have distinct pharmacological properties. Therefore, dimerization can be seen as a way for an organism to increase the functional variety of GPCRs. A large body of evidence for the existence of GPCRs as dimers and oligomers has accumulated in recent years. GPCR dimerization has been shown to be a physiological process that modifies receptor pharmacology and regulates function. Heterodimerization can generate receptors with characteristics distinct from either single receptor, leading to altered pharmacological properties (George et al., 2002). Within the opioid receptor family, DORs have been shown to interact with both KORs and MORs to form heterodimers (Gomes et al., 2000; Jordan and Devi, 1999). In the case of interactions between DORs and KORs, the heterodimers were found to have reduced affinities for highly selective KOR or DOR agonists. Cells co-expressing KOR and DOR also exhibited synergistic effects on agonist signaling. Additionally, dimerization was found to affect the trafficking of these receptors, as etorphine-induced trafficking of DOR was greatly reduced in cells expressing KOR/DOR dimers. Etorphine causes DOR to be internalized, but when DOR/KOR dimers were expressed, the DOR remained at the cell surface (Jordan and Devi, 1999). MOR/DOR dimers have also been demonstrated to have altered selective agonist binding affinities (George et al., 2000). In cells expressing MOR/DOR dimers, low doses of DOR selective ligands produced a significant increase in the binding of a MOR selective agonist, which enhanced MOR- mediated signaling (Gomes et al., 2004). Additionally, MOR/DOR dimers constitutively recruit beta-arrestin 2, which results in alterations in ERK1/2 signaling (Rozenfeld and Devi, 2007). More recently, MOR/KOR heterodimers expression in spinal cords of Sprague-Dawley rats was found to be dependent on the estrous cycle stage of females and was also less expressed in males (Chakrabarti et al., 2010). Opioid receptors have also been shown to dimerize with distantly related GPCRs (Table 1.3). These receptor heterodimers exhibit properties that are quite distinct from those of each individual
18 receptor. Unfortunately, the influence of SNPs on heterodimers formation has not been studied. This lack of study is likely due to the lack of data on SNPs in other receptors and the functional consequences of these differences.
Table 1.3: Opioid receptor dimers with related G protein-coupled receptors This table lists the known opioid receptor heterodimers that have been discovered and the proposed function of the interaction.
Dimer Function Reference
MOR and serotonin MOR-induced ERK1/2 (Cussac et al., 2012) receptor 5-HT(1A) phosphorylation was desensitized with 5-HT(1A) activation
MOR and α2A-adrenergic Activation of either receptor leads (Jordan et al., 2003) to an increase in signaling, but activation of both leads to a decrease in signaling
Apelin receptor and KOR Agonist stimulation of either (Li et al., 2012) receptor resulted in higher ERK1/2 phosphorylation
Chemokine CXCR2 and Antagonists of CXCR2 enhanced (Parenty et al., 2008) DOR the effects of DOR agonists
Opioid receptor-like 1 and Reduced potency of mu-agonist to (Wang et al., 2005) MOR inhibit adenylate cyclase
Substance P receptor (NK1) Co-internalization with mu- (Pfeiffer et al., 2003) and MOR agonist treatment
Somatostatin (SST2A) Mu-agonist treatment induced (Pfeiffer et al., 2002) receptor and MOR phosphorylation and desensitization of both receptors
19 1.2.5 Endogenous Opioid Peptides
Endogenous opioid peptides include enkephalins, dynorphins, endorphins, and nociceptin (Bloom, 1983). Early studies that identified and characterized these peptides suggested the possibility that they were derived from larger precursor proteins (Snyder and Childers, 1979), which have now been identified and cloned (Chavkin et al., 1983; Chretien et al., 1979; Mollereau et al., 1996). The enkephalins, endorphins, and dynorphins contain the same four amino acid residues at their amino-termini (Try-Gly- Gly-Phe). This sequence was thought to be necessary for binding to opioid receptors (Snyder and Childers, 1979); however, more recently identified opioid peptides, such as endomorphins, do not contain this sequence but still bind opioid receptors (Akil et al., 1998). The endogenous peptides have not been found to be selective to binding to the different opioid receptors, but there are differences in their binding affinities for the opioid receptors (Akil et al., 1998; Dhawan et al., 1996). Table 1.4 lists the endogenous opioid peptides, their precursor proteins, and their binding affinities.
20
Table 1.4: Endogenous opioid peptides Precursor proteins and resulting peptides are listed along with the relative differences in binding affinities of the peptide family for the opioid receptor subtypes. Adapted from (Corbett et al., 2006).
Precursor Gene Peptide Binding Affinity Proopiomelanocortin POMC Endorphins MOR, KOR, DOR
α-endorphin β-endorphin γ-endorphin δ-endorphin
Unknown Unknown Endomorphins MOR
endomorphin-1 endomorphin-2
Preproenkephalin Penk Enkephalins DOR MOR, KOR
met5-enkephalin leu5-enkephalin
Preprodynorphin Pdyn Dynorphins KOR MOR, DOR
dynorphin A dynorphin B α-neoendorphin β-neoendorphin
Prepronociceptin PPNOC Nociceptin NOR
21 1.3 Neurobiology of Opioid Exposure
Historically, addiction of any kind was thought to be a problem of weak-willed people. The general consensus was that the addicted person was not trying hard enough to break his or her bad habit. However, with the sustained scientific efforts of the last 40 years, “addiction” has begun to be recognized as a neuropsychiatric disease. Unfortunately, much of the social stigma associated with addiction still exists to this day (Hyman, 2007). Opioid addiction is triggered by a chemical substance that activates a specific set of receptors in the brain. These same receptors are also acted upon by endogenous ligands to regulate normal neurobiological processes. Since not all people that are exposed to opioid drugs develop addiction, it is difficult to conclude if the activation of these receptors is the sole underlying cause of the disease. Twin, family, and adoption studies have provided evidence that addiction is a complex genetic disorder that can also be influenced by environmental factors, such as parental support, home environment, level of education, and socioeconomic status (Lachman, 2006). This complexity suggests that there are additional mechanisms that help to determine whether or not a particular individual will develop addiction to an opioid drug.
1.3.1 Mechanisms of Opioid Addiction
Addiction to opioids is thought to be mediated by activation of MORs in areas of the brain that mediate reward, such as the ventral tegmental area (VTA) and the nucleus accumbens (NAc), which are the major constituents of the mesolimbic dopaminergic pathway (Le Merrer et al., 2009). The direct injection of morphine or endomorphins into these brain areas has been observed to mediate self-administration and conditioned place preference in animal models (Tang et al., 2005; van Ree and de Wied, 1980; Zangen et al., 2002). The potency of these exogenous opioid drugs leads to an over-activation of the dopaminergic pathway that reinforces the drug-taking behavior. Unfortunately, this explanation only pertains to the psychological and behavioral foundations of opioid addiction and does not address the biological adaptations that may contribute to this
22 condition. However, these behavioral studies have illustrated the central role of the MOR in mediating the rewarding effect of these drugs. In a study by David et al. (2008), wild- type mice and δ-opioid receptor (DOR) knockout mice self-administered morphine injected into the VTA, but MOR knockout mice did not. Along with the initial characterization of the MOR knockout mouse (Matthes et al., 1996), these results directly implicate the MOR as an essential component of the behavioral response to opioid exposure. Interestingly, almost all people who receive opioid drugs over a prolonged period of time will develop tolerance and physical dependence. Although the mechanisms that underlie the development of these changes are not clear, many studies hypothesize that the regulation of MORs at the cellular and molecular levels may be involved (Bailey and Connor, 2005; Harrison et al., 1998).
1.3.2 Animal Models of Addiction-Like Behavior
Animal studies have been crucial in understanding the biology and pathobiology of drug addiction. In contrast to clinical studies, the subject population can be more easily controlled for variables. Animal models often focus on the ability of the drugs to directly control the animal’s behavior. Animal studies have demonstrated that the rewarding effect is not dependent on preexisting conditions. Exposure to the drug alone is sufficient to motivate drug-taking behavior (Lynch et al., 2010). There are two major approaches to assessing addiction-like behavior; conditioned place preference (CPP) and self-administration. The CPP procedure exposes animals to a distinct environment in the presence of drug. If the rodents find this substance positively reinforcing, they will show preference for that environment when later given a choice (Tzschentke, 1998). CPP is widely used to study the tolerance and sensitization to the rewarding effects of drugs induced by pre- treatment regimens. This method can also be used to model certain aspects of relapse to drug use, as in extinction/reinstatement procedures; however, this aspect is more often investigated using self-administration paradigms (Myers et al., 2012; Tzschentke, 2007).
23 A more direct procedure to evaluate the reinforcing properties of a drug is to test whether animals will work (usually lever press) to obtain the drug. This model traditionally entails training an animal to self-administer a drug during daily sessions, usually 1-3 hours in length. Most self-administration procedures use a fixed ratio (FR) schedule of reinforcement. In this assessment, the animal must perform a fixed number of responses to receive an intravenous drug infusion. If the animal finds the drug rewarding, the number of responses that an animal will perform to obtain an infusion generally increases. This is clearly seen in progressive ratio studies where the number of responses required for the infusion is increased until the animal no longer responds (Moser et al., 2011). Results from these kinds of studies have revealed that drugs can serve as positive reinforcers and that there is correspondence between humans and animals in terms of drugs that are self-administered and the pattern of drug intake. For example, drugs that are abused by humans typically maintain responding in animals, whereas drugs that do not maintain responding in animals are usually not abused by humans (Collins et al., 1984). Similar patterns of drug intake have been reported in humans and animals for opioids and other abused drugs (Griffiths, 1980).
1.3.3 Opioid Receptor Knockout Studies
Each gene encoding a receptor or peptide that is involved in the endogenous opioid system has been knocked out in mice either singly or in combination (Gaveriaux- Ruff and Kieffer, 2002). The generation of a MOR knockout (KO) mouse confirmed the importance of the MOR in mediating the analgesic and rewarding properties of opioid drugs. These mice appeared normal morphologically, did not differ in weight when compared to wild-type littermates, bred normally, and showed no changes in maternal behavior (Matthes et al., 1996). However, MOR KO pups exhibited less attachment behavior when separated from their mothers and were less responsive to their own mother’s cues (Moles et al., 2004). The loss of MOR expression did not appear to significantly change other components of the endogenous opioid system, nor were other components compensating
24 for this loss (Matthes et al., 1996; Sora et al., 1997). Tail flick, tail immersion, and hot plate tests were used to confirm that MOR KO mice were insensitive to the analgesic effects of morphine and other agonists (Kitanaka et al., 1998; Matthes et al., 1996; Sora et al., 1997). Other responses to opioid drugs including respiratory depression and slowed intestinal transit were also abolished in the absence of MORs (Dahan et al., 2001; Matthes et al., 1998; Roy et al., 1998; Roy et al., 2001). Importantly, MOR KO mice were insensitive to the rewarding effects of morphine and heroin in conditioned place preference tests (Contarino et al., 2002; Matthes et al., 1996; Sora et al., 2001). A few studies also found that self-administration of morphine and withdrawal precipitated by naloxone was also abolished in these mice (Becker et al., 2000; Matthes et al., 1996; Sora et al., 2001). MOR KO mice have also provided a great deal of insight into the rewarding properties of other drugs of abuse. Conditioned place preference for the active ingredient of marijuana (Δ9-tetrahydrocannabinol) and nicotine were reduced when MORs were not expressed (Berrendero et al., 2002; Ghozland et al., 2002). The self-administration of cocaine and ethanol were also abolished in MOR KO mice (Mathon et al., 2005; Roberts et al., 2000). Taken together, these data implicate the MOR in processing the rewarding effects of other substances, not just opioid drugs. This also indicates that the endogenous opioid system is involved in the regulation of reward processes. Delta opioid receptor (DOR) knockout mice studies revealed that these mice displayed higher levels of anxiety than MOR or kappa opioid receptor (KOR) knockouts (Filliol et al., 2000). In a study by Roberts (2001), DOR KO mice were exposed to alcohol in a two-bottle choice test. Naïve mutant mice and wild-type mice were indistinguishable in their alcohol consumption. However, when tested in an operant paradigm, the KO mice self-administered more alcohol than the wild-type mice. Additionally, the elevated anxiety-like behavior in the DOR KO mice returned to wild- type levels after alcohol administration. Therefore, the DOR may be an important factor to explain the relationship between emotional states and the initiation and maintenance of drug abuse. Interestingly, DOR-deficient mice do not develop tolerance to morphine,
25 which indicates that it may be involved in some kind of adaptive response to chronic morphine exposure (Zhu et al., 1999). Kappa and mu opioid receptors have largely been shown to oppose each other’s effects. The KOR is involved in pain control, particularly visceral pain. KOR-deficient mice showed no obvious change in the perception of mechanical or thermal pain; however, they did show an enhanced response to peritoneal acetic acid injections. The administration of kappa agonists typically leads to dysphoria. When KOR KO mice were exposed to the kappa agonist, U 50,488H, conditioned place aversion was almost absent. These mutant mice also displayed an attenuation of naloxone-precipitated withdrawal syndrome when chronically treated with morphine (Simonin et al., 1998). This suggests that the KOR participates in adaptations to long-term exposure to opioid drugs. All three subtypes of opioid receptors appear to be potentially involved in some aspect of addiction. A summary of the opioid receptor knockout mice studies is presented in Table 1.5.
26 Table 1.5: Summary of opioid receptor knockout mice studies Summary of responses to drugs and spontaneous behaviors observed in opioid receptor knockout mice. Adapted from Gaveriaux-Ruff and Kieffer, 2002.
Knockout Drug Response Behavior in absence of drugs MOR Morphine Decreased analgesia, Decreased locomotion reward and dependence Increased thermal nociception Decreased anxiety and depression δ agonists Decreased analgesia, reward and dependence THC, alcohol Decreased reward DOR δ agonists Decreased analgesia Increased locomotion Increased anxiety, and depression Morphine Decreased tolerance Alcohol Increased intake THC No change in reward KOR κ agonists Decreased analgesia and No change in locomotion dysphoria Increased visceral nociception No change in anxiety or depression Morphine Decreased Dependence THC Decreased dysphoria
1.3.4 Physiological and Psychological Adaptations to Opioid Exposure
Our current understanding of opioid pharmacology suggests that activation of the MOR by agonists, either endogenous or exogenous, is the most effective way to modulate pain signals. However, DOR- and KOR-specific ligands have also demonstrated pain modulation, but not to the same degree as MOR agonists.
27 How these drugs cause the development of tolerance, physical dependence, and addiction is still poorly understood. Tolerance is the physiological process of adaptation, where increasing amounts of the same drug are needed to achieve the same therapeutic benefit. Physical dependence refers to the adaptive process whereby cessation of drug use causes the onset of unpleasant withdrawal symptoms. Physical dependence can be tested by treating individuals with an opioid antagonist like naloxone. One of the severe side effects of opioid drugs is the development of addiction or substance dependence. Addiction can be summarized as the inappropriate use of a drug despite negative consequences (i.e. loss of job, criminal activity, poor health). The concern over “creating” addicts has been shown to influence the prescribing habits of physicians, leading to under-treatment of severe pain. For this reason, much effort has been focused on a better understanding of these side effects and attempting to synthesize opioid agonists that provide analgesia, but do not lead to tolerance, physical dependence, or addiction. These efforts have resulted in an extensive library of agonists, antagonists, and mixed agonists-antagonists; however, no compound has been developed that relieves pain with no side effects. Prolonged exposure to opioid agonists has been shown to cause pronounced changes in the structure of neurons and the synaptic organization of the brain (Robinson and Kolb, 2004). These changes are long lasting and may contribute to the characteristics of addiction, including drug craving, compulsive drug use, and analgesic tolerance. For example, in the ventral tegmental area chronic morphine exposure via subcutaneous morphine pellets caused reductions in the overall size of the neurons in the mesolimbic dopaminergic reward pathway. This long-term exposure also caused less branching and fewer spines on the dendrites of medium spiny neurons in the shell of the nucleus accumbens and on pyramidal cells in the prefrontal and parietal cortex (Robinson and Kolb, 1999). The significance of these morphological changes is based on the understanding that memory, learning, and other behavioral functions are associated with the reorganization of synaptic connections in relevant neural circuits (Lamprecht and LeDoux, 2004). Therefore, exposure to opioid drugs may be altering or creating neural connections that potentiate drug-seeking behaviors. However, the susceptibility for
28 addiction is highly variable in individuals. Twin, family, and adoption studies have shown that addiction is a complex genetic disorder that can be influenced by environmental factors, such as home environment, parental support, socioeconomic status, and education level (Lachman, 2006). This variable susceptibility has also been documented in rats. Certain strains, such as Lewis, are more susceptible to drugs of abuse than other strains, such as Fischer 344 (Suzuki et al., 1988). In Lewis rats, morphine self-administration caused decreases in the branching and size of dendrites of pyramidal cells in the motor cortex (Ballesteros-Yanez et al., 2007), but not in the Fischer 344 rats (Ballesteros-Yanez et al., 2008). When these two strains were compared prior to morphine administration, Fischer 344 rats had neurons with shorter dendrites containing fewer spines and branches when compared to neurons in Lewis rats (Ballesteros-Yanez et al., 2008). These studies imply that significant changes to the structure of dendrites in response to long-term opioid agonist exposure may contribute to susceptibility to addiction. Apart from its general inhibitory effects on dendritic structure, morphine has also been shown to decrease neurogenesis in the hippocampus of adult rats (Eisch et al., 2000) and to inhibit long-term potentiation (LTP) of excitatory synapses on CA1 neurons in the hippocampus (Williams et al., 2001). Additionally, morphine has been shown to inhibit the release of γ-aminobutyric acid (GABA) from neurons in the VTA (Nugent et al., 2007). The exact role of neurogenesis in the hippocampus is not definitively known, but recent evidence suggest that it plays an important role in integrating memories of events that occur closely in time (Deng et al., 2010). Therefore, the inhibition of neurogenesis by chronic morphine treatment may have deleterious effects on the function of this brain region. The role of the MOR in mediating this effect was confirmed in a separate study where it was found that the numbers and maturation of neurons in the granule cell layer was increased in MOR knockout mice (Harburg et al., 2007). LTP is important for synaptic plasticity, which has been implicated as a potential cellular mechanism that the brain uses to encode activity- or experience-dependent events, such as learning and memory (Cooke and Bliss, 2006). Presently, there is no clear relationship between LTP and neurogenesis. Inhibition of LTP in the hippocampus and VTA may lead to abnormal
29 patterns of neural activity with unknown behavioral consequences. Thus, mechanisms that are responsible for the structural and synaptic plasticity of the brain may play important roles in the development of addiction. However, the specific mechanisms linking opioid agonist activity to neuronal alterations observed with chronic opioid exposure remain unknown.
1.3.5 Regulation of MOR
As GPCRs, MORs are regulated by the same mechanisms that regulate these receptors, including endocytosis, recycling, and degradation (Drake et al., 2006; Marchese et al., 2008). These regulatory mechanisms are usually initiated in response to agonist receptor activation (Gainetdinov et al., 2004). Activated MORs undergo internalization (or endocytosis) in response to most agonist drugs in most experimental systems (Johnson et al., 2005). In general, agonist binding to MOR leads to downstream signaling events mediated by Gαi/Gαo proteins. This now activated receptor can serve as a target of G protein-coupled receptor kinases, which phosphorylate receptors at specific threonine and serine residues. Phosphorylated receptors are then capable of binding to arrestins that bind to clathrin and AP2, the clathrin-adaptor protein, which promotes internalization of receptors by clathrin-coated pits. This process is known as homologous desensitization (Gainetdinov et al., 2004). Following internalization, MORs are recycled back to the surface during resensitization, while other opioid receptors, such as DORs, are targeted to lysosomes for degradation and down-regulation (Tanowitz and von Zastrow, 2003). Using chimeric MORs and DORs, a specific sequence present in the C- terminal tail of the MOR was found to be both necessary and sufficient for recycling as opposed to degradation. More recent studies have localized this sequence to the last 17 residues in the C-terminal tail called the MOR regulatory sequence (MRS) (Hislop et al., 2009). The MRS promotes MOR recycling after short-term agonist exposure. The essential trafficking of the MOR is controlled by the MRS, as mutant receptors lacking this sequence are rapidly down-regulated. Some 3’ splice variants of the MOR lack the MRS, which may account for increased tolerance to drugs that normally cause recycling
30 of the receptor (Pasternak et al., 2004). However, the MOR also undergoes agonist- stimulated ubiquitination that promotes the sorting of receptors from the endosome limiting membrane to the lumenal compartment. This ubiquitination of the MOR functions independently and downstream of the ‘recycling versus degradation’ decision of the MRS. More importantly, the ubiquitin-directed step is not required to direct internalized MORs to lysosomes or to initiate their proteolysis, but does play a role in the destruction of the MOR’s opioid binding site (Hislop et al., 2011). However, the prototypical opioid agonist, morphine, does not cause endocytosis of MORs in human embryonic kidney (HEK) 293 cells that have been stably transfected with the receptor (Keith et al., 1996). Interestingly, the efficacy of morphine to produce internalization is highly dependent on the system and brain region being examined (Johnson et al., 2005). Brain slices from transgenic mice expressing epitope-tagged MORs in neurons of the locus ceruleus showed no morphine-induced internalization of MORs (Arttamangkul et al., 2008) and neither did MORs heterologously expressed in neurons of the hippocampus (Bushell et al., 2002). In contrast, primary cultures of dissociated rat striatal neurons showed rapid endocytosis of MORs in response to morphine (Haberstock-Debic et al., 2005). These studies suggest that the expression of cellular components that regulate MORs, such as GRKs and arrestins, are likely cell-type specific. For example, GRK2 has been shown to phosphorylate MORs in HEK293 cell culture models (Schulz et al., 2004), but has not been clearly shown in vivo (Johnson et al., 2005). However, a recent study on the effect of beta arrestin on MOR regulation has shed some light on the reason for morphine’s poor internalization. Mouse embryonic fibroblasts that were deficient in beta arrestin 1, beta arrestin 2 or both were assessed for their ability to regulate MOR when treated with morphine or DAMGO. DAMGO was shown to recruit both beta arrestin 1 and beta arrestin 2 to the MOR and either was sufficient to promote internalization. DAMGO was also shown to promote ubiquitination of the MOR, which did not occur in the absence of beta arrestin 1. In contrast morphine recruited beta arrestin 2 to the MOR, but not beta arrestin 1. This interaction was sufficient to promote internalization of the MOR, but not ubiquitination of the MOR. Taken together these results imply that beta arrestin 1 is important in the ubiquitination of the MOR (Groer et al., 2011).
31 Despite these agonist-specific differences on MOR internalization, all agonists produce similar physiological responses short-term (euphoria and analgesia) and adaptation (tolerance and physical dependence) long-term. A seminal study from the lab of Marshall Nirenberg attempted to correlate the development of tolerance and physical dependence in animals with the specific effects that opioid agonists produce in cells. This study showed that repeated administration of an opioid agonist on neurons diminished their responsiveness to the drug as measured by the progressive dampening of agonist-mediated cyclic adenosine monophosphate (cAMP) inhibition. When the agonist was removed, the levels of cAMP rebounded above baseline (Sharma et al., 1975). This suggested that receptor desensitization and subsequent cellular compensatory mechanisms may underlie the development of tolerance and physical dependence. Although numerous studies have continued to explore the mechanisms of MOR regulation in an attempt to better understand the cellular context of tolerance, physical dependence, and addiction; no clear mechanistic explanation for these adaptations has been found (Bailey and Connor, 2005).
1.4 GPCR Interacting Proteins
The GPCR family is composed of a large and multifaceted group of cell surface proteins that transduce extracellular signals into intracellular responses (Hill, 2006). GPCR signaling is mediated by the activation of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins), which act as secondary messengers to activate or inhibit other proteins creating a cascade of events that produce a specific cellular response (Hamm and Gilchrist, 1996). The versatility of these receptors is illustrated by the varied physiological events they mediate, including sensory recognition (vision, taste, and smell), endocrine regulation, and complex behavior phenomena (Gainetdinov et al., 2004). In the central nervous system, these receptors serve as important targets for a number of neuromodulatory drugs, such as psychotics and opioid analgesics (Hill, 2006). Mounting evidence indicates that GPCRs bind to other proteins to form multiprotein signaling complexes or signalplexes (Kabbani and Levenson, 2007; Milligan, 2005). The
32 interacting proteins have the potential to mediate previously unknown downstream effects or to alter known mechanisms of receptor signaling and regulation (Bockaert et al., 2010). For example, neuronal calcium sensor-1 (NCS-1) was shown to interact with the D2 dopamine receptor (D2R) and interfere with its phosphorylation, which prevented its endocytosis and desensitization (Kabbani et al., 2002). Since the MOR is known to mediate the analgesic and rewarding properties of opioid drugs, proteins that interact with the MOR likely play important roles in the development of tolerance, physical dependence, and addiction to opioid drugs (Milligan, 2005). Identifying and characterizing these proteins may provide insight into the mechanisms that underlie these phenomena.
1.4.1 MOR Interacting Proteins
A comprehensive list of proteins that have been shown to interact with the MOR is shown in Table 1.6. Many of these proteins affect the trafficking of the MOR by promoting endocytosis (synaptophysin, PLD2, and M6a), helping to recycle and sort internalized receptors (filamin A and GASP), or interfering with internalization (RanBP9). Others affect the signaling properties of the MOR by inhibiting activation of G-proteins (CaM and periplakin) or by inhibiting the ability of the MOR to inhibit cAMP levels (PCK1) (Georgoussi et al., 2012). A novel signaling outcome for the MOR was found when it was discovered that the MOR interacts with STAT5A. STAT5A is a transcription factor and a member of the Signal Transducers and Activators of Transcription family. When the MOR was activated, downstream transcriptional activation of STAT-responsive reporter genes was detected in this study (Mazarakou and Georgoussi, 2005). Although the role of these MORIPs has not been extensively studied in terms of addiction, the identity of these proteins has contributed to the overall knowledge of the MOR life cycle and downstream signaling events.
33 Table 1.6: Previously identified MORIPs The location of the interaction (if known), a brief summary of the function of the interaction and the original reference that identified the interaction are listed.
Interacting Protein Location Function Reference on MOR Alpha 2A adrenergic ND Receptor signaling (Jordan et al., receptor 2003)
Beta-arrestin 1 ND Receptor desensitization and (Bohn et al., 2000) Beta-arrestin 2 endocytosis (Molinari et al., 2010) Calmodulin (CaM) IL3 Interferes with activation of G (Wang et al., 1999) proteins
Cannabinoid receptor ND Inhibition of signaling (Rios et al., 2006) (CB-1)
Delta opioid receptor ND Modulation of signaling and ligand (Gomes et al., (DOR) binding 2000)
Filamin A C-tail Regulation of receptor recycling (Onoprishvili et and degradation al., 2003)
Gai2/Gai3 IL3, C-tail Receptor signaling (Georgoussi et al., 1997) Glycoprotein M6a ND Endocytosis and recycling of (Wu et al., 2007) receptor
G protein-regulated IL-3 Regulates receptor distribution in (Ge et al., 2009) inducer of neurite lipid rafts outgrowth
Heat shock protein C-tail ND (Ancevska-Taneva HLJ1 et al., 2006)
Kappa opioid receptor ND Sex-dependent antinociception (Chakrabarti et al., 2010)
Opioid receptor-like 1 C-tail Inhibits receptor signaling (Wang et al., 2005) receptor (ORL1)
34 Periplakin C-tail Interferes with activation of G (Feng et al., 2003) proteins
Phospholipase D2 C-tail Regulates receptor endocytosis (Koch et al., 2003) (PLD2)
Protein kinase Ci C-tail Negatively regulates receptor (Guang et al., (PKCi) desensitization 2004)
Ran binding protein 9 C-tail Negatively regulates receptor (Talbot et al., (RanBP9) internalization 2009)
Regulator of G-protein C-tail Signaling and scaffolding (Georgoussi et al., signaling 4 (RGS4) 2006)
RGS9-2 ND Receptor signaling and endocytosis (Garzon et al., 2005a; Garzon et al., 2005b)
Somatostatin receptor ND Internalization and desensitization (Pfeiffer et al., 2002)
Spinophilin ND Modulates receptor signaling and (Charlton et al., endocytosis 2008)
STAT5A C-tail Regulates receptor mediated (Mazarakou and transcription Georgoussi, 2005)
Substance P receptor ND Receptor internalization and (Pfeiffer et al., (NK-1) resensitization 2003)
Synaptophysin IL3 Modulates receptor signaling and (Liang et al., 2007) endocytosis
Wntless (WLS), GPR IL2 Wnt secretion (Jin et al., 2010) 177
Of particular interest is the discovery of an interaction between MORs and spinophilin, a scaffolding protein enriched in dendritic spines. Overexpression of spinophilin in rat pheochromocytoma 12 (PC12) cells allowed the morphine-induced endocytosis of the MOR within 30 minutes, an effect that was not seen in control cells. In agreement with this finding, the loss of spinophilin expression in spinophilin KO mice
35 was found to increase measures of MOR signaling by delaying receptor internalization. More importantly, in behavioral studies, spinophilin KO mice exhibited greater sensitivity to the rewarding properties of morphine and developed a higher degree of physical dependence (Charlton et al., 2008). This was the first study to show that the behavioral response of an animal to opioid drugs could be affected by altering the expression of a MOR interacting protein (MORIP). As more interactions and multiprotein complexes are identified, researchers should be able to propose functional roles for new associations, identify novel therapeutic targets, and devise more effective strategies for encountering pathologies where the functionality of MORs is relevant (Georgoussi et al., 2012). Thus, the continued identification and characterization of MORIPs will be necessary to get a better understanding of the variety of functions that are mediated by the MOR and the roles that these proteins may play in the pathogenesis and treatment of complex neuropsychiatric diseases (Bockaert et al., 2010).
1.4.2 Identifying Candidate MORIPs
Many well-characterized methods exist to identify potential GPCR interacting proteins. The conventional yeast two-hybrid approach was first introduced by Fields and Song (1989) as a method for identifying an interaction between two proteins. This technique relies on the reconstitution of the GAL4 yeast protein (a transcription factor), which contains two distinct and separable functional domains: a DNA-binding domain (BD) and a transcription-activating domain (AD). To test for an interaction, one protein is fused with the BD and another is fused to the AD. Plasmid constructs are then transformed into yeast cells that have been modified to have certain nutritional requirements. If the proteins form an interaction, the binding and activating domains will come into close proximity to one another and reconstitute the functional activity of the GAL4 protein. This protein can then activate transcription and translation of the yeast lacZ gene, which encodes for the enzyme β-galactosidase. This enzyme catalyzes the chemical conversion of 5-bromo-4-chloro-3-indolyl-β-galactopyranoside to galactose and 5-bromo-4-chloro-3-hydroxyindole, which is visualized as a blue pigment when oxidized
36 to 5,5’-dibromo-4,4-dichloro-indigo. This method has been utilized as a screening platform by the development of various cDNA libraries fused to the AD of the GAL4 protein. The selected library can then be tested against any ‘bait’ protein fused to the BD of GAL4 (Miller and Stagljar, 2004). This approach is limited because the protein interactions must take place within the nucleus of yeast cells. This technical issue makes it difficult to use this conventional method with highly hydrophobic or membrane-bound proteins (Lentze and Auerbach, 2008). In order to overcome these limitations, a membrane-based yeast two-hybrid method was developed to enable the ability to detect interactions between proteins that occur at the plasma membrane (Iyer et al., 2005; Lentze and Auerbach, 2008). This method utilizes a split-ubiquitin molecule where the N-terminal half (fused to a transcription factor) is fused to one protein and the C-terminal half is fused to another protein. If an interaction occurs between the two proteins, the ubiquitin molecule will be reconstituted and lead to the cleavage of the transcription factor that is fused to the N-terminal half of the split-ubiquitin molecule.
1.5 Rationale and Hypothesis
Although opioid analgesics are efficacious for the treatment of pain, their use is limited due to the side effects that they produce. The most undesirable side effect that these drugs produce is the development of addiction. Unfortunately, the precise cellular and molecular mechanisms that cause this effect are not yet known. What is known is that these drugs activate the MOR and knockout studies have implicated the MOR as the single opioid receptor subtype that mediates both the analgesic and rewarding properties of these drugs. A growing body of evidence suggests that MORs are part of large signaling complexes containing various MOR interacting proteins. Previously identified MORIPs have been found to alter the regulation and signaling properties of the MOR. Therefore, we hypothesize that identifying and characterizing novel MORIPs will help us to better understand the cellular and molecular mechanisms that underlie the addiction process.
37 In the following chapters, we examine the identification and validation of interactions between the MOR and several novel MORIPs (Chapter 2), the functional significance of the interaction between the MOR and novel MORIPs, seven in absentia homologs 1 and 2 (Chapter 3), and the significance of the interactions between the MOR and several novel MORIPs with opioid drug exposure (Chapter 4). The final chapter discusses the potential relevance of the MOR-MORIP interactions in relation to MOR regulation, possible implications for addiction, and directions for future research.
38 Chapter 2
Identification of MOR Interacting Proteins
2.1 Introduction
Most clinically relevant opioid agonists provide analgesia by activating MORs
(Berrettini et al., 1994; Inturrisi, 2002). MOR activation produces a euphoric effect in many individuals, which is considered the primary motivational reward for abusing opioid drugs (Bailey and Connor, 2005). The importance of the MOR in analgesia and opioid dependence has been demonstrated by MOR knockout mice which show significantly reduced sensitivity to both the analgesic and rewarding properties of opioids
(Contarino et al., 2002; Matthes et al., 1996). Like most GPCRs, the MOR is regulated by multiple mechanisms, such as receptor desensitization, internalization, degradation, and recycling (Petruzzi et al., 1997; Raehal and Bohn, 2005). The desensitization and trafficking of MORs has been shown by a number of studies to represent key aspects in the development of opioid tolerance and dependence (Bailey and Connor, 2005; Corbett et al., 2006; von Zastrow et al., 2003; Waldhoer et al., 2004). Therefore, understanding the mechanisms that regulate MOR signaling and trafficking is crucial for determining the physiological basis of opioid dependence and enhancing opioid receptor pharmacology for the treatment of addiction and pain.
A number of studies have shown that MOR desensitization and receptor trafficking can increase the rewarding properties of opioid drugs, while reducing the development of opioid tolerance and addiction-like behaviors (Berger and Whistler,
39 2011; Finn and Whistler, 2001; He et al., 2002; He et al., 2009; Martini and Whistler,
2007; von Zastrow et al., 2003; Whistler et al., 1999). However, the specific molecular mechanisms that regulate these processes are largely unknown. Elucidating the mechanisms that regulate MOR signaling and trafficking is critical for determining the cellular response to opioid agonist drugs and for opening new avenues of investigation into the pharmacotherapy of pain management.
Evidence indicates that GPCR signaling is modulated by proteins that bind to form mulitcomponent protein assemblies, or signalplexes, involving interactions between themselves, other GPCRs, and with other interacting proteins (Kabbani and Levenson,
2007; Milligan, 2005). A number of proteins have been identified and shown to affect
MOR biogenesis, trafficking, and signaling. For example, β-arrestin, calmodulin, and synaptophysin have all been found to bind to the third intracellular loop of the MOR
(Georgoussi et al., 2011). Arrestins have been shown to be key components of MOR desensitization because they can block the receptor’s interaction with G-proteins and can promote recycling of the receptor through interactions with the clathrin assembly machinery and clathrin-dependent endocytosis (Bohn et al., 2000; Molinari et al., 2010).
Calmodulin is believed to compete with G-protein for binding to the receptor and may serve as an independent second messenger molecule released in response to MOR stimulation (Wang et al., 1999). Synaptophysin is a major integral membrane glycoprotein found in synaptic vesicles that interacts with a variety of nerve terminal proteins such as dynamin I and adaptor protein 1 (AP-1). It is believed that synaptophysin recruits dynamin to the plasma membrane to facilitate fusion of clathrin- coated vesicles so that MOR can be endocytosed (Liang et al. 2007). The complete list of
40 currently known MOR interacting proteins along with a short description of the functional significance of each interactor is shown in Table 1.6 of Chapter 1.
To better understand the potential role of MORIPs in the MOR life cycle, we have performed conventional and modified yeast two-hybrid (Y2H) studies designed to identify novel constituents of the MOR signalplex. Previous interaction screens for
MORIPs have primarily utilized the third intracellular loop (IL3) or the C-terminal tail
(Ctail) of the MOR as bait (Georgoussi et al., 2012). Previous studies in our laboratory identified several D2 dopamine receptor interacting proteins, using the second intracellular loop as bait in a conventional yeast two-hybrid screen (Kabbani and
Levenson, 2007). Therefore, we utilized the second intracellular loop as well as the entire MOR to screen human brain cDNA libraries in order to expand the growing list of
MORIPs. Using these approaches, we have identified ten novel MOR binding partners.
Functional characterization of these interacting proteins will serve to further our understanding of the mechanisms regulating MOR-mediated signaling and may help elucidate the underlying molecular basis of cellular response to opioid agonist drugs.
2.2 Experimental Procedures
2.2.1 Membrane Yeast Two-Hybrid Screen
A modified split-ubiquitin yeast two-hybrid (MYTH) screen was performed in collaboration with Igor Stagljar (University of Toronto) as described previously (Iyer et al., 2005; Kittanakom et al., 2009; Stagljar et al., 1998). Briefly, the full-length human
41 MOR (transcript MOR-1) cDNA was cloned into the bait vector pCCW-STE
(Dualsystems Biotech AG, Switzerland) and the human fetal brain cDNA library was cloned into the prey vector pRP3-N (Dualsystems). These constructs were then sequentially transformed into S. cerevisiae reporter strain THY.AP4. This transformation yielded 6 x 106 transformants/μg DNA on synthetic dropout (SD) agar plates (-Trp/-Leu/-
His/-Ade; Clontech, Palo Alto, CA) containing 3-amino-1,2,4-triazole (3AT). The identity of the putative MORIPs was determined by recovering the prey plasmid from the transformed yeast colonies. The cDNA clone in the plasmid was sequenced using primers designed against the regions flanking the cloning site. These sequences were then analyzed using Basic Local Alignment Search Tool (BLAST) to determine the identity of the clone.
2.2.2 Conventional Yeast Two-Hybrid Screen
Two conventional yeast two-hybrid screens were performed as described previously (Lin et al., 2001; 2005) using either the second intracellular loop (IL2; amino acids 166-187) or the C-terminal tail (C-tail; residues 361-420) of the MOR as bait to screen a fetal human brain cDNA library (Clontech). The MORIL2 and MOR C-tail were cloned into the yeast GAL4 DNA binding domain expression vector pAS2-1
(Clonetech), while the human fetal brain cDNA library was provided in the GAL4 activation domain vector pACT2 (Clontech). Bait and prey plasmids were successively transformed into yeast strain MaV103. Transformation of the yeast with MORIL2 and the fetal human brain library produced approximately 2 x 106 transformants/μg DNA on
42 quadruple dropout plates (-Leu/-Trp/-His/-Ura; Clontech) containing 3AT.
Transformation of the yeast with MOR C-tail and the fetal human brain library yielded no colonies on transformation efficiency plates; therefore the number of transformants/μg
DNA could not be determined. Interactions were assayed for β-galactosidase (β-gal) activity via the nitrocellulose lift method (Lin R, 2005). cDNAs were extracted from yeast colonies, sequenced, and subjected to BLAST analysis to determine their identities.
2.2.3 Mapping of Binding Regions
To map sites of interaction between the MOR and the newly identified MORIPs, each MOR intracellular loop (IL) was tested for interaction with individual MORIPs using the traditional Y2H method. MOR IL domains (IL1, amino acids 97-102; IL2, amino acids 166-187; IL3, amino acids 259-282; and C- tail residues 361-420) were separately ligated into pAS2-1 (Clontech) and assayed for interaction with candidate
MORIP cDNA clones in pACT2. Bait and prey plasmids were simultaneously co- transformed into S. cerevisiae strain MaV103 and interactions were assayed for β-gal activity as described above.
2.2.4 Glutathione S-transferase Pulldown Assay
MORIP-GST (glutathione S-transferase) fusion proteins were constructed by separately fusing cDNAs encoding human AUP1 (residues 1-410), human DOK4
(residues 1-326), human CSN5 (residues 1-335), or the second intracellular loop of the
43 mu-opioid receptor (MOR-IL2; residues 166-187) to GST in the expression vector pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ). The polymerase chain reaction
(PCR) primers used for this cloning can be found in Table 2.1. cDNAs encoding the
MOR-IL2 (residues 166-187), SIAH1 (residues 1-233) and RanBP9 (residues 170-381) were subcloned into the pET30a expression vector (Novagen, Madison, WI) containing an S-tag. Fusion proteins were induced in Escherichia coli strain BL21 (DE3) using the
ZYP-5052 auto-induction media as described previously (De Cotiis et al., 2008; Studier,
2005). MORIP-GST fusion proteins bound to glutathione sepharose beads (GE
Healthcare, Piscataway, NJ) were used to pull down S-tagged proteins from bacterial lysates as previously described (Jin et al., 2010; Lin et al., 2001). Eluted proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) filter for western blot analysis. The filter was probed with a horseradish peroxidase (HRP)- conjugated anti-S-tag antibody (1:5000 dilution, Novagen, Madison, WI), and immunoreactivity detected by enhanced chemiluminescence with an ECL Plus kit (GE
Healthcare).
44 Table 2.1: PCR primers used for cloning MORIPs This table lists the PCR primers that were used to clone MORIPs into pET-30a to produce S-tagged proteins or into pGEX-4T-1 to produce GST-fusion proteins for use in GST pull down experiments.
Construct Direction PCR primer sequence
SIAH2 S-tag Forward GAT ATC ATC AGG AAC CTG GCT ATG GAG AAG GTG Reverse CTC GAG GGC CAA CCC CAT TCA TCC CAG GT
SIAH1 S-tag Forward GAT ATC ATG AGC CGT CAG ACT GCT ACA GCA TTA CC Reverse CTC GAG TCA ACA CAT GGC AAT AGT TAC ATT GAT GCC
CSN5-GST Forward GGA TCC GTA AAG TTG CGT CTT GGT TGT Reverse GAA TTC TGT CTT TCA GGT AAA GTA CTT CTC AGA GAC TGT
DOK4-GST Forward GGA TCC ATG GCG ACC AAT TTC AGT GAC ATC GTC AAG Reverse GAA TTC CTG TGG TCA CTG GGA TGG GGT CTT
AUP1-GST Forward GAA TTC ATG GAG CTT CCC TCA GGG CCG Reverse CTC GAG GTT CCT TTG AGC TCA GTC AGC CTC
MORIL2 S-tag Forward GGG GAA TTC GAT CGA TAC ATT GCA GTC TGC CAC CCT MORIL2 GST Reverse GGG CTC GAG TTT GGC ATT TCG GGG AGT ACG GAA ATC
RanBP9-GST Forward GGA TCC GCC GTG GAC GAA CAA GAG ACG Reverse CTC GAG GGT CTG CCA TTC TCC TCG ATC
2.2.5 Cell Culture
Human embryonic kidney (HEK) 293 cells stably transfected with either FLAG- tagged mu-opioid receptor (HEK-MOR), delta opioid receptor (HEK-DOR), or kappa opioid receptor (HEK-KOR) were maintained in Dulbecco's Modified Eagle's Medium
45 (DMEM) supplemented with 10% fetal bovine serum (FBS) and 400 μg/mL G418.
HEK-MOR and HEK-DOR cell lines were generously provided by Dr. Mark van
Zastrow (University of California San Francisco). HEK-KOR cells were a gift from Dr.
Lee-Yuan Liu-Chen (Temple University School of Medicine).
2.2.6 Co-immunoprecipitation and Western blot Analysis
HEK-MOR, DOR, or KOR cells (stably expressing FLAG-tagged MOR, DOR, or
KOR, respectively) were separately transfected with constructs encoding full-length
MORIPs sub-cloned in the pCMV-Tag3B expression vector (Stratagene, La Jolla, CA) containing a myc-tag. Transfections were carried out using Effectene transfection reagent (Qiagen, Valencia, CA). Cells were cultured for 24 hours in DMEM and then lysed in 1X lysis buffer (20 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,
1% Triton X-100 and 1mg/mL leupeptin) supplemented with protease inhibitor cocktail
(Pierce, Rockford, IL). MOR, DOR, and KOR were immunoprecipitated from total cell lysates using a polyclonal rabbit anti-FLAG antibody (Sigma, St. Louis, MO) coupled to
Protein-G Dynabeads (Invitrogen). Western blot analysis of immunoprecipitated complexes was performed by using a monoclonal mouse anti-myc antibody (1:5000 dilution; Millipore, Billerica, MA) and a rabbit anti-mouse HRP-conjugated secondary antibody (1:20,000 dilution; Jackson ImmunoResearch, West Grove, PA).
Immunoreactivity was detected by ECL Plus.
46 2.2.7 Immunofluorescence Microscopy
Immunohistochemistry was performed by transfecting HEK-MOR as described for the co-immunoprecipitation. Cells were then split onto collagen coated coverslips
(BD Biosciences) and allowed to grow overnight. After 18 hours, cells were fixed with
4% paraformaldehyde and 5% sucrose. The cells were immunostained with monoclonal mouse anti-myc antibodies (1:1000 dilution; Millipore) and rabbit anti-MOR antibodies
(1:500; AB5511, Millipore). After this initial staining, the cells were washed with 1X
PBS, and exposed to FITC conjugated donkey anti-rabbit antibodies (1:500 dilution;
Jackson ImmunoResearch) and rhodamine conjugated donkey anti-mouse antibodies
(1:500 dilution; Jackson ImmunoResearch). Images were obtained with a confocal microscope (Zeiss LSM 510 Meta, Carl Zeiss Inc., Thornwood, NY), and digital images were captured and imported with the LSM 5 image browser (Carl Zeiss, Inc.).
2.3 Results
2.3.1 Identification of Novel MOR Interacting Proteins
In the MYTH screen, 104 positive clones were obtained from the 6 x 106 colonies screened. The putative MOR interacting proteins identified in this screen can be found in
Table 2.2. Sequence analysis identified GPR177 (WLS) (Jin et al., 2010), a putative multi-pass membrane-spanning protein that is the mammalian ortholog of Drosphila
Wntless/Evi/Sprinter (reviewed in Hausmann et al., 2007). WLS plays an important role in Wnt protein secretion, a process that is inhibited by the opioid agonist morphine (Jin et
47 al., 2010). Members of the Wnt pathway, such as WLS, are of particular interest because they have been shown to affect neuronal development (Salinas and Zou, 2008). Many of these Wnt-mediated effects mirror the effects induced by opioid agonist activity, but with opposite outcomes. For example, opioid drugs have been shown to inhibit neurogenesis in the adult rat hippocampus (Eisch et al., 2000), whereas Wnt proteins have been demonstrated to promote neurogenesis in the adult rat hippocampus (Lie et al., 2005).
Vesicle-associated membrane protein-A (VAPA or VAP33) plays a role in endoplasmic reticulum to Golgi transport (Peretti et al., 2008; Wyles et al., 2002). The number and activity of opioid receptors at the cell surface are important in determining their capacity to modulate downstream signaling. The receptor number at the cell surface can be regulated through the biosynthesis pathway, including transcription, translation, protein folding, and transport (Chen et al., 2006a; Chen et al., 2006b; Petaja-Repo et al., 2000).
Although the general consensus is that total opioid receptor protein does not change in response to most opioid drugs (Christie, 2008; Johnson et al., 2005; Patel et al., 2002;
Stafford et al., 2001). This is not always the case. In a study using rat nucleus accumbens, acute morphine treatment was shown to cause an increase in the amount of epitope-tagged MORs in neuronal processes (Haberstock-Debic et al., 2003). Therefore, proteins involved in the biosynthetic pathway may play an important role in responses to opioid drugs. Connexin 37 (Cx37), and voltage-gated potassium channel Kv5.1 were also identified in the MYTH screen (Table 2.1). These proteins play a role in, gap junction formation (Haefliger et al., 1992), and regulation of resting membrane potential
(Kramer et al., 1998), respectively. Gap junctions mediate electrical transmission between neurons by providing a low resistance pathway for the spread of electrical
48 currents and small metabolites (Bennett, 1997), while voltage-gated potassium channels help to repolarize neurons after an action potential has been generated. Changes in electrical transmission can contribute to synaptic plasticity in the form of long-term potentiation (strengthening of synapses) or long-term depression (weakening of synapses)
(Dacher and Nugent, 2011). Thus, these two proteins may be important in the synaptic plasticity observed with opioid addiction.
Table 2.2: Putative MORIPs identified in MYTH screen ADD3 Adducing gamma subunit GJA4 Gap junction protein, alpha 4 GPR177 Orphan g-protein coupled receptor KCNF1 Potassium channel subunit SLC31A2 Putative copper transporter SLC39A13 Putative zinc transporter SLC9A9 Putative Na+/H+ exchanger VAP33 VAMP-associated protein A WHSC2 Wolf-Hirschhorn syndrome candidate 2 YIPF3 Yipl domain family, member 3
In the conventional yeast two-hybrid screen using the second intracellular loop
(IL2) of the MOR as bait to screen a fetal human brain cDNA library, 20 positive clones were obtained from the 2 x 106 colonies screened. Sequence analysis identified several clones encoding components involved in the ubiquitin pathway and in signal transduction
(Table 2.3). These included COP9 subunit 5 (CSN5), a protein proposed to regulate
49 exosomal protein sorting in both a deubiquitinating activity-dependent and -independent manner (Liu et al., 2009), ancient ubiquitous protein 1 (AUP1), a protein that promotes ubiquitination of misfolded proteins (Mueller et al., 2008), and seven in absentia homologs 1 and 2 (SIAH1 and SIAH2), known really interesting new gene (RING) finger
E3 ubiquitin ligases (Hu et al., 1997a). The ubiquitin pathway has been shown to mediate synaptic plasticity, learning and memory, and neurogenesis (Bingol and Sheng,
2011; Tuoc and Stoykova, 2010). Thus, these proteins were of interest for further study.
Additionally, four putative scaffolding proteins, docking protein 4 (DOK4), docking protein 5 (DOK5), zyxin, and Ran binding protein 9 (RanBP9) were also identified as candidate mu-opioid receptor interacting proteins (Table 2.3). Scaffolding proteins bind and bring together two or more signaling proteins. In doing so, these proteins can direct the flow of information by activating, coordinating, and regulating signaling events
(Buday and Tompa, 2010). Drugs of abuse can alter MOR signaling (Bailey and Connor,
2005; Christie, 2008), so these types of proteins are of interest for further study.
50 Table 2.3: Putative MORIPs identified in conventional yeast two-hybrid screens MORIP NAME BAIT RESIDUES
AUP1 Ancient ubiquitous protein 1 IL2 321-410 CSN 5 Constitutive photomorphogenic signalosome 9 IL2 25-318 subunit 5 DOK4 Downstream of kinase 4 IL2 65-289 DOK5 Downstream of kinase 5 IL2 40-306 PLAGL1 Pleiomorphic adenoma gene-like 1 IL2 28-165 PLXNA4 Plexin A4 C-tail Non-coding RanBP9 RAN binding protein 9 IL2, C-tail 88-366 SIAH1 Seven in absentia homolog 1 IL2 1-233 SIAH2 Seven in absentia homolog 2 IL2 122-324 Zyxin Zyxin IL2 203-483
In the conventional yeast two-hybrid screen using the C-terminal tail of the MOR as bait to screen a fetal human brain cDNA library, 7 positive clones were obtained in the screen. The transformation efficiency plates did not produce yeast colonies, so the estimation of transformation efficiency could not be determined. Sequence analysis identified RanBP9, which was identified in the screen using MORIL2, and plexin A4, a protein that is involved with axonal guidance (Haklai-Topper et al., 2010) (Table 2.2).
However, the portion of plexin A4 pulled out in the screen was part of the non-coding region, and was not pursued further.
51 2.3.2 Mapping of Binding Regions
The split-ubiquitin screen used the entire coding region of MOR as bait; therefore, this approach provided no information regarding the physical location of MORIP binding sites on the MOR. To map the binding site of the MORIPs on the MOR, the conventional yeast two-hybrid system was used to interrogate interaction of individual
MORIPs with each of the MOR intracellular loops and the C-terminal cytoplasmic tail
(Ctail). Each of the MORIPs identified in the split-ubiquitin screen was found to interact with only the second intracellular loop of the MOR (Figure 2.1). This result was somewhat unexpected, as previously discovered MORIPs have been found to bind to IL3,
IL2, and the Ctail. Therefore, it is a little unsual that all of the MORIPs discovered in the
MYTH screen interacted only with the second intracellular loop. However, this data does suggest that the second intracellular loop of the MOR is an important site for protein- protein interactions.
The MORIPs identified in the screen using MORIL2 as bait were also tested, because some GPCR interacting proteins have been shown to interact with more than one intracellular region of GCPRs (Cen et al., 2001; Georgoussi et al., 2006). Each of the
MORIPs from the conventional screens interacted, as expected, with the second intracellular loop (IL2) of the MOR (Figure 2.1). Two of the MORIPs, CSN5 and
RanBP9, also interacted with the first intracellular loop (IL1), while AUP1, CSN5,
DOK4 and RanBP9 were found to interact with the C-terminal tail of the receptor. This is consistent with RanBP9 being pulled out in the screen using the MOR Ctail as bait.
The binding site for Cx37 was not tested in this assay. These results suggest that the
52 MORIPs identified interact with specific domains of the MOR in the yeast system, and that the second intracellular loop of the receptor is an apparent hot-spot for protein- protein interactions.
Figure 2.1: Mapping of MORIP binding regions Portions of each MORIP were tested in a directed Y2H assay for interaction with each of the intracellular loops (IL) and carboxyl-terminus (C-tail) of the MOR. Empty bait vector (pACT2) was used as a negative control. A positive interaction is indicated by the production of a blue colony in the β-galactosidase assay.
53 2.3.3 Validation of MOR/MORIP interaction by GST Pulldown
To further validate direct interaction between newly identified MORIPs and the
MOR, we tested the ability of selected MORIP-GST fusion proteins to associate with S- tagged MORIL2 in a pull-down assay. Western blots containing lysates from bacteria expressing an S-tagged MORIL2 cDNA produced an immunoreactive band of ~13 kDa when probed with anti-S-tag antibodies (Figure 2.2). This band corresponds to the expected size of the MORIL2 encoded by the cDNA construct. The same band was detected by pull-down after the bacterial lysate was incubated with either the CSN5-GST,
AUP1-GST, or DOK4-GST fusion proteins, but not when the lysate was absorbed onto beads alone or GST-coated beads. We also utilized the pull-down assay to test the ability of RANBP9, SIAH1, and zyxin to associate with a MORIL2-GST fusion protein. As shown in Fig. 2.2, S-tagged RANBP9 cDNA produced an immunoreactive band of ~34 kDa, S-tagged SIAH1 cDNA produced a band of ~32 kDa, and S-tagged Zyxin was detected at ~85 kDa when probed with anti-S-tag antibody. These bands correspond in size to the predicted sizes of the protein fragments encoded by the respective cDNA constructs, and were not detected when the lysates were absorbed onto beads alone or
GST-coated beads.
54
Figure 2.2: GST pull-down of MORIPs and MOR GST pull-down assays were performed to interrogate the interaction between selected full-length MORIPs and the MORIL2 domain. In the top three panels, MORIP-GST fusion proteins were used to pull down the S-tagged MORIL2. In the bottom three panels, MORIL2-GST fusion proteins were used to pull down S-tagged MORIPs. Pull- down products were purified on glutathione beads, separated by SDS-PAGE, and probed on Western blots using HRP-conjugated anti-S-tag antibodies. S-tagged MORIPs or MORIL2 domains produced in bacteria are shown in lysate lanes (Ly), while uncoated glutathione sepharose beads (Beads) or GST-coated glutathione sepharose beads (GST) incubated with S-tagged proteins served as negative controls. PD indicates pull-down lanes.
55 2.3.4 Interaction of MORIPs with MOR, DOR, and KOR in Cultured Mammalian Cells
The interaction between full-length MOR and selected MORIPs was verified using co-immunoprecipitation (co-IP) experiments. To demonstrate an interaction in cell culture, the ability of an anti-FLAG antibody to co-IP MORIPs from lysates prepared from HEK 293 cells stably expressing FLAG-tagged MORs (HEK-MOR) was tested.
Probing Western blots with anti-myc antibodies revealed immunoreactive bands of the expected sizes in lysate lanes (L) prepared from HEK-MOR cells transiently transfected with myc-tagged AUP1, CSN5, Cx37, DOK4, VAPA cDNAs (Figure 2.3B). Anti-
SIAH1, anti-SIAH2, and anti-WLS antibodies detected immunoreactive bands of the expected sizes in lysate lanes for SIAH1, SIAH2, and WLS, respectively, in untransfected cells. In co-IPs where no anti-FLAG antibody was added (mock, M), no such bands were detected; however, bands migrating at similar molecular weights were detected in the IP lanes. Y2H and co-IP experiments have recently confirmed an interaction between RANBP9 and the MOR (Talbot et al., 2009). Taken together, these results support the view that the MORIPs identified in our Y2H screens interact with full length MOR in the context of cultured mammalian cells.
56
Figure 2.3: Co-immunoprecipitation of MORIPs and MOR, KOR, and DOR (A) Amino acid sequence comparison between MOR, DOR, and KOR IL2 domains. (B) Co-immunoprecipitation of full length MORIPs with MOR, DOR, or KOR. Flag-tagged MOR, DOR, or KOR was immunoprecipitated from HEK-MOR, HEK-DOR, or HEK- KOR cells, respectively, using rabbit anti-flag antibodies. Mock immunoprecipitations were performed with Protein-G beads coated with non-specific rabbit IGG. Blots were probed with either anti-MORIP antibodies for the presence of endogenously expressed MORIPS (SIAH1, SIAH2 and WLS) or with anti-myc antibodies for transiently transfected myc-tagged MORIPs. Lysate lanes (L) contain 5% of the total protein compared to the mock (M) and immunoprecipitation (IP) lanes.
The Y2H and pull-down data indicate that each of the MORIPs identified in the screens interacts with the second intracellular loop of the MOR. Sequence comparisons indicate that the second intracellular loops of the MOR, DOR and KOR exhibit a high
57 degree of amino acid sequence homology (Figure 2.3A). Among the opioid receptor proteins, the second intracellular loop exhibits 85-90% amino acid sequence similarity, with the highest homology within the N-terminal portion of the loop (Figure 2.3A).
Because of this homology, we used co-IPs to determine whether any of the MORIPs also interacted with other opioid receptor family members. To do this, we tested the ability of anti-FLAG antibodies to co-IP MORIPs from lysates prepared from HEK-DOR and
HEK-KOR cells (stably expressing FLAG-tagged DORs and KORs, respectively). As shown in Figure 2.3B, AUP1-myc, Cx37-myc, SIAH1, SIAH2, VAPA-myc, and WLS were able to interact with both DOR and KOR. However, neither CSN5-myc nor DOK4- myc showed interaction with DOR or KOR, despite the fact that the proteins were expressed in the transfected cells, as indicated by the presence of immunoreactive bands of the appropriate size in the DOR and KOR lysate lanes (Figure 2.3B). Together, these results suggest that many of the MORIPs we identified are also capable of interaction with the DOR and KOR, at least within the context of transfected mammalian cells. The interaction of these proteins with each of the opioid receptor family members will depend in large part on whether an opioid receptor family member and a particular opioid receptor binding protein are co-expressed within the same cell (and cellular compartment) within the nervous system.
2.3.5 Immunofluorescence Microscopy
Immunofluorescence microscopy was performed to determine the cellular location of MORIPs and MOR in HEK-MOR cells. Due to the lack of available
58 antibodies to selected MORIPs or their use in immuohistochemistry, myc-tagged proteins were transfected into HEK-MOR cells. Cells were double labeled with mouse anti-myc and rabbit anti-FLAG antibodies, donkey anti-mouse FITC-conjugated, and donkey anti rabbit rhodamine-conjugated secondary antibodies to label the MORIPs green and MORs red. If the MOR and the MORIP signals overlap (orange or yellow), it suggests that the proteins occupy the same space within the cell, and therefore are able to interact.
However, the observed staining of the MOR is not entirely at the membrane, as would be expected (Figure 2.4). This may be due the overexpression of the MOR in these cells or to non-specific staining by the rabbit anti-MOR antibodies. Therefore, conclusions cannot be made in regards to the possible co-localization of the MOR with these MORIPs. The immunofluorescence does allow the approximate intracellular location of the MORIPs to be determined. DOK4 was observed primarily near the plasma membrane (Figure 2.4), which is consistent with a previous report (Uchida et al., 2006). AUP1, SIAH1, and
CSN5 were all predominantly expressed in the cytosol (Figure 2.4). As with DOK4, the cellular locations of AUP1, SIAH1, and CSN5 are consistent with previous reports (Hu and Fearon, 1999; Kato et al., 2002; Luo et al., 2006).
59
Figure 2.4: Co-localization of MOR and MORIPs MOR was labeled with rabbit anti-MOR antibody and donkey anti-rabbit FITC. Myc fusion proteins were visualized using mouse anti-myc antibody and donkey anti-mouse- rhodamine. Images are confocal. Overlay shows the merging of the labels and is indicated by a yellow color.
2.4 Discussion
GPCR signaling is modulated by proteins that bind to form signalplexes, involving interactions between themselves, other GPCRs, and with other interacting proteins. As a GPCR, the MOR is expected to have an extensive signalplex (Kabbani and Levenson, 2007; Milligan, 2005). Utilization of conventional and modified yeast two-hybrid (Y2H) methods identified ten novel and two previously reported components of this complex. Directed Y2H and GST-PD studies were used to confirm interaction
60 and map the binding site for several MORIPs to the second intracellular loop of MOR.
Many MORIPs were demonstrated to interact with full length MOR in the context of cultured human cells. Due to high sequence homology in intracellular loop two, many
MORIPs also interact with the KOR and DOR, suggesting that these proteins may be universal regulators of opioid function
Previous conventional Y2H screens have identified many of the known MOR interactors including periplakin, protein kinase C I (PKCI), phospholipase D2 (PLD2), synaptophysin, glycoprotein M6a, filamin A, and DnaJ-like 1 protein (Hlj) (Georgoussi et al., 2012). The conventional Y2H presented here was unique in that it utilized the second intracellular loop of MOR, a region of the receptor that was previously unstudied in terms of interacting proteins. This screen yielded six novel interactors and two previously reported MORIPs (wntless and Ran binding protein 9). In addition, a modified split-ubiquitin Y2H screen identified four interactors of full length MOR. The advantage of this type of screen is that full length membrane proteins can be used as bait, and the screen is able to identify membrane-localized interactors which may be missed in the conventional Y2H screen.
Although these screens were successful in discovering novel MORIPs, the Y2H method can result in false negative and false positive results. False positive results are usually eliminated during subsequent analysis, such as GST pull-down and co-IP. False negative results can occur for a variety of reasons. For example, if the interaction depends on posttranslational modifications, such as glycosylation or phosphorylation, these modifications may not occur properly or at all in the yeast system. Therefore, these interactions would not be detected in the Y2H. Since the fusion proteins must be targeted
61 to the nucleus, hydrophobic proteins (such as membrane bound proteins) or proteins with strong targeting signals may not reach the nucleus to activate the reporter gene and would also be missed in the Y2H. Additionally, very weak or transient interactions are not usually detected using this method and would likely be missed in a screen. These disadvantages in using the Y2H method are important, because they help explain why more interacting proteins were not found.
These studies have shown via directed Y2H that several MORIPs are capable of interacting with multiple loops of the MOR. This is not surprising as some previously identified interactors of MOR and DOR have been shown to be able to associate with more than region of the receptor. Regulators of G protein signaling 4 protein (RGS4) and beta arrestin have been demonstrated to interact with both the third intracellular loop and the C-terminal tail of the DOR (Cen et al., 2001; Georgoussi et al., 2006).
Heterotrimeric G proteins (Gi) bind to the MOR at the third intracellular loop and the C- terminal tail (Georgoussi et al., 1997).
The MORIPs identified in this and other studies are functionally diverse, but many share common functions including protein processing, cellular localization, signal regulation, receptor desensitization, ubiquitination, scaffolding, WNT secretion, and axon guidance. Several of the novel MORIPs identified in the current screens belong to the ubiquitin proteasome pathway. Seven in absentia homolog 1 and 2 (SIAH 1 and 2) are
RING finger E3 ubiquitin ligases that mediate ubiquitination and subsequent proteasomal degradation of target proteins (Hu et al., 1997a). COP9 subunit 5 (COP9S5 or CSN5) is one subunit of the large COP9 signalosome (CSN) which is responsible for the deneddylation and activation of Cullin ring E3 ubiquitin ligases that ubiquitinate targets
62 for degradation (Choo et al., 2011). These MORIP/MOR interactions are surprising considering that MOR is typically downregulated by lysosomal degradation and not the proteasome. However, ubiquitination of MOR does occur and is required for trafficking events that downregulate receptor ligand binding (Hislop et al., 2011). Since the E3 ligase for ubiquitination of MOR is unknown, it will be important to determine whether
SIAH1, SIAH2, or CSN5 promotes ubiquitination of the opioid receptors. An alternative hypothesis is that MOR can act to scaffold these proteins for ubiquitination of other
MORIPs. In fact, synaptophysin, a previously identified MORIP is a target of both
SIAH1 and SIAH2 (Wheeler et al., 2002). There is also mounting evidence that the ubiquitin pathway is involved in a variety of neuronal activities, including dendritic morphogenesis and synaptic plasticity (Bingol and Sheng, 2011; Tuoc and Stoykova,
2010; Yang et al., 2008). Similarly, chronic opioid exposure has been shown to lead to changes in dendritic arborization and synaptic plasticity (Robinson and Kolb, 2004). To date there is no direct evidence to connect these observations.
Two proteins from the downstream of kinase (DOK) family of adaptor proteins were also identified as interactors of MORIL2. This family of proteins has a broad range of functions. In the nervous system, DOK4 has been shown to be required for axon myelination and the regulation of GDNF-dependent neurite outgrowth via the activation of the Rap1-ERK1/2 pathway (Uchida et al., 2006). DOK5 was demonstrated to interact with TrkB and TrkC neurotrophin receptors and is involved in the activation of the
MAPK pathway induced by neurotrophin stimulation (Shi et al., 2006). Like opioid receptors, neurotrophins have a strong link to pain perception, reward, and synaptic plasticity (Pardon, 2010; Pezet and McMahon, 2006). For example, BDNF (ligand of
63 TrkB) has been implicated in substance dependence as well as antinociception (Ghitza et al., 2010; Marcol et al., 2007; Obata and Noguchi, 2006; Ren and Dubner, 2007). It will certainly be of interest to determine whether there is functional interplay between the
MOR and neurotrophic factors in MAPK-mediated signaling, pain processing, reward, and memory formation.
The results presented in this chapter highlight the use of multiple screening methods to expand our knowledge of proteins that interact with, and may contribute to the regulation of opioid receptor-mediated signaling in brain. Identification of the full panorama of MORIPs represents a critical step in understanding the normal regulation of the receptor life-cycle, as well as how receptor signaling or trafficking may be hijacked in response to drugs of abuse. The
MORIPs identified in this and other studies may also represent key players involved in other opioid-mediated processes such as neural development, nociception, aversion, and synaptic plasticity. As the list of MORIPs grows, and their contributions to receptor function become better understood, it is possible that some of these proteins will also become targets for new drug development to prevent and/or treat opioid addiction.
64 Chapter 3
Functional Analysis of the MOR/SIAH Interaction
3.1 Introduction
In Chapter 2, two E3 ligases, SIAH1 and SIAH2, were identified as novel
MORIPs. Since the identity of ubiquitin ligases that regulate the MOR are unknown, these proteins were chosen for further study.
E3 ligases are the last enzymes utilized by the cell to modify proteins with ubiquitin. Ubiquitin is attached to lysine residues of substrate proteins covalently and requires the action of a cascade of enzymes. First, an ubiquitin-activating enzyme (E1) forms an ATP-dependent high energy thiol ester bond with ubiquitin. Then the activated ubiquitin is transferred to an ubiquitin-conjugating enzyme (E2). Some E2s are capable of ubiquitinating the substrate directly; however, this is usually done by an ubiquitin ligase (E3) and often confers substrate specificity (Myung et al., 2001).
Seven in absentia was first described as a gene required for the specification of R7 cell fate in the Drosophila melanogaster eye (Carthew and Rubin, 1990). Three years later, the murine homologues were discovered and designated Siah (seven in absentia homolog) and were found to exist as three genes, Siah-1a, Siah-1b, and Siah-2 (Della et al., 1993). The first report and cloning of a human Siah gene suggested a role for Siah in apoptosis and tumor suppression (Nemani et al., 1996). The human SIAH1 is homologous to the mouse Siah1a, whereas the human SIAH2 is homologous to the mouse
Siah2. Humans do not have a homologus Siah1b gene (Hu et al., 1997a). The first report
65 of the involvement of Siah proteins in the ubiquitin-proteasome pathway was found when the deleted in colorectal cancer (DCC) protein was shown to be regulated by Siah and that Siah interacted with ubiquitin-conjugating enzymes (Hu et al., 1997b). Since these initial studies, SIAH1 and SIAH2 have been shown to be RING (Really Interesting New
Gene)-finger E3 ligases capable of interacting with a variety of proteins and targeting them for degradation via the ubiquitin-proteasome pathway.
Knockout mice have been generated for each of the Siah proteins. Knock out of
Siah1a (analogous to knock out of human SIAH1) causes postnatal growth retardation and premature death, with few surviving beyond 3 months. Surviving males are infertile and females are subfertile (Dickins et al., 2002). In contrast, Siah2 knockout mice do not exhibit growth retardation or early mortality and both males and females are fertile, but have abnormal bone marrow. Knock out of both Siah1 and Siah2 in combination results in pups dying within hours of birth (Frew et al., 2003).
Both SIAH1 and SIAH2 have a protein-protein interacting domain in the C- terminal region (amino acids 90-282 for SIAH1; amino acids 116-325 for SIAH2) and the RING-finger (catalytic domain) in the N-terminal region (amino acids 1-89 for
SIAH1; amino acids 1-115 for SIAH2) (House et al., 2003; Polekhina et al., 2002). It has been shown that these two domains can be separated, where mutations in the RING- finger do not abrogate the binding of the proteins to their binding partners. The Siah proteins have been shown to have auto-ubiquitinating activity and can form homo- and heterodimers with each other and other E3 ligases (Hu and Fearon, 1999). They mediate a variety of different pathways by regulating proteins via the ubiquitin-proteasome pathway. Some of the reported targets of Siah-mediated degradation include
66 adenomatous polyposis coli protein (APC) (Liu et al., 2001), kinesin like DNA binding protein (Kid) (Germani et al., 2000), synaptophysin (Wheeler et al., 2002), group 1 metabotropic glutamate receptor (Ishikawa et al., 1999), Pard3A (Famulski et al., 2010), and beta-catenin (Dimitrova et al., 2010). SIAH2 has been reported to interact with Vav, a GDP-exchange factor for Rac/Rho, but does not cause its degradation. SIAH2 was shown to negatively regulate Vav-induced JNK activation (Germani et al., 1999). SIAH1 interacts with BAG1, an Hsp70/Hsc70-binding protein that modulates cell proliferation and death, but does not cause its degradation. In fact, BAG1 inhibits the activity of
SIAH1 in p53-mediated cell cycle arrest (Matsuzawa et al., 1998). Therefore, not all
Siah-binding proteins are targets for Siah-mediated degradation.
In addition to the roles the Siah proteins play in protein-protein interactions and in
Siah-mediated degradation, these proteins have been implicated as players in neurological disease and in addiction. Specifically, SIAH2 has been demonstrated to be elevated in the blood of patients with psychosis and this elevation corresponded to greater symptom severity (Bousman et al., 2010). SIAH1 has been shown to exhibit decreased expression in the brains of patients with a history of alcohol abuse and/or dependence although the functional consequence of this change was not pursued (Sokolov et al.,
2003).
The role of ubiquitin has largely been characterized to function as a sorting signal for lysosomal degradation of GPCRs. Ubiquitin is best known to target agonist-activated
GPCRs to lysosomes for degradation via the endosomal-sorting complex required for transport (ESCRT) pathway (Marchese et al., 2008). This process is important for disposing of irreversibly activated receptors and for ridding cells of receptors after
67 chronic agonist stimulation (Marchese et al., 2008; Trejo et al., 1998). Interestingly, delta opioid receptors are ubiquitinated following agonist stimulation, but ubiquitin is not required for lysosomal sorting, indicating an additional undiscovered role for ubiquitin in
GPCR regulation (Hislop et al., 2009). More recent studies have demonstrated a role for ubiquitination in the sorting of MORs from the endosome limiting membrane to the lumenal compartment, but not in determining the recycling or degradation of the receptors by the lysosome or proteasome (Hislop et al., 2011).
The identity of the ubiquitin ligases that mediate these responses is largely unknown for the opioid receptors. However, an E3 ligase, atrophin-interacting protein 4
(AIP4), has been reported for the DOR. AIP4 was shown to control the down-regulation, measured as loss of ligand binding, of DORs without affecting the delivery of the receptor to lysosomes. This effect required the direct ubiquitination of receptors (Hislop et al., 2009). The identity of E3 ligases that regulate either the KOR or the MOR have not yet been identified.
Ubiquitination can serve as a signal for many cellular processes, including degradation, trafficking, signal transduction, and endocytosis (Hicke and Dunn, 2003;
Marchese et al., 2008; Myung et al., 2001). Many of these same cellular processes regulate the MOR and have been shown to play a role in the development of addiction
(Tuoc and Stoykova, 2010). E3 ligases are believed to confer substrate specificity for ubiquitination; therefore, I investigated the functional role of the interaction between the
MOR and SIAH1. SIAH1 was chosen for further study over SIAH2, due to its reported role in the ubiquitination and degradation of the metabotrophic glutamate receptor 1
(mGluR1), a GPCR (Moriyoshi et al., 2004). SIAH2 has no reported role in GPCR
68 regulation. Using overexpression and siRNA knockdown experiments, I show that
SIAH1 appears to form an interaction with SIAH2, and that cooperation between the Siah proteins affects MOR ubiquitination and MOR protein levels.
3.2 Experimental Procedures
3.2.1 Mapping of binding sites
SIAH1 truncation (TR) deletion mutants (SIAH1-TR, residues 91-282; SIAH1-
TR2; residues 91-157, and SIAH1-TR3; residues 151-217) were ligated into pGEX-4T-1 to create GST fusion proteins. The PCR primers used in the cloning of the mutants can be found in Table 3.1. The MORIL2-S-tag fusion protein was constructed as described in
Chapter 2. Fusion proteins were induced in Escherichia coli strain BL21 (DE3) using the
ZYP-5052 autoinduction media as described previously (De Cotiis et al., 2008; Studier,
2005). SIAH1 deletion mutant GST fusion proteins were used to pull down S-tagged
MORIL2 using glutathione sepharose beads (GE Healthcare) as previously described
(Lin et al., 2001). Eluted proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) filters for western blot analysis. The filter was probed with a horseradish peroxidase (HRP)-conjugated anti-S-tag antibody (1:5000 dilution,
Novagen, Madison, WI), and immunoreactivity was detected by enhanced chemiluminescence with an ECL Plus kit (GE Healthcare).
69
Table 3.1: PCR primers used for cloning of SIAH1 deletion mutants This table lists the PCR primers that were used to clone SIAH1 mutants into pGEX-4T-1 to produce GST-fusion proteins for use in GST pull down experiments.
Construct Direction Sequence
SIAH1-TR-GST Forward GGA TCC AAT TCA GTA CTT TTC CCC TGT Amino Acids 91-282 AAA Reverse CTC GAG TCA ACA CAT GGC AAT AGT TAC ATT GAT GCC
SIAH1-TR2-GST Forward GAA TTC AAT TCA GTA CTT TTC CCC TGT Amino Acids 91-157 AAA TAT Reverse CTC GAG TAG GGT TGT AAT GGA CTT ATG
SIAH1-TR3-GST Forward GAA TTC AAG TCC ATT ACA ACC CTA CAG Amino Acids 151-217 Reverse CTC GAG AAA ATT TTC AGC TTG CTT
3.2.2 Cell culture and transfections
Human embryonic kidney (HEK) 293 cells stably transfected with either FLAG- tagged mu-opioid receptor (HEK-MOR), delta-opioid receptor (HEK-DOR), or kappa- opioid receptor (HEK-KOR) were maintained in Dulbecco's Modified Eagle's Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and 400 μg/mL G418. Full- length SIAH1 and SIAH1 deletion mutant (SIAH1-TR, residues 91-282) were cut from pGEX-4T-1 with EcoRI and XhoI restriction endonucleases and ligated into pCMV- tag3B to create myc-tagged constructs. These constructs were transfected into HEK-
MOR cells using Effectene per manufacturer instructions (Qiagen, Valencia, CA).
70 3.2.3 Treatment of cells with inhibitors of the proteasome and lysosome
Human embryonic kidney (HEK) 293 cells stably transfected with FLAG-tagged mu-opioid receptor (HEK-MOR) were treated for 30 minutes, 1 hour, 2 hours, and 4 hours with 50 μM cyclohexamide (protein synthesis inhibitor; Sigma) or 30 μM MG132
(proteasome inhibitor; Sigma). HEK-MOR cells were also treated for 30 minutes, 1 hour,
2 hours, 4 hours, and 6 hours with 50 μM of chloroquine (lysosome inhibitor; Sigma).
After the specified time point cells were collected and lysed in lysis buffer (20 mM
Na2HPO4, 20 mM NaH2PO4, 2 mM EDTA, 50mM sodium fluoride, 5 mM tetrasodium pyrophosphate, 10mM β-glycerophosphate, 1% NP-40, 1 mM Pefabloc, 1 mM DTT, 1.2 mg/ml protease inhibitors). Crude cell lysates were run on SDS-PAGE and transferred to
PVDF membrane. To visualize the MOR blots were incubated with rabbit anti-FLAG antibodies (1:500 dilution; Sigma) and goat anti-rabbit HRP-conjugated antibodies
(1:20,000 dilution; Jackson ImmunoResearch).
3.2.4 Detection of ubiquitinated MOR using TUBEs
HEK-MOR cells were transfected with SIAH1-myc or SIAH1-TR-myc as described above. After 24 hours cells were lysed in lysis buffer containing 200 μg/mL of tandem ubiquitin binding entities fused to GST (TUBEs; Life Sensors, Malvern, PA).
TUBEs contain multiple ubiquitin binding domains, which recognize and bind to ubiquitin and protect the bound proteins from degradation via the proteasome. Cell lysate was allowed to nutate for 30 minutes before the addition of glutathione sepharose beads
(Invitrogen, Grand Island, NY). Eluted proteins were resolved on SDS-PAGE and
71 transferred to PVDF membrane for western blotting. Blots were probed with rabbit anti-
MOR antibodies (1:5000 dilution AB5511; Millipore, Billerica, MA) and goat anti-rabbit
HRP conjugated antibodies (1:20,000 dilution; Jackson ImmunoResearch).
3.2.5 siRNA knockdown of SIAH proteins
HEK-MOR, HEK-DOR, or HEK-KOR cells were cultured in 100 cm2 plates to
50% confluency and then transfected with 120 pmol of Stealth siRNA for either SIAH1
(5’-GAUGCAUCAGCAUAAGUCCAUUACA-3’) or SIAH2 (5’-
ACUGGGUGAUGAUGCAGUCAUGUUU-3’) (Invitrogen) using RNAiMAX per manufacturer instructions (Invitrogen). Cells were incubated for 72 hours and then harvested in lysis buffer (20 mM Na2HPO4, 20 mM NaH2PO4, 2 mM EDTA, 50mM sodium fluoride, 5 mM tetrasodium pyrophosphate, 10mM β-glycerophosphate, 1% NP-
40, 1 mM Pefabloc, 1 mM DTT, 1.2 mg/mL protease inhibitors) with 50μg/mL of
MG132 (Sigma, St. Louis, MO). MG132 was added to prevent protein degradation via the proteasome during lysis. Western blotting was performed to confirm knockdown of proteins.
3.2.6 Co-immunoprecipitation and western blot analysis
Crude cell lysate was prepared as described in siRNA knockdown of SIAH proteins. Protein G magnetic agarose beads (GE Healthcare) incubated with polyclonal rabbit anti-FLAG antibodies (Sigma) were used to immunoprecipitate receptors from
72 HEK-MOR, HEK-DOR, and HEK-KOR cell lysate. Immunoprecipitations (IPs) using rabbit immunoglobulin g were performed as negative controls. IPs were resolved by
SDS-PAGE and transferred to PVDF membranes. For the detection of ubiquitin, blots were first treated with 0.5% gluteraldehyde (as recommended by the antibody manufacturer) (Sigma), then immunoblotted using mouse anti-ubiquitin antibody (1:5000 dilution of VU-1, LifeSensors) and rabbit anti-mouse HRP conjugated secondary antibody (1:20,000 dilution, Jackson ImmunoResearch). The amount of endogenous
SIAH1 in crude lysate was determined by resolving lysates by SDS-PAGE and immunoblotting using goat anti-SIAH1 (1:5000 dilution, Abnova, Taipei City, Taiwan) antibody and bovine anti-goat HRP conjugated secondary antibody (1:20,000 dilution,
Jackson ImmunoResearch). The amount of SIAH2 in crude lysate was determined by resolving lysates by SDS-PAGE , treating blots with Pierce Western Blot Enhancer Kit per manufacturer instructions (Pierce), and immunoblotting using goat anti-SIAH2 antibody (1:500 dilution, N-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and bovine anti-goat secondary antibody (1:20,000 dilution, Jackson ImmunoResearch). The amount of opioid receptors in crude lysate was determined by immunoblotting with rabbit anti-FLAG antibody (1:5000 dilution, Sigma) and goat anti-rabbit HRP conjugated antibody (1:20,000 dilution, Jackson ImmunoResearch). To control for loading, blots were also probed for GAPDH (1:5000 dilution, Sigma) using chicken anti-GAPDH antibodies and donkey anti-chicken antibody (1:20,000 dilution, Jackson
ImmunoResearch) or tubulin (1:10,000 dilution, Novus) using mouse anti-tubulin antibodies and goat anti-mouse HRP conjugated antibody (1:20,000 dilution, Jackson
ImmunoResearch).
73 3.2.7 Quantitation of Western blots
All blots were run in duplicate or triplicate. Blots were scanned using a back-lit scanner and quantitation was performed using ImageJ software (Abramoff et al., 2004).
Expression was normalized to total protein for siRNA experiments (as measured by
GAPDH), averaged between replicates, and subjected to a two-tailed Student’s t-test.
3.3 Results
3.3.1 Mapping of the MOR binding site on SIAH1
In Chapter 2, the binding site for SIAH1 on the MOR was found to be the second intracellular loop. However, the location where MORIL2 binds to SIAH1 has not been determined. The SIAH1 protein can be divided into an N-terminal region (residues 1-
89), which includes the RING domain (residues 40-75), and the C-terminal region (amino acids 90-282), which is involved in substrate binding (Hu and Fearon, 1999). The RING domain is the catalytic domain of the protein and is required for ubiquitination of target proteins (Hu and Fearon, 1999). I therefore hypothesized that the C-terminal region of
SIAH1 would bind to the second intracellular loop of the MOR. To determine a more specific binding region, SIAH1 deletion mutants were made in the C-terminal region of the protein (Figure 3.1B). As expected, the C-terminal region but not the N-terminal region of SIAH1 was required for SIAH1 to bind to the MOR (Figure 3.1A). Upon the truncation of the C-terminal region, amino acids 91-157 (SIAH1-TR2-GST) were able to
74 pull down MORIL2, while amino acids 151-282 (SIAH1-TR3-GST) were no longer able to pull down MORIL2 (Figure 3.1A). This indicates that the region of the C-terminus that includes amino acids 91-157 is required for the interaction between SIAH1 and
MORIL2.
Figure 3.1: Mapping of SIAH1 binding site (A) Deletion mutants of SIAH1 were used in GST pull down experiments to localize the MOR binding site on SIAH1. SIAH1-TR-GST (a. a 91-282), SIAH1-TR2-GST (a. a 91- 157), and SIAH1-TR3-GST (a. a 151-217) were used to pull down S-tagged MORIL2. Pull-down products were purified on glutathione beads, separated by SDS-PAGE, and probed on western blot using HRP-conjugated anti-S-tag antibodies. S-tagged MORIL produced in bacteria are shown in the lysate lane (LY), while uncoated glutathione sepharose beads (BEADS) or GST-coated beads (GST) incubated with S-tagged MORIL2 served as controls. (B) Map of constructs. The green represents the N-terminal region, which includes the RING-finger domain in gold (RF). The pink represents the C- terminal region, which includes the substrate binding domain in pink (SBD).
75 3.3.2 SIAH1 regulates the amount of MOR modified with ubiquitin
As an E3 ligase, SIAH1 has the ability to conjugate ubiquitin to its binding partners. I wanted to determine if SIAH1 changes the ubiquitination of the MOR. To test this, HEK-MOR cells were transfected with SIAH1-myc or SIAH1-TR-myc.
SIAH1-TR-myc has the RING domain (catalytic domain) removed and should not be able to participate in ubiquitination, but retains the ability to bind to the MOR. Tandem ubiquitin binding entities (TUBEs) fused to GST were used to pull down the total ubiquitinated protein from cell lysates. HEK-DOR cells were mock transfected and used as a negative control to demonstrate the specificity of the MOR antibody (Figure 3.2 A).
MOR protein modified with ubiquitin often resolves as a broad high molecular weight band or smear of 75 -250 kDa on western blot (Groer et al., 2011). Due to this broad band, quantitation cannot be performed for this kind of analysis and is not performed by other researchers in the field. In HEK-MOR cells transfected with empty vector, the pull down lane shows ubiquitinated MOR as an immunoreactive band between 100 and 200 kDa (Figure 3.2, top panel, lane 2). Unexpectedly, when SIAH1 was overexpressed, the intensity of the immunoreactive band between 100 and 200 kDa decreased slightly
(Figure 3.2 A, lane 4). This indicates that overexpression of SIAH1 may cause a reduction in the amount of MOR that is ubiquitinated. In contrast, when the RING mutant was overexpressed, the intensity of this band increased (Figure 3.2 A, lane 6).
To confirm the results seen with the TUBEs, I next immunoprecipitated the MOR and probed the eluted material for ubiquitin. If SIAH1 is an E3 ligase for the MOR, the overexpression of SIAH1 would be expected to cause the amount of ubiquitinated MOR
76 to increase. Although not as robust as the TUBEs experiment, the amount of MOR that is ubiquitinated is slightly decreased when SIAH1 is overexpressed as compared to mock transfected cells (Figure 3.2B, top panel, lanes 3 and 5). Conversely, when the RING mutant SIAH1 is overexpressed, there is a slight increase in the amount of ubiquitinated
MOR (Figure 3.2B, top panel, lane 7). Without the ability to quantitate these results, it is difficult to determine the significance of the effect of SIAH1 on ubiquitination of the
MOR. However, SIAH1-TR overexpression did cause a noticeable increase in the ubiquitination of the MOR in the TUBEs experiment, which indicates that the RING- finger is not responsible for the increase in the observed ubiquitination. Therefore,
SIAH1 is likely not an E3 ligase for MOR.
77
Figure 3.2: SIAH1 decreases the ubiquitination of the MOR (A) HEK-MOR cells were transfected with empty vector (pCMV-tag3B), SIAH1 with a myc tag (SIAH1-myc), SIAH1 RING mutant with a myc tag (SIAH1-TR-myc). The amount of MOR eluted from the beads was determined by immunoblotting with anti- MOR antibodies and is shown in the IP lanes. HEK-DOR cells were used as a control to show that the TUBEs do not cause non-specific banding patterns. (B) HEK-MOR cells were transfected as in (A) and cell lysate was subjected to immunoprecipitation with anti- FLAG antibodies to IP the MOR and then immunoblotted with mouse anti-ubiquitin antibodies. IPs using rabbit IgG was used as a negative control (M). Crude cell lysates were immunoblotted to show that the constructs were expressed in the cells used in pull down or immunoprecipitation experiments (Bottom panels A and B). Blots are representative of three separate experiments.
78 3.3.3 siRNA knockdown of SIAH1 increases the ubiquitination of the opioid receptors
Since the overexpression of SIAH1 caused a decrease in the amount of detectable ubiquitinated MOR, I expected that knocking down endogenous SIAH1 would result in increased ubiquitination of MOR. HEK-MOR cells were transfected with siRNA against
SIAH1. Crude cell lysate was immunoblotted to detect the amount of SIAH1 and
GAPDH (Figure 3.3B). Knockdown of SIAH1 resulted in an 80% reduction in the amount of endogenous Siah1 protein (Figure 3.3C). To determine the ubiquitination level of the MOR, MOR was immunoprecipitated from lysates of cells that were transfected with scrambled or SIAH1 siRNA, subjected to SDS-PAGE, and immunoblotted with anti-ubiquitin antibodies. As expected, Siah1 knockdown resulted in an increase in the amount of detectable ubiquitinated MOR (Figure 3.3A). These results are consistent with observations made by overexpression of SIAH1-TR-myc, which suggests that SIAH1-TR-myc acts as a dominant negative mutant. It is possible that this assay is not measuring MOR ubiquitination, but the ubiquitination of another MORIP.
This assay is indirect as any protein that immunoprecipitates with the MOR and is also ubiquitinated may be observed on the blot. However, this assay can be used to determine whether or a particular protein is a putative E3 ligase or not. If SIAH1 is an E3 ligase for the MOR, its knockdown should cause the ubiquitination of the MOR to decrease, not increase. Therefore, SIAH1 most likely is not an E3 ligase for the MOR.
The opioid receptors show considerable similarity in the second intracellular loop
(Chapter 2, Figure 2.3A) and SIAH1 has been shown to co-immunoprecipitate with all three receptors (Chapter 2, Figure 2.3B). Therefore, it seems plausible that SIAH1 might
79 affect the ubiquitination of DOR and/or KOR. To investigate this possibility, siRNA against SIAH1 was transfected into HEK-DOR cells and HEK-KOR cells. Siah1 protein was knocked down by 94% in HEK-DOR cells and by 82% in HEK-KOR cells (Figures
3.3B and 3.3C). As with the MOR, knockdown of SIAH1 caused an increase in the detectable ubiquitination of DOR and KOR (Figure 3.3A). These results indicate that
SIAH1 is likely not an E3 ligase for either DOR or KOR.
80
Figure 3.3: Knockdown of SIAH1 increases ubiquitination of MOR, DOR, and KOR HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against SIAH1 (KD) or scrambled siRNA (SC). (A) Cells were lysed and the opioid receptors were immunoprecipitated using rabbit anti-FLAG antibodies. Blots were probed with mouse anti-ubiquitin. The ubiquitinated opioid receptors yield immunoreactive smears between 75-200 kDa. IPs using normal rabbit IgG were used as a negative control (Mock). (B) Western blots showing the amount of SIAH1 in lysate was used to confirm the knockdown, while GAPDH was used as a loading control. (C) Western blot bands were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
81 3.3.4 Knockdown of SIAH2 results in decreased ubiquitination of MOR, but increased ubiquitination of DOR and KOR.
Another E3 ligase, SIAH2, was found to interact with all three opioid receptors
(Chapter 2). Since SIAH1 did not seem to be a candidate E3 ligase for any of the opioid receptors, I wanted to determine if SIAH2 had any effect on the ubiquitination of the opioid receptors. To test this HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA targeting SIAH2.
Crude cell lysate was immunoblotted to detect the amount of SIAH2, and
GAPDH (Figure 3.4B). Knockdown of SIAH2 resulted in a 98% reduction in the amount of endogenous Siah2 protein (Figure 3.4C). To determine the ubiquitination level of the
MOR, MOR was immunoprecipitated from lysates of cells that were transfected with scrambled or SIAH2 siRNA, subjected to SDS-PAGE, and immunoblotted for ubiquitin.
SIAH2 knockdown resulted in a slight decrease in the amount of detectable ubiquitinated
MOR (Figure 3.4A). These results are more consistent with SIAH2 being an E3 ligase involved in MOR ubiquitination because the ubiquitination of MOR would be expected to decrease. Therefore, SIAH2 may be a putative E3 ligase of the MOR.
Siah2 protein was knocked down by 96% in HEK-DOR cells and 97% in HEK-
KOR cells (Figuress 3.4B and 3.4C). In contrast to the MOR, knockdown of SIAH2 caused an increase in the detectable ubiquitination of DOR and KOR (Figure 3.4A). If
SIAH2 is an E3 ligase for the DOR and/or KOR, the knockdown would be expected to result in a decrease in the ubiquitination of DOR. Therefore, these results indicate that
SIAH2 is most likely not an E3 ligase for this receptor. However, if the results of SIAH1 and SIAH2 knockdown in HEK-DOR and HEK-KOR cells are considered together, they
82 suggest that the Siah proteins may compensate for each other and therefore, cannot be ruled out as putative E3 ligases for these receptors.
Figure 3.4: Knockdown of SIAH2 decreases ubiquitination of MOR, but increases ubiquitination of DOR and KOR HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against SIAH2 (KD) or scrambled siRNA (SC). (A) Cells were lysed and the opioid receptors were immunoprecipitated using rabbit anti-FLAG antibodies. Blots were probed with mouse anti-ubiquitin. IPs using rabbit IgG were used as negative controls (Mock). The ubiquitinated opioid receptors yield immunoreactive smears between 75-200 kDa. (B) Western blots showing the amount of SIAH2 in cell lysate was used to confirm the knockdown, while GAPDH was used as a loading control. (C) Western blot bands were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
83 3.3.5 The MOR undergoes degradation via the proteasome and lysosome
The degradation of the MOR has been demonstrated to occur primarily through the lysosomal pathway, but has also been demonstrated to occur via the proteasome pathway in overexpression cell culture systems. However, both pathways have been observed in to occur in overexpression cell culture systems (Chaturvedi et al., 2001;
Hislop and von Zastrow, 2011). Since E3 ligases can target proteins to either pathway in the HEK-MOR cells, I wanted to determine if the MOR was capable of undergoing degradation via the proteasome and/or the lysosome. This was tested by treating HEK-
MOR cells with cyclohexamine, MG132, or chloroquine for varying amounts of time. A protein that undergoes proteasomal degradation should accumulate in the cell as the length of proteasome inhibitor, MG132, exposure increases. Likewise, a protein that undergoes lysosomal degradation should accumulate in the cell as the length of lysosomal inhibitor, chloroquine, exposure increases. Cyclohexamide, a protein synthesis inhibitor, was used to determine the half-life of the MOR. The levels of a protein that are degraded (lysosomal or proteasomal) will decrease in the cell overtime if protein synthesis is inhibited. The MOR has previously been shown to resolve on Western blot as two major bands, one at 50 kDa and one at 75 kDa due to varying amounts of glycosylation (Huang and Liu-Chen, 2009). Interestingly, only the low molecular weight
(50 kDa band) form of the MOR was observed to change with either cyclohexamide
(Figure 3.5A) or MG132 (Figure 3.5B) treatment. The half-life of the low molecular
MOR band is short, as this band is no longer detectable after 1 hour of cyclohexamide treatment (Figure 3.5A; 50 kDa band). This result suggests that the low molecular weight
84 form of MOR is either subject to degradation or further post-translational modification
(perhaps maturing into high molecular weight form of MOR). It is likely that a portion of low molecular weight MOR is subject to proteasomal degradation because the levels of this protein species increased with lengthening MG132 exposure (Figure 3.5B; 50 kDa band). The high molecular weight form of the MOR (75 kDa band) did not change with either cyclohexamide or MG132 treatment (Figures 3.5A and B) indicating that this protein species is stable and not degraded within the time points measured. Therefore, no conclusions on the involvement of the proteasome in degradation of the high molecular weight form of MOR can be made from this experiment.
Contrary to the results seen with the proteasome block, both the low molecular weight band (50 kDa band) and the high molecular weight band (75 kDa band) of MOR increased over time with chloroquine treatment (Figure 3.5C). Thus, it appears that lysosomal degradation has a more prominent role than proteasomal degradation for the
MOR.
85
Figure 3.5: Inhibitor treatment of HEK-MOR cells HEK-MOR cells were treated with (A) 50 μM cyclohexamide, a protein synthesis inhibitor, (B) 30 μM MG132, a proteasome inhibitor, or (C) 50 μM of chloroquine, a lysosomal inhibitor for 30 minutes, 1 hour, 2 hours, and 4 hours. Cells treated with chloroquine were also treated for 6 hours. Cells were treated with vehicle (0) as a control. After treatment cells were lysed and crude cell lysate was resolved. Blots are representative of three individual experiments.
3.3.6 Overexpression of SIAH1 causes a decrease in MOR protein levels
Since both the proteasomal and lysosomal degradation pathways can be mediated by ubiquitination, I wanted to determine the effects of overexpressing SIAH1 on MOR protein levels in HEK-MOR cells. Although it appears that SIAH1 does not ubiquitinate the MOR, it may still play a role in its degradation. If SIAH1 is involved in MOR degradation by either pathway, overexpression of the protein would be expected to cause the amounts of detectable MOR to decrease. HEK-MOR cells were transfected with full- length SIAH1 fused to myc using increasing microgram concentrations of the SIAH1- myc construct or with vector as a negative control. Transfecting increasing amounts of
SIAH1-myc resulted in decreasing amounts of both the low molecular weight (50 kDa
86 band) and the high molecular weight (75 kDa band) species of MOR protein with increasing concentrations of SIAH1-myc (Figure 3.6). These results indicate that SIAH1 may regulate MOR protein levels in mammalian cells.
Figure 3.6: SIAH1 induces MOR degradation HEK-MOR cells were transfected with increasing amounts of SIAH1-myc DNA. Vector DNA (V) was added to make the total amounts of DNA transfected equal. 2.0 μg of vector DNA was transfected as a negative control. The amount of MOR-FLAG, SIAH1- myc, and GAPDH in cell lysates was determined by immunoblotting with anti-FLAG, anti-myc, and anti-GAPDH antibodies, respectively. GAPDH was used as a loading control. Blot images are representative of three experiments.
3.3.7 siRNA knockdown of SIAH1 increases MOR protein levels, but decreases DOR and KOR protein
Since the overexpression of SIAH1 caused a decrease in MOR protein levels, I expected that knocking down endogenous SIAH1 would result in increased MOR protein levels. To test this, siRNA against SIAH1 was transfected into HEK-MOR cells. The reduction in Siah1 protein levels caused a 16-fold increase in the amount of low
87 molecular weight MOR (50 kDa band) (Figures 3.7A and D), while no significant change was detected for the high molecular weight version of MOR (75 kDa band) (Figures 3.7A and 3.7C). This suggests that SIAH1 may play a role in the regulation of MOR protein levels.
Since SIAH1 had been shown to co-immunoprecipitate with all three receptors, and knockdown of SIAH1 caused an increase in ubiquitination of the DOR and KOR, it seems possible that SIAH1 might affect DOR and/or KOR protein levels. To investigate this possibility, siRNA against SIAH1 was transfected into HEK-DOR cells and HEK-
KOR cells. In contrast to the results for MOR, the levels of both the DOR low molecular weight protein (45 kDa band) (Figures 3.7A and D) and the high molecular weight DOR
(55 kDa band) decreased with knockdown of SIAH1 (Figuress 3.7A and 3.7C). The
DOR is also differentially glycosylated and the observation of multiple bands is due to this modification (Petaja-Repo et al., 2000). The levels of KOR low molecular weight protein (45 kDa) (Figuress 3.7A and 3.7D) and high molecular weight protein (55 kDa)
(Figures 3.7A and 3.7C) also decreased, as was the case with DOR. Like the MOR and the DOR these differences in molecular weight are due to glycosylation (Chen et al.,
2006). Therefore, these results indicate that SIAH1 may play a role in the regulation of
DOR and KOR protein levels.
88
Figure 3.7: Knockdown of SIAH1 changes MOR, DOR, and KOR protein levels HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing the levels of receptor in cell lysate. (B) Western blot showing the levels of SIAH1 protein to confirm the knockdown. GAPDH was used as a loading control. (C) High molecular weight bands on Western blots were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05). (D) Low molecular weight bands on Western blots were quantitated as in (C).
89 3.3.8 siRNA knockdown of SIAH2 decreases MOR protein levels, but increases DOR and KOR protein
Since SIAH2 knockdown resulted in decreased ubiquitination of the MOR, I wanted to determine if SIAH2 was involved in degradation of the receptor. To test this,
HEK-MOR cells were transfected with siRNA targeting SIAH2. The reduction in Siah2 protein levels caused a significant decrease in the amount of both the low molecular weight MOR (band of 50 kDa) (Figures 3.8A and 3.8D) and the high molecular weight
MOR (band of 75 kDa) (Figures 3.8A and 3.8C). However, if SIAH2 is the E3 ligase that targets the MOR for degradation, the knockdown of Siah2 protein should result in an increase in MOR protein levels. Therefore, SIAH2 may be a putative E3 ligase of the
MOR, but its tagging of the MOR with ubiquitin may not result in degradation of the
MOR.
In contrast to the results for MOR, the ubiquitination of DOR and KOR increased with SIAH2 knockdown. Therefore, I wanted to determine if this ubiquitination lead to degradation of these receptors. Knockdown of Siah2 protein, caused the levels of the
DOR low molecular weight protein (45 kDa band) (Figures 3.8A and 3.8D) to increase significantly, but did not significantly change the high molecular weight DOR (55 kDa band) protein levels (Figures 3.8A and3.8C). Contrary to the results seen in HEK-DOR and HEK-MOR cells, the levels of the KOR low molecular weight protein (45 kDa band)
(Figures 3.8A and 3.8D) and the high molecular weight KOR (55 kDa band) both significantly increased with knockdown of SIAH2 (Figures 3.8A and 3.8C). Together these results suggest that SIAH2 may play a role in the degradation of DOR and KOR.
90
Figure 3.8: Knockdown of SIAH2 changes MOR, DOR, and KOR protein levels HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing the levels of receptor in cell lysate. (B) Western blots showing the levels of SIAH1 protein to confirm the knockdown. GAPDH was used as a loading control. (C) High molecular weight bands on Western blots were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05). (D) Low molecular weight bands on Western blots were quantitated as in (C).
91 3.3.9 SIAH1 and SIAH2 interact in HEK-MOR cells
If SIAH1 is an E3 ligase for the MOR, its overexpression would be expected to cause an increase in the amount of ubiquitinated receptor; however, this was not the case.
In Chapter 2, SIAH2 was also identified as an interactor with the MOR. The Siah1 and
Siah2 proteins share more than 85% amino acid identity in the C-terminal region (amino acids 80-324) and 77% overall identity. Essentially, they only differ from one another in the length and sequence of the extreme N-terminus, which includes a portion of the ring domain (Hu et al., 1997b). Therefore, the antibodies used in this study were to epitopes in the N-terminus of the proteins. There is evidence that SIAH1 and SIAH2 can form heterodimers with each other and in some cases regulate each other’s function (Depaux et al., 2006; Hu and Fearon, 1999). Additionally, the Siah proteins have been shown to interact with some of the same proteins, such as ALL-1 fused gene on chromosome 4
(Af4) (Bursen et al., 2004; Oliver et al., 2004), phospholipase C eplison (Yun et al.,
2008) and alpha synuclein (Szargel et al., 2009). Since SIAH2 was also found to be a
MOR interactor, I wanted to determine if SIAH1 and SIAH2 interact in HEK-MOR cells as this may affect ubiquitination and degradation studies of MOR. Due to the lack of commercial antibodies that can immunoprecipitate either Siah protein, HEK-MOR cells were transfected with SIAH2-myc. Anti-myc antibodies were used to immunoprecipitate
SIAH2-myc and anti-SIAH1 antibodies were used to detect SIAH1 in eluted proteins via western blot. As shown in the IP lane of Figure 3.9, SIAH2-myc was able to co- immunoprecipitate with SIAH1. These results indicate that SIAH1 and SIAH2 may interact with each other in HEK-MOR cells.
92
Figure 3.9: Co-immunoprecipitation of SIAH1 with SIAH2 HEK-MOR cells were transfected with SIAH2-myc. Myc-tagged SIAH2 was immunoprecipitated using mouse anti-myc antibodies. Mock immunoprecipitations were performed with Protein-G beads coated with normal rabbit IgG. Blot was probed with goat anti-SIAH1 antibodies. Lysate lane contains 5% of the total protein compared to the mock (M) and immunoprecipitation lands (IP).
3.3.10 SIAH1 regulates the levels of SIAH2 in HEK cells
After determining that SIAH1 and SIAH2 can interact in HEK-MOR cells, I wanted to determine if either Siah protein had an effect on the protein level of the other.
Knockdown of SIAH1 resulted in a two-fold increase in Siah2 protein in HEK-MOR,
HEK-DOR, and HEK-KOR cells (Figures 3.10A and 3.10B). This result was somewhat unexpected, as SIAH2 has been shown to be capable of regulating SIAH1 levels, but the opposite has not (Depaux et al., 2006). However, my results suggest that SIAH1 regulates SIAH2 levels.
93
Figure 3.10: SIAH1 knockdown causes an increase in SIAH2 protein levels HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing levels of SIAH1 (to confirm knockdown), SIAH2, and GAPDH (loading control). (B) Western blots were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
Although SIAH1 appears to regulate the levels of SIAH2 protein, the knockdown of SIAH2 did not result in an increase in SIAH1 protein levels in any of the cell lines tested (Figures 3.11A and 3.11B). Again this was unexpected as SIAH2 has been shown to regulate SIAH1 levels (Depaux et al., 2006). However, it does not appear that SIAH2 regulates the protein level of SIAH1.
94
Figure 3.11: Knockdown of SIAH2 does not alter SIAH1 protein levels HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against SIAH2 (KD) or scrambled siRNA (SC). (A) Western blots showing levels of SIAH2 (to confirm knockdown), SIAH1, and GAPDH (loading control). (B) Western blots were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).
3.3.11 Knockdown of both SIAH1 and SIAH2 results in a decrease in the ubiquitination of the MOR and an increase in MOR protein levels
Knockdown of an E3 ligase involved in the ubiquitination and subsequent degradation of the MOR would be expected to cause both a decrease in ubiquitination of the MOR and an increase in MOR protein levels. Since neither SIAH1 nor SIAH2 knockdown alone had that effect on the MOR, I wanted to determine the effect of a double knockdown on protein levels and ubiquitination. HEK-MOR cells were
95 transfected with siRNA targeting SIAH1 and SIAH2 in combination. There were difficulties in knocking both proteins in combination; therefore the experiments were performed in duplicate and no statistical analysis was performed. However, it is clear that SIAH1 protein was knocked down, as was SIAH2 (Figure 3.12B). Due to the lack of replicates, the effect of the double knockdown on MOR protein levels cannot be determined. Interestingly, the detection of ubiquitinated MOR was abolished when both
SIAH1 and SIAH2 were knocked down in combination (Figure 3.12A). When these results are considered with the results of the knockdown of the Siah proteins singly, they indicate that the Siah proteins may cooperate to regulate the levels of MOR protein and the ubiquitination the MOR.
96
Figure 3.12: Knockdown of both SIAH1 and SIAH2 decreases MOR ubiquitnation HEK-MOR cells transfected with siRNA against SIAH1 and SIAH2 in combination (DKD) or scrambled siRNA as a control (SC). (A) Crude cell lysate was immunoprecipitated with rabbit anti-FLAG antibodies and probed with mouse anti- ubiquitin antibodies to detect ubiquitinated MOR. (B) Cell lysate was immunoblotted with rabbit anti-FLAG antibodies, goat anti-SIAH1 and goat anti-SIAH2 to detect MOR, SIAH1, and SIAH2, respectively. GAPDH was used as a loading control.
3.4 Discussion
The functional relevance of the SIAH1/MOR interaction was investigated using a series of experiments designed to test the hypothesis that SIAH1 is the E3 ligase that mediates the ubiquitination and subsequent degradation of the MOR. If this hypothesis is
97 correct, overexpression of SIAH1 should result in a decrease in MOR protein levels and
MOR ubiquitination. In line with this hypothesis, when myc-tagged SIAH1 was transfected in increasing concentrations, the amount of MOR decreased. However, this observed decrease was found to be different for the two species of MOR detected on
Western blots. The high molecular weight species (band of 75 kDa) was not as sensitive to the effect of SIAH1 overexpession as the low molecular weight species (band of 50 kDa). These differences in molecular weight have been reported to be attributable to the amount of N-linked glycosylation present on the receptor as the predicted size of the unmodified MOR protein is approximately 43 kDa. These two species of the receptor correspond to highly glycosylated plasma membrane associated receptors (60-100 kDa) and slightly glycosylated or unglycosylated intracellularly associated (40-60 kDa) receptors (Huang and Liu-Chen, 2009). These results indicate that SIAH1 may be acting on the immature form of the MOR, possibly at the endoplasmic reticulum or some other intracellular location.
Treatment of HEK-MOR cells with the proteasome inhibitor, MG132, shows that the intracellular MOR (low molecular weight, 50 kDa band) is sensitive to degradation via the proteasome, but that the plasma membrane MOR is not (high molecular weight,
75 kDa band) within the time frame tested. These results are consistent with recent evidence showing that the plasma membrane MOR is degraded via the lysosome and that ubiquitination does not serve as a signal for degradation via the proteasome (Hislop et al.,
2011). When HEK-MOR cells were treated with the lysosomal inhibitor, chloroquine, both species of the receptor increased over time. Taken together, these results suggest
98 that the immature, intracellular form of the MOR is sensitive to proteasomal degradation and that this degradation may be mediated by SIAH1.
Contrary to the proposed hypothesis, overexpression of myc-tagged SIAH1 caused a decrease in the amount of ubiquitinated MOR in both the TUBEs experiments and MOR immunoprecipitation experiments, while overexpression of a RING-deficient mutant (SIAH1-TR-myc) caused an increase in detectable ubiquitinated MOR. Although these results are contradictory to the hypothesis that SIAH1 is an E3 ligase for MOR, they are confirmed by the SIAH1 siRNA knockdown experiments in HEK-MOR cells and suggest that the RING domain is not required for this increase in ubiquitination of the
MOR. A summary of results from the overexpression and siRNA knockdown experiments is shown in Table 3.2. The siRNA knockdown of SIAH1 and the overexpression of myc-tagged RING-deficient SIAH1 show similar results which is consistent with the previous observation that the RING domain mutant functions as dominant negative (Hu and Fearon, 1999). Both the knockdown of SIAH1 and the overexpression of SIAH1-TR-myc resulted in an increase in the amount of ubiquitinated
MOR, which is not in line with SIAH1 playing a role as an E3 ligase which conjugates ubiquitin to the MOR.
99 Table 3.2: Summary of SIAH overexpression and knockdown experiments For experiment column, upward arrows indicate overexpression, whereas downward arrows indicate siRNA knockdown. For effect on Siah proteins, ubiquitination, and protein levels, upward arrows indicate an increase and downward arrows indicate a decrease. ND indicates that the experiment was not performed.
Cell Line Experiment Effect on Siah Effect on Effect on proteins ubiquitination protein levels HEK-MOR SIAH1 ↑ ND ↓ Both species ↓ HEK-MOR SIAH1-TR ↑ ND ↑ ND HEK-MOR SIAH1↓ SIAH2 ↑ ↑ Low species ↑ HEK-MOR SIAH2 ↓ No change in ↓ Both species ↓ SIAH1 HEK-MOR SIAH1 ↓ and Not applicable ↓ Low species ↑ SIAH2 ↓ HEK-DOR SIAH1 ↓ SIAH2 ↑ ↑ Both species ↓ HEK-DOR SIAH2 ↓ No change in ↑ Low species ↑ SIAH1 HEK-KOR SIAH1 ↓ SIAH2 ↑ ↑ Both species ↓ HEK-KOR SIAH2 ↓ No change in ↑ Both species ↑ SIAH1
The knockdown of SIAH1 caused a significant increase in the amount of another
MORIP, SIAH2, which is an E3 ligase that can interact with SIAH1 and MOR in these cells. One could argue that the results obtained in the SIAH1 knockdowns could be due to the upregulation of SIAH2. This is partially supported by the observation that MOR ubiquitination and protein expression levels are reduced in SIAH2 knockdowns in HEK-
MOR cells, a result that is opposite from those obtained from SIAH1 knockdown.
However, when both SIAH1 and SIAH2 were knocked down in combination in HEK-
MOR cells, the amount of detectable ubiquitinated MOR was abolished.
In summary, the results obtained from the knockdown of either SIAH1 and
SIAH2 alone are not consistent with the hypothesis that one of these individually acts as
100 both an E3 ligase and an inducer of degradation for the MOR. On the contrary, a combinatorial knockdown of SIAH1 and SIAH2 did provide results that would be consistent with the hypothesis that these two proteins may work in concert to ubiquitinate
MOR. Unfortunately, no conclusions can be drawn about the effect of the double knockdown on the levels of MOR protein as not enough replicates were able to be used for statistical analysis.
The effect of Siah protein knockdown on the amount of low and high molecular
MOR species is unexpected. SIAH1 knockdown caused the amount of the low molecular weight (50 kDa band), intracellular MOR to increase significantly but did not significantly affect the high molecular weight (75 kDa band), plasma membrane- associated form of MOR (Huang and Liu-Chen, 2009). This suggests that SIAH1 may have a role in the regulation of intracellular MOR protein levels, but does not significantly affect MOR at the plasma membrane. In contrast, SIAH2 knockdown caused a decrease in both species. The knockdown of SIAH2 may allow the activity of
SIAH1 to increase, but this increased activity is not due to increases in Siah1 protein levels, as the amount of Siah1 protein did not increase as shown via Western blot analysis. Alternatively, SIAH2 may regulate another protein that is involved in regulation of MOR, resulting in decreased levels of MOR protein. However, the results of the double knock down experiments indicate that the Siah proteins are working together, as there is a reduction in the ubiquitination of MOR. These results also indicate that SIAH2 may participate in ubiquitination of the MOR, but that SIAH1 is required for degradation of the receptor. Models for the role of the Siah proteins in the ubiquitination and regulation of the MOR are discussed in more detail in Chapter 5.
101 Knockdown of either SIAH1 or SIAH2 in HEK-DOR or HEK-KOR cells resulted in an increase in the ubiquitination of DOR or KOR, respectively. These results may indicate that both Siah proteins play a role in the ubiquitination of DOR and KOR.
Additionally, SIAH1 knockdown caused a decrease in the amount both low and high molecular weight DOR (45 kDa and 55 kDa bands) and KOR (45 kDa and 55 kDa bands). While SIAH2 knock down caused significant increase in the amount of low molecular weight DOR both species of KOR were increased. As has been demonstrated with the MOR, the DOR and KOR also exist as differentially glycosylated species. For the DOR and KOR, the 45 kDa protein has been shown to remain in an intracellular compartment and is not transported to the cell surface (Chen et al., 2006b; Petaja-Repo et al., 2000), whereas the 55 kDa protein is the fully mature receptor found at the plasma membrane (Petaja-Repo et al., 2001). Without performing a double knockdown of
SIAH1 and SIAH2 in these cell lines, there are several interpretations that fit the data collected from knockdown of SIAH1 or SIAH2 in DOR-HEK and KOR-HEK cells.
These interpretations are discussed in more detail in Chapter 5. One explanation is that
SIAH1 and SIAH2 could possibly have an overlapping role in receptor ubiquitination.
Knockdown of either protein could result in upregulation or overactivation of the other which would lead to the observed increase in receptor uiquitination. Indeed as with all cell lines observed knockdown of SIAH1 caused an increase in SIAH2 levels. Another possibility is that another unknown protein is involved in the ubiquitination of DOR and
KOR and that the SIAH proteins play a role in regulating this unknown protein. Double knockdowns of both Siah proteins were attempted for both DOR and KOR cell lines, but
102 were unsuccessful. Optimization of the double siRNA transfection in these cell lines may clarify the role these E3 ligases play in the MOR life cycle.
These interactions between the Siah proteins and the opioid receptors are surprising considering that opioid receptors are typically downregulated by lysosomal degradation and not the proteasome (Marchese et al., 2008). However, ubiquitination of plasma membrane-bound MOR does occur in response to receptor activation and is required for trafficking events that downregulate receptor ligand binding (Hislop et al.,
2011). This is the first study to address basal (unstimulated) ubiquitination of the MOR.
My results indicate that SIAH1 and SIAH2 may play a role in the ubiquitination the intracellular form of the MOR under unstimulated conditions. The exact location of the the low molecular weight, intracellular MOR is not well established, but it is suspected that some resides within the biosynthetic pathway, possibly in the endoplasmic reticulum
(ER) (Huang and Liu-Chen, 2009). As a glycoprotein, the MOR must be properly folded and modified within the ER before being translocated to the plasma membrane. If the
MOR misfolds, it is subjected to endoplasmic reticulum-associated degradation (ERAD).
ERAD requires poly-ubiquitination and retro-translocation of the misfolded protein from the ER lumen to the ER membrane to the cytosol. Once in the cytosol, these proteins are subjected to degradation via the proteasome (Feldman and van der Goot, 2009).
Interestingly, misfolded delta opioid receptors have been shown to be transported to the cytoplasmic side of the ER membrane, where they are deglycosylated and conjugated with ubiquitin before being degraded by the proteasome (Petaja-Repo et al., 2001). It seems reasonable to assume that the other opioid receptors may be treated similarly. If
Siah proteins are involved in the biosynthesis of MORs, then they may regulate the
103 number and activity of opioid receptors at the cell surface, which is important in the modulation downstream signaling (Chen et al., 2006b; Petaja-Repo et al., 2000).
Presently, the precise intracellular locations of the Siah proteins are not known.
Evidence continues to mount suggesting that the ubiquitin pathway is involved in a variety of neuronal activities, including dendritic morphogenesis and synaptic plasticity which have been implicated in the development of addiction (Robinson and Kolb, 2004;
Tuoc and Stoykova, 2010; Yang et al., 2008). Thus, it will be interesting to determine how the SIAH proteins regulate the opioid receptors in cells where the both the receptors and the SIAH proteins are expressed at endogenous levels. Additionally, tissue specific or conditional knockouts of SIAH1 or SIAH2 could be tested in addiction paradigms to determine whether this regulation plays a role in the neuronal changes observed in addiction.
Chapter 4
Effect of Opioid Exposure on MORIPs
4.1 Introduction
The mu-opioid receptor (MOR), which belongs to the Class A family of G protein coupled receptors (GPCRs), mediates a variety of functions in the body, including modulation of pain perception, cell proliferation, cell survival, neural development, and synaptic plasticity (Christie, 2008; Tegeder and Geisslinger, 2004). This receptor couples preferentially to Gi/o types of G proteins and therefore inhibits adenylyl cyclase activity and regulates a number of other signaling intermediates such as mitogen activated kinase
(MAPK), phospholipase C (PLC), and various ion channels. The MOR is activated by endogenous and exogenous opioids, the latter of which include some of the most effective analgesics, but also the most highly addictive drugs of abuse.
Abuse of drugs or addiction is an abnormal behavior characterized by compulsive, out-of-control drug use despite negative consequences. The development of these behavioral abnormalities occurs gradually and progressively during repeated exposure to the abused drug. In some cases, these abnormalities can persist for a long period of time after the cessation of use (Nestler, 2004). Thus, drug addiction is seen as a form of drug- induced plasticity (Russo et al., 2010). Multiple studies have shown that repeated
105 exposure to an addictive drug can alter the types or the levels of genes/proteins expressed in the brain. In addition, these alterations mediate the function of individual neurons and related neural circuits which may be responsible for the observed behavioral abnormalities seen in addicted individuals (Nestler, 2001). Therefore, the identification of the genes/proteins associated with drug dependence is vital to understanding the molecular mechanisms underlying addiction.
Many different techniques have been utilized to investigate the gene/protein expression changes associated with drug exposure and addiction. Proteomic approaches allow for the study of these changes without the need for a prior hypothesis. Since drug exposure and addiction involve a large array of interacting proteins, it is well suited for this type of analysis. However, the lack of a hypothesis, also presents problems. Without a hypothesis, any found changes have little meaning unless further studies are implemented. One reason for this is that changes observed in the proteome may not be a direct consequence of the drug itself, but could represent downstream changes that are caused by but are far removed from the cellular pathways that are directly affected. For example, a drug might act directly on the mesolimbic dopaminergic tract. Changes in proteins in the prefrontal cortex may result as an indirect effect of altered signaling from the mesolimbic pathway. While this might give you information about the disease state overall, it provides little evidence as to how the drug itself is working on its receptors.
Another approach is to use a candidate gene(s) approach, where genes/proteins with known functions or interactions associated with genes or proteins that have already been shown to be involved in drug exposure or abuse are interrogated for expression changes during drug exposure or in addiction models.
106 The first proteins shown to interact functionally with the MOR were G proteins, which modulate canonical G protein-dependent signaling. However, it is now clear that modulation of MOR signaling and regulation are far more complex (Georgoussi et al.,
2012). Using approaches such as yeast-two hybrid screens, immunoprecipitation, immunofluorescence, or bioluminescence resonance energy transfer in live cells, it has been shown that a diverse group of other interacting partners can participate in the regulation of virtually every aspect of MOR activity. Observations from these studies have indicated that the MOR can form mulitcomponent protein assemblies, or signalplexes, involving interactions between themselves, other GPCRs, and with other interacting proteins. These interactions likely contribute to the process of signaling downstream of the receptor (Alfaras-Melainis et al., 2009; Georgoussi et al., 2012;
Kabbani et al., 2002; Milligan, 2005).
Few previously identified mu-opioid receptor interacting proteins have been investigated with regards to altered expression when cells or animals are treated with opioid drugs. Spinophilin was found to have decreased expression with acute but not with chronic exposure to morphine in the nucleus accumbens of mice. Additionally, spinophilin knockout mice exhibited decreased morphine analgesia, but increased tolerance, physical dependence, and place conditioning to morphine (Charlton et al.,
2008). Acute morphine administration has been demonstrated to cause an increase in
RGS9-2 protein levels in the nucleus accumbens and spinal cord of C57BL/6 mice.
However, mice chronically treated with morphine, showed decreases in RGS9-2 proteins levels in the same brain regions (Zachariou et al., 2003). These studies suggest that
MORIP expression levels can change with drug exposure and that these changes are
107 likely to have functional consequences, which may contribute to the development of addiction. Therefore, investigation of MORIP expression upon drug exposure may lead to the discovery of additional molecular components that may be involved in the development of the addicted state.
In this chapter, we examine the effect of drug exposure on the SIAH1/MOR interaction and the expression of selected MORIPs in specific brain regions of morphine treated mice. We demonstrate that the SIAH1/MOR interaction is altered in response to morphine and that changes in the expression of several MORIPs may be correlated with morphine exposure.
4.2 Experimental Procedures
4.2.1 Cell culture and drug treatment
Human neuroblastoma cells (SYH5Y) were cultured in Dulbecco's Modified
Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Human embryonic kidney (HEK) 293 cells stably transfected with FLAG-tagged mu-opioid receptor (HEK-MOR), FLAG-tagged delta-opioid receptor (HEK-DOR), or FLAG- tagged kappa-opioid receptor (HEK-KOR) were maintained in DMEM supplemented with 10% FBS and 400 μg/mL G418. HEK-MOR and HEK-DOR cell lines were generously provided by Dr. Mark van Zastrow (University of California San Francisco).
HEK-KOR cells were a gift from Dr. Lee-Yuan Liu-Chen (Temple University School of
108 Medicine). For drug treatment, either 10 μM morphine or 10 μM [D-Ala2, NMe-Phe4,
Gly-ol5] (DAMGO) was added to cell culture media and cells were incubated for specified time periods.
4.2.2 Co-immunoprecipitation and Western blot analysis
Human neuroblastoma cells (SHSY-5Y) were exposed to either 10 μM morphine or 10 μM DAMGO for 30 minutes and 1 hour, then lysed in 1X lysis buffer (20 mM Tris-
HCl pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1% Triton X-100 and
1mg/mL leupeptin) supplemented with protease inhibitor cocktail (Pierce, Rockford, IL).
The MOR was immunoprecipitated from total cell lysates using a polyclonal rabbit anti-
MOR antibody (AB 1580, Millipore, Billerica, MA) coupled to Protein-G Dynabeads
(Invitrogen, Grand Island, NY). Human embryonic kidney cells (HEK) stably transfected with FLAG-tagged MOR, DOR, or KOR (HEK-MOR, HEK-DOR, HEK-KOR, respectively) were treated for 1 hour and 21 hours with 10μM morphine. Cells were collected and lysed in 1X lysis buffer supplemented with protease inhibitor cocktail. The opioid receptors were immunoprecipitated using polyclonal rabbit anti-FLAG antibody
(Sigma, St. Louis, MO) bound to Protein-G Dynabeads (Invitrogen). Western blot analysis of immunoprecipitated complexes was performed by using a polyclonal goat anti-SIAH1 antibody (1:5000 dilution; Abnova, Taipei City, Taiwan) and a bovine anti- goat HRP-conjugated secondary antibody (1:20,000 dilution; Jackson ImmunoResearch).
Blots were also immunoblotted using Guinea pig anti-MOR antibodies (1:5000 dilution;
109 Neuromics, Ednia, MN) or monoclonal mouse anti-FLAG antibodies (1:5000 dilution;
F1804; Sigma) as a positive control and donkey anti-Guinea pig horseradish peroxidase
(HRP)-conjugated antibodies or goat anti-mouse-HRP conjugated secondary antibodies
(Jackson ImmunoResearch). Mock immunoprecipitations using normal rabbit IgG (Santa
Cruz Biotechnology) were used as negative controls. Immunoreactivity was detected by
ECL Plus.
4.2.3 Animal Care and Drug Treatment
All mouse studies were performed in collaboration with Tom Ferraro (University of Pennsylvania) in the research laboratories at the Veteran’s Administration Medical
Center (VAMC) in Coatsville, PA, and were approved by the University of Pennsylvania
Institutional Animal Care and Use Committee. C57BL/6J mice were bred in-house, maintained on a 14hr/10hr light/dark schedule and had food and water available ad libitum throughout the course of the experiment.
Female mice, 8-9 weeks of age, were implanted sub-cutaneously with a 25 mg morphine (n=5) or placebo (n=4) pellet. Morphine and placebo pellets were obtained from the NIDA Drug Supply Program. Mice were anesthetized with ketamine and zylazine (80 and 10 mg/kg, ip, respectively, then euthanized by cervical dislocation under carbon dioxide anesthesia four days (96 hr) after pellet implantation. Brains were harvested and dissected on ice under 5x magnification into 6 regions including prefrontal cortex, nucleus accumbens, dorsal striatum, midbrain, hippocampus, and cerebellum.
Specimens were frozen on dry ice and stored at -80C.
110 4.2.4 Tissue Preparation and Protein Analysis
Frozen brain tissue obtained from drug or sham treated mice was utilized for protein expression analysis. Fresh whole-brain tissue from untreated mice was obtained for co-IP experiments. All tissue was suspended in lysis buffer (50mM Tris-HCl, 1mM EDTA, 150mM
NaCl, 1% NP40, 0.25% deoxycholate, 5mM NaF, 2mM NaVO3) containing protease inhibitors
(cOmplete MINI EDTA free, Roche), homogenized using a microcentrifuge pestle for 2 minutes, sonicated using a probe sonicator, then centrifuged at 13,000 RPM to remove cellular debris.
For protein expression analysis, samples were analyzed by electrophoresis on 10% SDS- containing polyacrylamide gels, followed by Western blotting. In addition to antibodies utilized for co-IP, guinea pig anti-MOR (GP10106, Neuromics, Edina, MN) and Rabbit anti-DOK4
(SAB1300112, Sigma-Aldrich) antibodies were also used to probe brain lysates. For quantitative analysis of MORIP expression in drug treatment studies, lysates from sham and morphine-treated mice (20 μg/brain region) were analyzed in quadruplicate. To rule out transfer artifacts, samples from each brain region were loaded in a different order on each of the four blots. Blots were scanned using a back-lit scanner and quantitation was performed using IMAGEJ software
(Abramoff et al., 2004). Expression was normalized to total protein (as measured by Ponceau stain), averaged between replicates, and subjected to a two-tailed Student’s t-test.
111 4.3 Results
4.3.1 Drug exposure alters strength of interaction between SIAH1 and opioid receptors
In an effort to better understand the effect of the MOR/SIAH1 interaction on the regulation of the MOR under stimulated conditions, I wanted to investigate the effect of opioid agonists on MOR/SIAH1 complex formation. To determine whether opioid drugs promote MOR/SIAH1 complex formation, human neuroblastoma cells (SHSY-5Y) were exposed to either 10 μM DAMGO or 10μM morphine for 30 minutes or 1 hour. These two drugs were chosen because of their different effects on the trafficking and signaling of the MOR (Groer et al., 2011). DAMGO causes robust internalization and recycling of the MOR, while morphine causes markedly slow internalization and no recycling of the
MOR (Christie, 2008). The doses chosen have been shown to saturate receptors and activate downstream signaling as measured by adenylyl cyclase activity. The treatment times represent acute exposure that should theoretically capture internalization and downstream events (such as recycling and ubiquitination), but should not be long enough to capture degradation of the MOR (Christie, 2008).
To investigate the effects of acute DAMGO and morphine exposure on the
MOR/SIAH1 interaction, SH-SY5Y cells were treated with drug for the designated time periods. The MOR was immunoprecipitated from equal microgram concentration of total protein in the lysate (900 μg), resolved by SDS-PAGE, and immunoblotted using goat anti-SIAH1 antibodies. As seen in figure 4.1A, DAMGO caused an increase in the amount of SIAH1 that co-immunoprecipitated with the MOR in SH-SY5Y cells after 1 hour of exposure when compared to DMSO treated cells. In contrast, morphine did not
112 cause a change in the amount of co-immunoprecipitated SIAH1 at any time point tested
(Figure 4.1B). These results indicate that DAMGO induced MOR signaling or internalization enhances the MOR/SIAH1 interaction, which in turn could result in altered ubiquitination of the receptor.
Figure 4.1: DAMGO treatment of SH-SY5Y cells promotes MOR/SIAH1 complex formation (A) SHSY-5Y cells were treated with 10 μM DAMGO for 30 minutes and 1 hour. After the designated time period cells were lysed and the MOR was immunoprecipitated from 900 μg of total protein using rabbit anti-MOR antibodies bound to Protein G beads. Eluted proteins were immunoblotted for SIAH1 using goat anti-SIAH1 antibodies. (B) SHSY-5Y cells were treated with 10 μM morphine for 30 minutes and 1 hour. The MOR was immunoprecipitated from 900 ug of total protein using rabbit anti-MOR antibodies bound to Protein G beads. Eluted proteins were immunoblotted for SIAH1 using goat anti-SIAH1 antibodies. (A and B) Mock IPs using normal rabbit IgG were used as negative controls. Crude cell lysate (Ly) was used as a positive control and represents approximately 10% of protein used in IPs.
113 The SHSY cells express both MOR and DOR, which have been shown to heterodimerize and affect each other’s trafficking and function (Prather et al., 1994). I therefore wanted to assess opioid drug effects on the interaction of SIAH1 with the individual opioid receptors in the HEK cell models each expressing a single opioid receptor subtype. Although, acute morphine exposure did not alter the interaction of
SIAH1 and MOR in SHSY cells, this agonist will act at all three receptors (albeit with different affinities) and is a known clinically relevant and addictive substance (Neil,
1984). DAMGO is a selective agonist for MOR and would not be useful for the DOR and
KOR studies. HEK-DOR, HEK-KOR, and HEK-MOR cells were exposed to 10 μM of morphine for 1 hour and 21 hours. For this experiment both acute (1hr) and chronic effects (21hr) were measured as there were no changed in the MOR/SIAH1 interaction with acute exposure of morphine in the SHSY cells. Chronic treatment (21hr) has previously been determined to allow for degradation of these receptors and, therefore may be a more likely time point to observe a change in the OR/SIAH1 interaction (Afify,
2002; Chen et al., 2006b). Similar to the results with morphine treatment in SHSY-5Y cells, the MOR/SIAH1 interaction did not change with 1 or 21 hours of treatment in
HEK-MOR cells (Figure 4.2, lanes 11-13). The DOR/SIAH1 interaction, increased with both acute and chronic morphine treatment (Figure 4.2, lanes 3-5), whereas the
KOR/SIAH1 interaction did not change. These results indicate that acute morphine exposure may only alter the interaction of SIAH1 with the DOR. However, the IP lanes appear to have a double band for SIAH1 when immunoprecipitated with DOR and KOR.
This double band could be due to a number of things, such as degradation of SIAH1 or posttranslational modification. Taken together these results suggest that morphine may
114 have a selective effect on the DOR/SIAH1 interaction by promoting the formation of this complex.
Figure 4.2: Morphine treatment promotes DOR/SIAH1 complex formation (A) HEK-DOR, HEK-KOR, and HEK-MOR cells were treated with 10 μM morphine for 1 hour and 21 hours. After the designated time period cells were lysed and the DOR, KOR, or MOR was immunoprecipitated using rabbit anti-FLAG antibodies bound to Protein G beads. Eluted proteins were immunoblotted for SIAH1 using goat anti-SIAH1 antibodies. Mock IPs using normal rabbit IgG were used as negative controls. Crude cell lysate (Ly) was used as an additional positive control and represents approximately 5% of protein used in IPs.
4.3.2 Interaction of MOR and MORIPs in Rodent Brain
To determine whether the interaction between the MOR and MORIPS may occur in vivo, we analyzed the expression of MOR and several MORIPS in various mouse brain regions. Not all commercially available antibodies were reliable when used to detect
MORIPs in the brain. For example, antibodies available for SIAH2, zyxin, and DOK5 were not able to detect proteins of the proper size in brain lysates. Other antibodies, such as AUP1 could detect the protein in cell lysates, but yielded many non-specific bands in brain lysates. Therefore, only MORIPs whose commercially available antibodies detected a protein of the correct molecular weight in brain lysates were chosen for further
115 study. As shown in Fig. 4.3A, the MOR and its interacting partners DOK4, SIAH1,
VAPA, and WLS were each expressed in cerebellum, hippocampus, nucleus accumbens, midbrain, prefrontal cortex, and dorsal striatum. Co-expression of the MOR and each of the MORIPs in all brain regions tested is consistent with the idea that the MOR and its binding partners have the potential to interact in multiple brain regions. To verify interaction, the MOR was immunoprecipitated from a whole mouse brain lysate, and immunocomplexes probed for the presence of several MORIPs. As shown in Fig. 4.3B,
SIAH1, VAPA, and WLS were able to co-IP with MOR but not with rabbit-IgG alone, suggesting that these MORIPs are capable of interacting with MOR in mouse brain.
DOK4 was not tested due to crossreactivity of the antibody with rabbit IGG used for immunoprecipitations.
116
Figure 4.3: MORIP expression and interaction with MOR in mouse brain (A) Expression of MOR and MORIPS in mouse brain regions. Western blots containing lysates prepared from mouse cerebellum (C), hippocampus (H), midbrain (M), nucleus accumbens (N), prefrontal cortex (P), and striatum (S) were probed with Guinea pig anti- MOR, rabbit anti-DOK4, goat anti-SIAH1, mouse anti-VAPA, and chicken anti-WLS antibodies. (B) Co-immunoprecipitation. The MOR was immunoprecipitated from whole mouse brain lysates using rabbit anti-MOR antibodies. Immunocomplexes were probed for the presence of SIAH1, VAPA, or WLS using MORIP-specific antibodies. Lysate lanes (L) contain 5% of the total protein prepared from whole mouse brain lysate compared to the mock (M, rabbit IGG) and immunoprecipitation (IP) lanes. Experiments performed by Jessica Petko at The Pennsylvania State University College of Medicine.
117
4.3.3 The Effect of Morphine on MORIP Expression
To determine whether chronic morphine exposure alters the protein levels of
MORIPs, we performed Western blot analysis of selected MORIPs (DOK4, SIAH1, and
WLS) in brain regions thought to be involved in response to opioid drug exposure. MOR levels are not reported for this experiment, as the antibody lot utilized resulted in high background and very light bands that were unable to be quantified. Mice were implanted subcutaneously with either saline (sham n=4) or morphine (n=5) pellets for 96 hours, then euthanized, and the brains dissected.
As shown in Figure 4.4, no significant changes were detected in any of the
MORIPs tested in either cerebellum or nucleus accumbens. However, in response to morphine exposure, DOK4 levels were decreased by ~50% in hippocampus (p=0.02) and midbrain (p=0.003). In morphine-treated animals, SIAH1 expression was reduced by
48% in hippocampus (p=0.008) but showed a 55% increase in midbrain (p=0.03) (Figure
4.4). WLS levels were decreased by 49% in midbrain (p=0.006) and 39% in striatum
(p=0.02) following drug exposure (Figure 4.4). Although every effort was made to ensure equal loading of proteins, some samples consistently contained higher amounts of total protein than others (e.g., sham versus morphine treated samples in hippocampus).
Despite these differences in protein loading, our data suggest that morphine treatment may lead to significant alterations in MORIP protein expression in various regions of mouse brain.
118
Figure 4.4: MORIP expression in brain regions of morphine treated mice
Mice were treated for 96 hours with either morphine-containing (n=5) or placebo (n=4) pellets. Animals were sacrificed, brain regions dissected, and Western blots of selected MORIPs probed with MORIP-specific antibodies. Each panel contains a representative blot for a MORIP in the specified brain region (n=4 blots/MORIP/brain region). Total protein was quantitated by Ponceau stain of the blot prior to antibody probing. Bar graphs represent the average pixel density (as determined by IMAGEJ) of four blots for each brain region normalized to total protein and placebo treatment. Data was analyzed using a two-tailed Student's t-test. Error is expressed as standard error of the mean. * indicates a statistically significant difference (p<.05) between sham and morphine treatment. Experiments performed by Jessica Petko at The Pennsylvania State University College of Medicine.
119 4.4 Discussion
My results indicate that the strength of the interaction between SIAH1 and the MOR changes with acute exposure to DAMGO, but not with acute or chronic morphine treatment. In line with this finding, DAMGO has been shown to cause ubiquitination of mu opioid receptors that peaks at 1 hour of agonist stimulation (Groer et al., 2011). An acute treatment with
DAMGO causes robust internalization of the MOR within 30 minutes which may make MOR available in the cytosol for an interaction with SIAH1. In contrast, morphine causes slow internalization and no ubiquitination of the MOR which could explain why no increase in
SIAH1/MOR interaction was detected. Alternatively, the differences seen in MOR/SIAH1 complex formation could be due to proteins that are brought to the signaling complex during activation of the MOR. For example, DAMGO stimulation of MOR brings β-arrestin 2 to the
MOR signaling complex, but morphine does not. Additionally, loss of β-arrestin 2 prevents the
DAMGO-stimulated ubiquitination of the MOR (Groer et al., 2011). Degradation of the MOR in response to agonist (DAMGO or morphine) requires several hours of agonist binding (Afify,
2002; Chen et al., 2006b). Although DAMGO was not studied under chronic conditions, no change in the MOR/SIAH1 interaction was detected with morphine. Together, these results may indicate that SIAH1 may be involved in agonist stimulated ubiquitination (by DAMGO) of the
MOR as well as unstimulated ubiquitination (chapter 3).
Morphine was used to determine the effects of acute and chronic receptor activation on the interaction of the individual opioid receptors and SIAH1. With chronic morphine treatment, the interaction between SIAH1 and DOR increased while the KOR/SIAH1 interaction did not change. These results are interesting as morphine has a lower binding affinity for DOR and
120 KOR and likewise only weakly activates these receptors (Keith et al., 1996; Trescot et al., 2008).
While DOR and KOR can be ubiquitinated in response to selective agonists, nothing is known about the ubiquitination these receptors in regards to morphine stimulation (Henry et al., 2011;
Li et al., 2008). Morphine does not cause ubiquitination of MOR, but this does not rule out this possibility for DOR or KOR. In addition, little is known about the trafficking of these receptors in response to morphine exposure. Therefore, we cannot make any conclusions as to whether trafficking events are changing the DOR or KOR interaction with SIAH1.
The study of MORIP levels in morphine treated mice, utilized the inbred strain
C57BL/6J. This strain has been found to be more sensitive to the rewarding effects of morphine than other strains, such as DBA/2J, in self-administration and conditioned place preference paradigms (Elmer et al., 2010). However, our study did not use a reward paradigm (self- administration or conditioned place preference), but was designed to detect differences between those individuals exposed to morphine and naïve individuals. Although behavior was not measured in this study, 96 hour morphine treatment is known to lead to changes in dendritic morphology and neurogenesis associated with synaptic plasticity and addiction (Eisch et al.,
2000). Additionally, these changes are found to occur, regardless of the method of administration (pellet, experimenter, or self-administration) (Robinson and Kolb, 2004).
Therefore this kind of study can be a useful tool when combined with behavioral paradigms to tease out which changes are due to the effect of the drug and which may be due to behavior.
An inbred strain of mouse was utilized in this analysis, because it should control for genetic variability between animals as they should be homozygous at almost all loci. Even with this homogeneity of mice, there are some differences in the level of MORIPs detected within treatment groups. The explanation for this interanimal variability is unknown. However, it may
121 result from genetic variation, such as single nucleotide polymorphisms (SNPs) between animals.
According to the Mouse Genome Informatics database, the C57BL/6J strain can have SNPs in
SIAH1 (Siah1a of mouse), DOK4, and WLS. If these SNPs are found in some individuals and not others in this study, it may help to explain the observed variation in the level of expression of a particular MORIP in a mouse. However, no studies have investigated the role of single nucleotide polymorphisms or splice variants in SIAH1, DOK4, or WLS in regards to the expression of these proteins.
Knockout mouse experiments have shown that MOR is the primary mediator of the analgesic and rewarding properties of opioid drugs. Since MORIPs regulate the functional output of the MOR, they represent potential candidates for proteins that may contribute to the underlying molecular mechanisms leading to drug addiction. Protein expression analysis of selected brain regions from morphine-treated mice indicate that the expression of DOK4, SIAH1, and WLS are altered in distinct brain regions following chronic morphine exposure. Morphine has been shown to decrease WNT secretion in a cell culture system, and that WLS shifts from a predominantly cytosolic to a more plasma membrane-associated distribution in cultured cells as well as rat striatal neurons after morphine treatment (Jin et al., 2010a; Reyes et al., 2012). The decrease in WLS expression observed in midbrain and striatum of mice after morphine administration could possibly lead to long term decreases in WNT secretion within the CNS. It is possible that decreased Wnt secretion could be responsible for many of the observed effects of chronic opioid exposure including decreased neurogenesis and decreased dendritic spine density that are known to be affected by WNT signaling (Eisch et al., 2000; Liao et al., 2005).
The implications of decreased SIAH1 expression in the hippocampus and its increased expression in the midbrain could be enhanced by looking at MOR levels. As mentioned in the
122 results this was attempted, but changes in antibody performance (change of Lot number) did not allow the completion of this analysis. Although the functional relevance of changes in DOK4 and SIAH1 expression after morphine exposure is currently unknown, it is of interest that these changes in protein expression were observed within specific brain regions, rather than throughout the entire brain. Thus the changes in protein expression we detected may play an important role in the response to opioid drug exposure mediated by these brain regions. In the future, it should become possible to examine the role of these MORIPs in behavioral aspects of opioid addiction using animal models of opioid self-administration.
In summary the findings presented in this chapter indicate that while the non- internalizing agonist morphine does not affect the MOR/Siah1 interaction, the internalizing agonist DAMGO enhances this interaction. This indicates that although the SIAH proteins appear to be involved in a basal level of ubiquitination and receptor degradation (as described in chapter 3), there may also be a role for SIAH1 in agonist stimulated MOR regulation. In addition, several newly identified MORIPS including SIAH1 are co-expressed with and interact with MOR in the mouse brain. Finally, several MORIPs display altered expression in the mouse brain during chronic exposure to morphine. This finding not only suggests a role for these proteins in the drug adaptation process, but also validates the interaction screen/candidate gene approach to identifying potential players in development of addiction.
Chapter 5
Overall Discussion
The overall aim of this thesis was to characterize novel mu opioid receptor interacting proteins (MORIPs) in an attempt to provide new understandings into the cellular and molecular mechanisms that underlie the development of opioid addiction.
This goal was founded upon several lines of evidence, including the central role of the
MOR in mediating both the analgesic and rewarding effects of opioid agonists (Matthes et al., 1996), the growing body of evidence suggesting that G protein-coupled receptors
(GPCRs) are part of large signaling complexes whose protein constituents have not all been identified (Kabbani and Levenson, 2007), and that we still do not have a clear understanding of the basic biological underpinnings of this disease. In working towards this aim, several novel MORIPs were discovered, the function of the MOR/SIAH1/2 interaction was explored, and changes in MORIP protein levels were detected in morphine treated mice.
Possible roles of Novel MORIPs in morphine exposure
Utilization of traditional and modified yeast two-hybrid (Y2H) screens identified
10 novel and two previously discovered components of the MOR signaling complex.
Some of these MORIPs were shown to interact with other opioid receptors, which
124 suggests that they may be universal regulators of opioid function. The conventional Y2H was unique as it utilized the second intracellular loop of the MOR, which had not previously been studied in terms of interacting proteins. Several of the novel MORIPs identified in these screens have been shown to share common cellular functions that may correlate with changes that have been observed in the development of addiction, such as synaptic plasticity and neurogenesis. A summary of the cellular function of MORIPs found in this study and in previous studies is depicted in Figure 5.1.
Figure 5.1: MOR interaction map Web of newly identified and previously known MOR interacting proteins. MORIPs were grouped based on previously characterized functional properties. MORIPs identified in the current study are depicted by green shaded circles, while previously identified MORIPs are represented by white circles. Many of the newly identified MORIPs also interact with DOR and KOR (see Chapter 2 Results).
125 Since MORIPs have the potential to regulate the functional output of the MOR, they represent potential candidates for proteins that may contribute to the underlying molecular mechanisms leading to drug addiction. Protein expression analysis of selected brain regions from morphine-treated mice indicate that the expression of DOK4, SIAH1, and WLS are altered in distinct brain regions following chronic morphine exposure. Morphine has been shown to decrease WNT secretion in a cell culture system, and WLS shifts from a predominantly cytosolic to a more plasma membrane-associated distribution in cultured cells as well as rat striatal neurons after morphine treatment (Jin et al., 2010a; Reyes et al., 2012). The decrease in WLS expression observed in midbrain and striatum of mice after morphine administration could possibly lead to long term decreases in WNT secretion within the CNS. The midbrain contains the ventral tegmental area (VTA) nuclei that are the main sources of dopamine (DA) in the brain, whereas other regions, such as the nucleus accumbens, striatum, and prefrontal cortex are the target regions for DA released from the VTA. It is widely accepted that addictive drugs cause functional changes these brain regions. These functional changes may manifest as changes in neuron firing, strengthening or weakening of synapses, changes in neuron morphology, and neurogenesis (Nestler, 2004; Nugent et al., 2007; Robinson and Kolb, 2004; Russo et al., 2010).
Coincidentally, dendritic spine density and neurogenesis are known to be affected by WNT signaling (Eisch et al., 2000; Liao et al., 2005). Therefore, it has been hypothesized that decreased WNT secretion could be responsible for many of the observed structural and functional effects of chronic opioid exposure (Jin et al., 2010).
In the nervous system, DOK4 has been shown to be required for axon myelination
(Blugeon et al., 2011) and the regulation of growth derived neurotropic factor (GDNF)- dependent neurite outgrowth (through activation of the Rap1-ERK1/2 pathway) (Uchida et al.,
126 2006). Neurite outgrowth has been proposed as a possible mechanism for plasticity in the hippocampus (Ring et al., 2006). A hypothetical explanation for the decreased levels of DOK4 in the hippocampus is that the reduction in DOK4 protein may contribute to reduced neurite outgrowth and consequently reduced plasticity in this brain region. Determining the function of the MOR/DOK4 interaction in relation to neuronal morphology, MAPK-mediated signaling, and other processes associated with MOR activation and opioid addiction may clarify the consequences of reduced DOK4 in response to morphine exposure in mice.
The expression of SIAH1 mRNA has been found to be decreased in the middle temporal gyrus (includes the hippocampus) of patients with a history of alcohol abuse (Sokolov et al.,
2003). Our study found a decrease in SIAH1 protein levels in the hippocampus of morphine treated mice. The method of morphine treatment performed in this study has previously been shown by an independent group to cause structurally changes in the hippocampus that are associated with the development of addiction (Liao et al., 2005; Nestler, 2004). Additionally, other proteins involved in the ubiquitin proteasome pathway have been shown to be altered in addiction (Bingol and Sheng, 2011; Tuoc and Stoykova, 2010). Based on the results from
Chapter 3, it is tempting to speculate that SIAH levels play an important role in the addiction process by direct regulation of MOR ubiquitination, trafficking, and/or degradation. Further characterization of the roles of SIAH E3 ligases in ligand-mediated MOR regulation will be crucial to determining whether this speculation may be possible.
Although the functional relevance of changes in WLS, DOK4 and SIAH1 expression after morphine exposure is currently unknown, it is of interest that these changes in protein expression were observed within specific brain regions, rather than throughout the entire brain.
Thus, the changes in protein expression we detected may play an important role in the response
127 to opioid drug exposure mediated by these brain regions. In the future, it should become possible to examine the role of these MORIPs in behavioral aspects of opioid addiction using animal models of opioid self-administration. Knockout animals of WLS, SIAH1, or DOK4 could be used in self-administration paradigms to determine the possible role that they play in the addiction process. This type of approach allows for the measurement of drug seeking, taking, and reinstatement. The affect on these addiction-like behaviors due to the loss of a particular
MORIP, has already been used for spinophilin (a previously identified MORIP). The loss of spinophilin in mice resulted in a greater sensitivity to the rewarding properties of morphine and the development of a higher degree of physical dependence (Charlton et al., 2008). The use of a similar approach for WLS, SIAH1, and DOK4 may be able to clarify the roles of these proteins in behavior. Additionally, the role of degradation could be explored through the use of lysosomal and proteasomal inhibitors in conjunction with a self-administration paradigm with knockout animals.
The DOR/SIAH1/2 and KOR/SIAH1/2 interactions
The MOR is known to form functional dimers with the other two major opioid receptors (Chakrabarti et al., 2010; Gomes et al., 2000), and all three of these receptors have been demonstrated to interact with both SIAH1 and SIAH2 (Chapter 2). Thus, it was important to investigate the role of the Siah proteins in the regulation of the DOR and KOR. MOR will be discussed in more detail; therefore, DOR and KOR will be discussed first. In chapter 3, SIAH1 and SIAH2 were knocked down in HEK-DOR and
HEK-KOR cells. The results from those experiments yield several possible models for
128 the function of the Siah proteins in the basal (unstimulated) regulation of DOR and KOR.
Two of these models will be discussed below.
In the first model, both SIAH1 and SIAH2 are capable of conjugating ubiquitin to the DOR and the KOR (Figure 5.2A). In other words, the Siah proteins may compensate for the loss of the other. This ubiquitination of DOR and KOR by the Siah proteins leads to different fates for the receptors. Ubiquitination by SIAH2 appears to signal for degradation of these receptors, as knockdown of SIAH2 caused an increase in the protein levels of both DOR and KOR. In contrast, ubiquitination by SIAH1 does not appear to signal for degradation, as knockdown of SIAH1 caused a decrease in the protein levels of both DOR and KOR. In the second model, the Siah proteins regulate the ability of another E3 ligase to conjugate ubiquitin to DOR and KOR. SIAH 1 may regulate
SIAH2, SIAH2 may regulate SIAH1, or the regulation may require a heterodimers of
SIAH1/SIAH2, which regulate a third E3 ligase. This E3 ligase would then conjugate the
DOR or KOR with ubiquitin and signal for degradation or trafficking (Figure 5.2B). This model predicts increased ubiquitination with SIAH1 or SIAH2 knockdown. Consistent with this model, knockdown of either SIAH1 or SIAH2 resulted in increased receptor ubiquitination. Without the benefit of double knockdown experiments in HEK-DOR and
HEK-KOR cells, it is difficult to discern which model may best represent what is occurring in these cells with regards to DOR and KOR regulation.
The experiments performed in this dissertation did not look at the effects of opioid agonist stimulation in combination with SIAH1 or SIAH2 knockdown. However, the effect of acute and chronic morphine treatment on DOR/SIAH1 and KOR/SIAH1 complex formation was examined. Although morphine weakly binds and activates DOR
129 and KOR, chronic morphine treatment resulted in an increase in the formation of
DOR/SIAH1 complexes. Therefore, it is possible that SIAH1 may play an additional role in the regulation of DOR and/or KOR stimulated with agonist. Experiments utilizing
DOR- and KOR-specific agonists in combination with knockdown of SIAH1 and SIAH2 may help clarify the roles of the Siah proteins in receptor regulation.
Lastly, it will be important to determine the functional consequences of the ubiquitination of these receptors both basally and upon activation. My results suggest that ubiquitination in the absence of SIAH1 allows for receptor degradation, whereas the absence of SIAH2 does not. Since both the lysosomal and proteasomal pathways can degrade the DOR and KOR, it will be important to determine which pathway is being used (Chaturvedi et al., 2001; Henry et al., 2011; Li et al., 2008). This could be accomplished by blocking the lysosome and the proteasome with inhibitors in combination with the knockdown of SIAH1 and SIAH2.
130
Figure 5.2: Models for SIAH1 and SIAH2 function in HEK-DOR and HEK-MOR cells (A) SIAH1 and SIAH2 are capable of ligating ubiquitin to the DOR and/or KOR. This ubiquitination signal has three possible consequences, degradation by the lysosome, degradation by the proteasome, or a trafficking event. (B) SIAH1 and SIAH2 or a SIAH1/SIAH2 heterodimer may regulate another E3 ligase, which acts to ubiquitinate DOR and/or KOR. This ubiquitination signal has three possible consequences, degradation by the lysosome, degradation by the proteasome, or trafficking of the receptor(s).
Model for the Interaction between the MOR and the Siah proteins
The discovery that SIAH1 and SIAH2 interact with the MOR was surprising, considering that the MOR is usually downregulated by lysosomal degradation and not via the proteasome. However, ubiquitination of the MOR does occur and has been shown to be required for trafficking events that downregulate receptor ligand binding after agonist
131 stimulation (Hislop et al., 2011). Additionally, this ubiquitination has been shown to be ligand dependent, as DAMGO but not morphine, will cause ubiquitination of the MOR
(Groer et al., 2011).
The work presented in this dissertation suggests a previously unrecognized role for ubiquitination in the regulation of unstimulated MOR expression via interaction with
SIAH1 and SIAH2. The combined overexpression data and Siah proteins knockdowns, yield several possible models for their role in MOR regulation. In both models, SIAH1 acts to regulate the levels of SIAH2 protein, either directly (Figure 5.3A) or indirectly
(Figure 5.3B). This is consistent with the observation that Siah2 proteins levels increased with SIAH1 knockdown in all cell lines tested. Both models also suggest that SIAH2 is an E3 ligase for the MOR, as SIAH2 knockdown resulted in a decrease in receptor ubiquitination. However, SIAH2 knockdown did not result in increased levels of MOR protein, suggesting that SIAH2-mediated ubiquitination of the MOR alone is not sufficient for degradation of the receptor. It is possible that another protein is required to escort the ubiquitinated MOR to the site of degradation (lysosome or proteasome). It is tempting to speculate that this shuttle protein may be another novel MORIP, such as
AUP1 or CSN5. The effect of the knockdown of these proteins on ubiquitination and receptor protein levels has not yet been investigated and may shed some light on the role of these ubiquitin pathway proteins in the MOR life cycle. Another alternative is that
SIAH1 may serve a dual role as the SIAH2 regulator and a shuttle protein. The double knockdown of both SIAH1 and SIAH2 yielded results that suggest that both SIAH1 and
SIAH2 are required for the ubiquitination of the MOR.
132
Figure 5.3: Simplified model of Siah protein-mediated MOR degradation (A) SIAH1 inhibits SIAH2, which ubiquitinates MOR. Degradation via either the lysosomal or proteasomal pathway requires a shuttle protein to bring the ubiquitinated MOR to the site of degradation. (B) SIAH1 may inhibit another protein that is responsible for activation of SIAH2, which ubiquitinates the receptor. Either the lysosomal or proteasomal degradation requires a shuttle protein to bring the ubiquitinated MOR to the site of degradation.
Treatment of SH-SY5Y cells with DAMGO and morphine, showed different effects on the SIAH1/MOR interaction. DAMGO has been shown to cause rapid internalization, recycling, and ubiquitination of the MOR, while morphine treatment results in much slower internalization and no recycling or ubiquitination of receptors
(Christie, 2008; Hislop et al., 2011). The interaction between SIAH1 and the MOR was strengthened with acute DAMGO treatment, but did not change with either acute or chronic morphine treatment. This observation indicates that DAMGO, but not morphine promotes the formation of MOR/SIAH1 complexes. One hypothesis is that the increased
133 interaction detected between MOR and SIAH1 with DAMGO stimulation, results in the ubiquitination of the MOR. In contrast to the role of SIAH1 in unstimulated MOR regulation, DAMGO stimulated MOR regulation suggests that SIAH1 may be the putative E3 ligase responsible for the ubiquitination of the MOR observed by Groer and colleagues (2011). Unfortunately, probing the Western blots from these experiments with anti-ubiquitin antibodies was unsuccessful. An alternative explanation is that
DAMGO treatment increases SIAH2 activity resulting in MOR ubiquitination and that the ubiquitination signals SIAH1 to form an interaction with the MOR. Determining the affect of DAMGO treatment on the MOR/SIAH2 interaction was attempted, but was unsuccessful. Unfortunately re-probing the Western blots from these experiments with the anti-SIAH2 antibody did not produce bands. In the future it will be important to determine the effect of DAMGO and morphine treatment on the MOR/SIAH2 interaction. In addition, acute and chronic DAMGO treatment of cells in which the Siah proteins have been knocked down may clarify the roles of the Siah proteins in regulation of agonist stimulated MOR.
Interestingly, not all MOR species were sensitive to the effects of SIAH1, as only the low molecular weight species (50 kDa band) significantly changed. This low molecular weight, intracellular species has been postulated to represent MOR in the biosynthetic pathway (Huang and Liu-Chen, 2009). Therefore, it is possible that some of this MOR species is associated with the endoplasmic reticulum or another intracellular compartment. Proteins destined for insertion into the plasma membrane enter the endoplasmic reticulum (ER) in an unfolded state and generally leave after they have reached their native states. However, folding in the ER is often slow and inefficient and a
134 substantial fraction of polypeptides fail to reach their native state. The cell must continuously scrutinize these newly synthesized proteins and remove those that are terminally misfolded (Smith et al., 2011). High fidelity of the ER quality control is crucial for the functionally integrity of the cell. To illustrate this point, even subtle mutations that would not dramatically affect protein function can lead to retention of the mutant protein within the ER, thereby preventing its physiologically relevant activity.
This has been shown for a variety of proteins, including cystic fibrosis transmembrane conductance regulator (CFTR), V2-vasopressin receptor, and many GPCRs, including the
MOR (Chaipatikul et al., 2003). However, retention and degradation of incorrectly folded proteins may not be restricted to products of mutated genes. It has been proposed that as much as 30% of newly synthesized proteins in various cell types never reach their correct native structure (Schubert et al., 2000). In fact, it has been demonstrated that only
40% of newly synthesized delta opioid receptors are transported to the cell surface in human embryonic kidney cells (Petaja-Repo et al., 2000). The number and activity of opioid receptors on the cell surface are important factors in determining the capacity of these receptors to modulate downstream signaling molecules (Law et al., 2000).
The involvement of the Siah proteins as potential E3 ligases involved in ERAD is one explanation for the interaction between SIAH1 and 2 and MOR under non-stimulated conditions. A number of ERAD ligases have been discovered in mammalian cells. Most of them, like RNF5/RMA1, TEB4, RFP2, Kf-1, and TRC8 are poorly characterized with only a few targets identified for each of them. Those that have been investigated in greater detail, such as Hrd1 (synoviolin) and gp78 (autocrine motility factor, AMFR) have numerous single and overlapping substrates (Mehnert et al., 2010). The majority of
135 these ERAD ligases have a transmembrane domain which tethers them to the ER membrane. Analysis of the Siah proteins using PRED-TMR, a program designed to predict transmembrane domains, did not detect any putative transmembrane domains
(Pasquier et al., 1999). In fewer cases, cytosolic ligases, such as Parkin, have been demonstrated to promote turnover of selected proteins via ERAD (Imai et al., 2002;
Yoshida et al., 2002). Thus, the Siah proteins may be additional cytosolic E3 ligases that participate in the ERAD pathway. Determining the precise location where the interaction between the Siah proteins and the MOR occurs could help determine whether or not this interaction may play a role in the ER-associated degradation of the MOR. This could be tested using immunogold labeling of MOR and Siah proteins and electromicrography.
Currently, there is a lack of data on the expression profile of SIAH1 and SIAH2 and the contribution of these E3 ligases to ERAD. The criteria that drive individual polypeptides into distinct ERAD pathways also remain unknown (Kostova et al., 2007;
Mehnert et al., 2010). However, one could hypothesize that the activity of individual ligases or their cofactors may be controlled spatially, temporally, or in a tissue-specific manner to allow cells to adapt to varying physiological conditions. In addition, the amount of some of these ligases and cofactors may be regulated by the proteolysis of other ERAD ligases (Mehnert et al., 2010). In fact, the ERAD ligase Hrd1 has been shown to promote gp78 ubiquitination and degradation under certain conditions, thereby affecting the degradation of gp78 target proteins (Shmueli et al., 2009). Thus, the regulation of SIAH2 by SIAH1 may be important in preventing the targets of SIAH2 from ubiquitination under certain conditions, such as basal MOR turnover.
136 In the model of MOR degradation via ERAD, misfolded MOR would be recognized by ER chaperones, which would bring it to an ER-resident E3 ligase for ubiquitination (Figure 5.4A). Once ubiquitinated, the misfolded MOR would be translocated through a channel protein to the cytosol. Cytosolic de-ubiquitinating enzymes (DUBs) would then remove the ubiquitin from the MOR (Figure 5.4B).
Cytosolic chaperones would bind the misfolded MOR and SIAH2 would ubiquitinate the
MOR to signal for degradation (Figure 5.4C). The SIAH2-mediated ubiquitination of
MOR, would facilitate the shuttling of the misfolded protein to the proteasome by SIAH1
(Figure 5.4D). However, further evidence is needed to support or refute this model. One method of determining the involvement of an E3 ligase in ERAD, is to induce ER stress in cells. Sustained ER stress eventually leads to a stress response, unfolded protein response (UPR). In an attempt to relieve this stress, cells will increase the synthesis of
ER-resident chaperones and other proteins involved in ER-associated protein degradation. Therefore, if the Siah proteins are involved in ERAD, exposing cells to an
N-linked glycosylation inhibitor, such as tunicamycin, may result in increased expression of the Siah proteins.
137
Figure 5.4: Model of the role of SIAH1 and SIAH2 in ER-associated degradation of the MOR (A) MOR (black line) is recognized by ER chaperones and is partially translocated through a protein conducting channel complex/retrotanslocon (brown). MOR is conjugated with chains of ubiquitin (gray circles) by an ER-resident ubiquitin ligase (E3) and its cognate ubiquitin conjugating enzyme (E2) on the cytosolic face of the ER membrane. (B) The ubiquitin chains on the MOR serve as an exit signal and MOR moves out of the ER. This movement is facilitated by the removal of ubiquitin molecules by de- ubiquitinating enzymes (DUB). (C) The dislocated MOR is ubiquitinated a second time by SIAH2. (D) SIAH1 recognizes the ubiquitnated MOR and shuttles it to the proteasome for degradation. Adapted from (Tsai and Weissman, 2011).
Taken together my results suggest that the roles for the Siah proteins in ubiquitination of the opioid receptors are different. The majority of my results suggest
SIAH2 is a putative E3 ligase for all three of the opioid receptors. Testing the ability of
SIAH2 to conjugate ubiquitin to the individual opioid receptors in in vitro ubiquitination assays would confirm or refute SIAH2 as an E3 ligase for the opioid receptors. In contrast, SIAH1 does not appear to be a putative E3 ligase for the unstimulated MOR, but instead may be a shuttle protein or chaperone that escorts the MOR. However, my results do not eliminate SIAH1 as a putative E3 ligase for DOR and KOR.
138 The precise role that ubiquitination by SIAH1 or SIAH2 may be playing in the life cycle of the opioid receptors is still unknown. For the MOR, ubiquitination by
SIAH2 appears to eventually lead to receptor degradation. In the future it will be important to determine whether SIAH2- mediated ubiquitination results in lysosomal or proteasomal degradation of the MOR. For the DOR and KOR, the role of SIAH1 and/or
SIAH2 ubiquitination is much less clear. Ubiquitination of DOR and KOR may also lead to degradation, but it may also result in trafficking of these receptors. Tracking the intracellular location of opioid receptors under both unstimulated and agonist-stimulated conditions may shed some light on a more precise role for ubiquitination in the life cycle of these receptors.
In summary, utilization of traditional and modified yeast two-hybrid (Y2H) methods identified ten novel and two previously reported MORIPs. Directed Y2H and
GST-PD studies were used to confirm interaction and map the binding site for several
MORIPs to the second intracellular loop of MOR. Many MORIPs were demonstrated to interact with full length MOR, as well as the DOR and KOR in the context of cultured human cells. Additionally, chronic morphine treatment was shown to affect the protein levels of WLS, SIAH1, and DOK4 in specific brain regions of mice. Functional characterization of two of these novel MORIPs, SIAH1 and SIAH2, has suggested distinct roles for these proteins in the basal (unstimulated) regulation of the MOR.
SIAH2 is a putative E3 ligase for the MOR, whereas SIAH1 appears to play a role in the trafficking of the MOR to the site of degradation. The Siah proteins also may ubiquitinate and degrade or traffic the DOR and KOR. Identification and characterization of the full panorama of MORIPs represents a critical step in
139 understanding the normal regulation of the receptor life-cycle, as well as how receptor signaling or trafficking may be hijacked in response to drugs of abuse. The MORIPs identified in this and other studies may represent key players involved in other opioid- mediated processes such as neural development, nociception, aversion, and synaptic plasticity. As the list of MORIPs grows, and their contributions to receptor function become better understood, it is possible that some of these proteins will also become targets for new drug development to prevent and/or treat opioid addiction.
REFERENCES
Abbadie, C., Gultekin, S.H., and Pasternak, G.W. (2000a). Immunohistochemical localization of the carboxy terminus of the novel mu opioid receptor splice variant MOR- 1C within the human spinal cord. Neuroreport 11, 1953-1957.
Abbadie, C., Pan, Y., Drake, C.T., and Pasternak, G.W. (2000b). Comparative immunohistochemical distributions of carboxy terminus epitopes from the mu-opioid receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat CNS. Neuroscience 100, 141-153.
Abbadie, C., Pan, Y.X., and Pasternak, G.W. (2000c). Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1- like immunoreactivity: evidence for region-specific processing. J Comp Neurol 419, 244- 256.
Abbadie, C., Pan, Y.X., and Pasternak, G.W. (2004). Immunohistochemical study of the expression of exon11-containing mu opioid receptor variants in mouse brain. Neuroscience 127, 419-430.
Abbadie, C., Pasternak, G.W., and Aicher, S.A. (2001). Presynaptic localization of the carboxy-terminus epitopes of the mu opioid receptor splice variants MOR-1C and MOR- 1D in the superficial laminae of the rat spinal cord. Neuroscience 106, 833-842.
Abramoff, M.D., Magalhaes, P.J., and Ram, S.J. (2004). Image Processing with ImageJ. Biophotonics International 11, 36-42.
Afify, E.A. (2002). Turnover of mu-opioid receptors in neuroblastoma cells. Brain Res Mol Brain Res 106, 83-87.
Akil, H., Owens, C., Gutstein, H., Taylor, L., Curran, E., and Watson, S. (1998). Endogenous opioids: overview and current issues. Drug Alcohol Depend 51, 127-140.
Alfaras-Melainis, K., Gomes, I., Rozenfeld, R., Zachariou, V., and Devi, L. (2009). Modulation of opioid receptor function by protein-protein interactions. Front Biosci 14, 3594-3607.
Ambrosio, E., Goldberg, S.R., and Elmer, G.I. (1995). Behavior genetic investigation of the relationship between spontaneous locomotor activity and the acquisition of morphine self-administration behavior. Behav Pharmacol 6, 229-237.
141 Ancevska-Taneva, N., Onoprishvili, I., Andria, M.L., Hiller, J.M., and Simon, E.J. (2006). A member of the heat shock protein 40 family, hlj1, binds to the carboxyl tail of the human mu opioid receptor. Brain Res 1081, 28-33.
Arttamangkul, S., Quillinan, N., Low, M.J., von Zastrow, M., Pintar, J., and Williams, J.T. (2008). Differential activation and trafficking of micro-opioid receptors in brain slices. Mol Pharmacol 74, 972-979.
Aubrun, F., Langeron, O., Quesnel, C., Coriat, P., and Riou, B. (2003). Relationships between measurement of pain using visual analog score and morphine requirements during postoperative intravenous morphine titration. Anesthesiology 98, 1415-1421.
Bailey, C.P., and Connor, M. (2005). Opioids: cellular mechanisms of tolerance and physical dependence. Curr Opin Pharmacol 5, 60-68.
Ballesteros-Yanez, I., Ambrosio, E., Benavides-Piccione, R., Perez, J., Torres, I., Miguens, M., Garcia-Lecumberri, C., and DeFelipe, J. (2007). The effects of morphine self-administration on cortical pyramidal cell structure in addiction-prone lewis rats. Cereb Cortex 17, 238-249.
Ballesteros-Yanez, I., Ambrosio, E., Perez, J., Torres, I., Miguens, M., Garcia- Lecumberri, C., and DeFelipe, J. (2008). Morphine self-administration effects on the structure of cortical pyramidal cells in addiction-resistant rats. Brain Res 1230, 61-72.
Bare, L.A., Mansson, E., and Yang, D. (1994). Expression of two variants of the human mu opioid receptor mRNA in SK-N-SH cells and human brain. FEBS Lett 354, 213-216.
Bart, G., Heilig, M., LaForge, K.S., Pollak, L., Leal, S.M., Ott, J., and Kreek, M.J. (2004). Substantial attributable risk related to a functional mu-opioid receptor gene polymorphism in association with heroin addiction in central Sweden. Mol Psychiatry 9, 547-549.
Bayerer, B., Stamer, U., Hoeft, A., and Stuber, F. (2007). Genomic variations and transcriptional regulation of the human mu-opioid receptor gene. Eur J Pain 11, 421-427.
Becker, A., Grecksch, G., Brodemann, R., Kraus, J., Peters, B., Schroeder, H., Thiemann, W., Loh, H.H., and Hollt, V. (2000). Morphine self-administration in mu-opioid receptor- deficient mice. Naunyn Schmiedebergs Arch Pharmacol 361, 584-589.
Befort, K., Filliol, D., Decaillot, F.M., Gaveriaux-Ruff, C., Hoehe, M.R., and Kieffer, B.L. (2001). A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling. J Biol Chem 276, 3130-3137.
Bennett, M.V. (1997). Gap junctions as electrical synapses. J Neurocytol 26, 349-366.
142 Berger, A.C., and Whistler, J.L. (2011). Morphine-induced mu opioid receptor trafficking enhances reward yet prevents compulsive drug use. EMBO Mol Med 3, 385-397.
Berrendero, F., Kieffer, B.L., and Maldonado, R. (2002). Attenuation of nicotine-induced antinociception, rewarding effects, and dependence in mu-opioid receptor knock-out mice. J Neurosci 22, 10935-10940.
Berrettini, W.H., Alexander, R., Ferraro, T.N., and Vogel, W.H. (1994). A study of oral morphine preference in inbred mouse strains. Psychiatr Genet 4, 81-86.
Bingol, B., and Sheng, M. (2011). Deconstruction for reconstruction: the role of proteolysis in neural plasticity and disease. Neuron 69, 22-32.
Bloom, F.E. (1983). The endorphins: a growing family of pharmacologically pertinent peptides. Annu Rev Pharmacol Toxicol 23, 151-170.
Blugeon, C., Le Crom, S., Richard, L., Vallat, J.M., Charnay, P., and Decker, L. (2011). Dok4 is involved in Schwann cell myelination and axonal interaction in vitro. Glia 59, 351-362.
Bockaert, J., Perroy, J., Becamel, C., Marin, P., and Fagni, L. (2010). GPCR interacting proteins (GIPs) in the nervous system: Roles in physiology and pathologies. Annu Rev Pharmacol Toxicol 50, 89-109.
Bohn, L.M., Gainetdinov, R.R., Lin, F.T., Lefkowitz, R.J., and Caron, M.G. (2000). Mu- opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 408, 720-723.
Bolan, E.A., Pan, Y.X., and Pasternak, G.W. (2004). Functional analysis of MOR-1 splice variants of the mouse mu opioid receptor gene Oprm. Synapse 51, 11-18.
Bond, C., LaForge, K.S., Tian, M., Melia, D., Zhang, S., Borg, L., Gong, J., Schluger, J., Strong, J.A., Leal, S.M., et al. (1998). Single-nucleotide polymorphism in the human mu opioid receptor gene alters beta-endorphin binding and activity: possible implications for opiate addiction. Proc Natl Acad Sci U S A 95, 9608-9613.
Bousman, C.A., Chana, G., Glatt, S.J., Chandler, S.D., May, T., Lohr, J., Kremen, W.S., Tsuang, M.T., and Everall, I.P. (2010). Positive symptoms of psychosis correlate with expression of ubiquitin proteasome genes in peripheral blood. Am J Med Genet B Neuropsychiatr Genet 153B, 1336-1341.
Buday, L., and Tompa, P. (2010). Functional classification of scaffold proteins and related molecules. FEBS J 277, 4348-4355.
143 Bursen, A., Moritz, S., Gaussmann, A., Dingermann, T., and Marschalek, R. (2004). Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins: indication for t(4;11) pathobiology? Oncogene 23, 6237-6249.
Bushell, T., Endoh, T., Simen, A.A., Ren, D., Bindokas, V.P., and Miller, R.J. (2002). Molecular components of tolerance to opiates in single hippocampal neurons. Mol Pharmacol 61, 55-64.
Carthew, R.W., and Rubin, G.M. (1990). seven in absentia, a gene required for specification of R7 cell fate in the Drosophila eye. Cell 63, 561-577.
Cen, B., Yu, Q., Guo, J., Wu, Y., Ling, K., Cheng, Z., Ma, L., and Pei, G. (2001). Direct binding of beta-arrestins to two distinct intracellular domains of the delta opioid receptor. J Neurochem 76, 1887-1894.
Chaipatikul, V., Erickson-Herbrandson, L.J., Loh, H.H., and Law, P.Y. (2003). Rescuing the traffic-deficient mutants of rat mu-opioid receptors with hydrophobic ligands. Mol Pharmacol 64, 32-41.
Chakrabarti, S., Liu, N.J., and Gintzler, A.R. (2010). Formation of mu-/kappa-opioid receptor heterodimer is sex-dependent and mediates female-specific opioid analgesia. Proc Natl Acad Sci U S A 107, 20115-20119.
Chang, A., Emmel, D.W., Rossi, G.C., and Pasternak, G.W. (1998). Methadone analgesia in morphine-insensitive CXBK mice. Eur J Pharmacol 351, 189-191.
Charlton, J.J., Allen, P.B., Psifogeorgou, K., Chakravarty, S., Gomes, I., Neve, R.L., Devi, L.A., Greengard, P., Nestler, E.J., and Zachariou, V. (2008). Multiple actions of spinophilin regulate mu opioid receptor function. Neuron 58, 238-247.
Chaturvedi, K., Bandari, P., Chinen, N., and Howells, R.D. (2001). Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors. J Biol Chem 276, 12345-12355.
Chavkin, C., Bakhit, C., Weber, E., and Bloom, F.E. (1983). Relative contents and concomitant release of prodynorphin/neoendorphin-derived peptides in rat hippocampus. Proc Natl Acad Sci U S A 80, 7669-7673.
Chen, C., Li, J.G., Chen, Y., Huang, P., Wang, Y., and Liu-Chen, L.Y. (2006a). GEC1 interacts with the kappa opioid receptor and enhances expression of the receptor. J Biol Chem 281, 7983-7993.
Chen, Y., Chen, C., Wang, Y., and Liu-Chen, L.Y. (2006b). Ligands regulate cell surface level of the human kappa opioid receptor by activation-induced down-regulation and pharmacological chaperone-mediated enhancement: differential effects of nonpeptide and peptide agonists. J Pharmacol Exp Ther 319, 765-775.
144 Choo, Y.Y., Boh, B.K., Lou, J.J., Eng, J., Leck, Y.C., Anders, B., Smith, P.G., and Hagen, T. (2011). Characterization of the role of COP9 signalosome in regulating cullin E3 ubiquitin ligase activity. Mol Biol Cell 22, 4706-4715.
Chretien, M., Benjannet, S., Gossard, F., Gianoulakis, C., Crine, P., Lis, M., and Seidah, N.G. (1979). From beta-lipotropin to beta-endorphin and 'pro-opio-melanocortin'. Can J Biochem 57, 1111-1121.
Christie, M.J. (2008). Cellular neuroadaptations to chronic opioids: tolerance, withdrawal and addiction. Br J Pharmacol 154, 384-396.
Collins, R.J., Weeks, J.R., Cooper, M.M., Good, P.I., and Russell, R.R. (1984). Prediction of abuse liability of drugs using IV self-administration by rats. Psychopharmacology (Berl) 82, 6-13.
Contarino, A., Picetti, R., Matthes, H.W., Koob, G.F., Kieffer, B.L., and Gold, L.H. (2002). Lack of reward and locomotor stimulation induced by heroin in mu-opioid receptor-deficient mice. Eur J Pharmacol 446, 103-109.
Cooke, S.F., and Bliss, T.V. (2006). Plasticity in the human central nervous system. Brain 129, 1659-1673.
Corbett, A.D., Henderson, G., McKnight, A.T., and Paterson, S.J. (2006). 75 years of opioid research: the exciting but vain quest for the Holy Grail. Br J Pharmacol 147 Suppl 1, S153-162.
Crystal, H.A., Hamon, S., Randesi, M., Cook, J., Anastos, K., Lazar, J., Liu, C., Pearce, L., Golub, E., Valcour, V., et al. (2012). A C17T polymorphism in the mu opiate receptor is associated with quantitative measures of drug use in African American women. Addict Biol 17, 181-191.
Cussac, D., Rauly-Lestienne, I., Heusler, P., Finana, F., Cathala, C., Bernois, S., and De Vries, L. (2012). mu-opioid and 5-HT(1A) receptors heterodimerize and show signalling crosstalk via G protein and MAP-kinase pathways. Cell Signal 24, 1648-1657.
Dacher, M., and Nugent, F.S. (2011). Opiates and plasticity. Neuropharmacology 61, 1088-1096.
Dahan, A., Sarton, E., Teppema, L., Olievier, C., Nieuwenhuijs, D., Matthes, H.W., and Kieffer, B.L. (2001). Anesthetic potency and influence of morphine and sevoflurane on respiration in mu-opioid receptor knockout mice. Anesthesiology 94, 824-832.
David, V., Matifas, A., Gavello-Baudy, S., Decorte, L., Kieffer, B.L., and Cazala, P. (2008). Brain regional Fos expression elicited by the activation of mu- but not delta- opioid receptors of the ventral tegmental area: evidence for an implication of the ventral thalamus in opiate reward. Neuropsychopharmacology 33, 1746-1759.
145 De Cotiis, D.A., Woll, M.P., Fox, T.E., Hill, R.B., Levenson, R., and Flanagan, J.M. (2008). Optimized expression and purification of myristoylated human neuronal calcium sensor 1 in E. coli. Protein Expr Purif 61, 103-112.
Della, N.G., Senior, P.V., and Bowtell, D.D. (1993). Isolation and characterisation of murine homologues of the Drosophila seven in absentia gene (sina). Development 117, 1333-1343.
Deng, W., Aimone, J.B., and Gage, F.H. (2010). New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11, 339-350.
Depaux, A., Regnier-Ricard, F., Germani, A., and Varin-Blank, N. (2006). Dimerization of hSiah proteins regulates their stability. Biochem Biophys Res Commun 348, 857-863.
Dhawan, B.N., Cesselin, F., Raghubir, R., Reisine, T., Bradley, P.B., Portoghese, P.S., and Hamon, M. (1996). International Union of Pharmacology. XII. Classification of opioid receptors. Pharmacol Rev 48, 567-592.
Dickins, R.A., Frew, I.J., House, C.M., O'Bryan, M.K., Holloway, A.J., Haviv, I., Traficante, N., de Kretser, D.M., and Bowtell, D.D. (2002). The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice. Mol Cell Biol 22, 2294-2303.
Dimitrova, Y.N., Li, J., Lee, Y.T., Rios-Esteves, J., Friedman, D.B., Choi, H.J., Weis, W.I., Wang, C.Y., and Chazin, W.J. (2010). Direct ubiquitination of beta-catenin by Siah-1 and regulation by the exchange factor TBL1. J Biol Chem 285, 13507-13516.
Doyle, G.A., Rebecca Sheng, X., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., and Berrettini, W.H. (2007a). Identification of three mouse mu-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 388, 135-147.
Doyle, G.A., Sheng, X.R., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N., and Berrettini, W.H. (2007b). Identification of five mouse mu-opioid receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively spliced exon. Gene 395, 98-107.
Drake, M.T., Shenoy, S.K., and Lefkowitz, R.J. (2006). Trafficking of G protein-coupled receptors. Circ Res 99, 570-582.
Drakenberg, K., Nikoshkov, A., Horvath, M.C., Fagergren, P., Gharibyan, A., Saarelainen, K., Rahman, S., Nylander, I., Bakalkin, G., Rajs, J., et al. (2006). Mu opioid receptor A118G polymorphism in association with striatal opioid neuropeptide gene expression in heroin abusers. Proc Natl Acad Sci U S A 103, 7883-7888.
146 Eisch, A.J., Barrot, M., Schad, C.A., Self, D.W., and Nestler, E.J. (2000). Opiates inhibit neurogenesis in the adult rat hippocampus. Proc Natl Acad Sci U S A 97, 7579-7584.
Elmer, G.I., Pieper, J.O., Hamilton, L.R., and Wise, R.A. (2010). Qualitative differences between C57BL/6J and DBA/2J mice in morphine potentiation of brain stimulation reward and intravenous self-administration. Psychopharmacology (Berl) 208, 309-321.
Famulski, J.K., Trivedi, N., Howell, D., Yang, Y., Tong, Y., Gilbertson, R., and Solecki, D.J. (2010). Siah regulation of Pard3A controls neuronal cell adhesion during germinal zone exit. Science 330, 1834-1838.
Feldman, M., and van der Goot, F.G. (2009). Novel ubiquitin-dependent quality control in the endoplasmic reticulum. Trends Cell Biol 19, 357-363.
Feng, G.J., Kellett, E., Scorer, C.A., Wilde, J., White, J.H., and Milligan, G. (2003). Selective interactions between helix VIII of the human mu-opioid receptors and the C terminus of periplakin disrupt G protein activation. J Biol Chem 278, 33400-33407.
Fields, S., and Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340, 245-246.
Fillingim, R.B., Kaplan, L., Staud, R., Ness, T.J., Glover, T.L., Campbell, C.M., Mogil, J.S., and Wallace, M.R. (2005). The A118G single nucleotide polymorphism of the mu- opioid receptor gene (OPRM1) is associated with pressure pain sensitivity in humans. J Pain 6, 159-167.
Fillingim, R.B., Wallace, M.R., Herbstman, D.M., Ribeiro-Dasilva, M., and Staud, R. (2008). Genetic contributions to pain: a review of findings in humans. Oral Dis 14, 673- 682.
Filliol, D., Ghozland, S., Chluba, J., Martin, M., Matthes, H.W., Simonin, F., Befort, K., Gaveriaux-Ruff, C., Dierich, A., LeMeur, M., et al. (2000). Mice deficient for delta- and mu-opioid receptors exhibit opposing alterations of emotional responses. Nat Genet 25, 195-200.
Finn, A.K., and Whistler, J.L. (2001). Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal. Neuron 32, 829-839.
Frew, I.J., Hammond, V.E., Dickins, R.A., Quinn, J.M., Walkley, C.R., Sims, N.A., Schnall, R., Della, N.G., Holloway, A.J., Digby, M.R., et al. (2003). Generation and analysis of Siah2 mutant mice. Mol Cell Biol 23, 9150-9161.
Gainetdinov, R.R., Premont, R.T., Bohn, L.M., Lefkowitz, R.J., and Caron, M.G. (2004). Desensitization of G protein-coupled receptors and neuronal functions. Annu Rev Neurosci 27, 107-144.
147 Garzon, J., Rodriguez-Munoz, M., de la Torre-Madrid, E., and Sanchez-Blazquez, P. (2005a). Effector antagonism by the regulators of G protein signalling (RGS) proteins causes desensitization of mu-opioid receptors in the CNS. Psychopharmacology (Berl) 180, 1-11.
Garzon, J., Rodriguez-Munoz, M., and Sanchez-Blazquez, P. (2005b). Morphine alters the selective association between mu-opioid receptors and specific RGS proteins in mouse periaqueductal gray matter. Neuropharmacology 48, 853-868.
Gaveriaux-Ruff, C., and Kieffer, B.L. (2002). Opioid receptor genes inactivated in mice: the highlights. Neuropeptides 36, 62-71.
Ge, X., Qiu, Y., Loh, H.H., and Law, P.Y. (2009). GRIN1 regulates micro-opioid receptor activities by tethering the receptor and G protein in the lipid raft. J Biol Chem 284, 36521-36534.
Gelernter, J., Kranzler, H., and Cubells, J. (1999). Genetics of two mu opioid receptor gene (OPRM1) exon I polymorphisms: population studies, and allele frequencies in alcohol- and drug-dependent subjects. Mol Psychiatry 4, 476-483.
George, S.R., Fan, T., Xie, Z., Tse, R., Tam, V., Varghese, G., and O'Dowd, B.F. (2000). Oligomerization of mu- and delta-opioid receptors. Generation of novel functional properties. J Biol Chem 275, 26128-26135.
George, S.R., O'Dowd, B.F., and Lee, S.P. (2002). G-protein-coupled receptor oligomerization and its potential for drug discovery. Nat Rev Drug Discov 1, 808-820.
Georgoussi, Z., Georganta, E.M., and Milligan, G. (2012). The other side of opioid receptor signalling: regulation by protein-protein interaction. Curr Drug Targets 13, 80- 102.
Georgoussi, Z., Leontiadis, L., Mazarakou, G., Merkouris, M., Hyde, K., and Hamm, H. (2006). Selective interactions between G protein subunits and RGS4 with the C-terminal domains of the mu- and delta-opioid receptors regulate opioid receptor signaling. Cell Signal 18, 771-782.
Georgoussi, Z., Merkouris, M., Mullaney, I., Megaritis, G., Carr, C., Zioudrou, C., and Milligan, G. (1997). Selective interactions of mu-opioid receptors with pertussis toxin- sensitive G proteins: involvement of the third intracellular loop and the c-terminal tail in coupling. Biochim Biophys Acta 1359, 263-274.
Germani, A., Bruzzoni-Giovanelli, H., Fellous, A., Gisselbrecht, S., Varin-Blank, N., and Calvo, F. (2000). SIAH-1 interacts with alpha-tubulin and degrades the kinesin Kid by the proteasome pathway during mitosis. Oncogene 19, 5997-6006.
148 Germani, A., Romero, F., Houlard, M., Camonis, J., Gisselbrecht, S., Fischer, S., and Varin-Blank, N. (1999). hSiah2 is a new Vav binding protein which inhibits Vav- mediated signaling pathways. Mol Cell Biol 19, 3798-3807.
Ghitza, U.E., Zhai, H., Wu, P., Airavaara, M., Shaham, Y., and Lu, L. (2010). Role of BDNF and GDNF in drug reward and relapse: a review. Neurosci Biobehav Rev 35, 157- 171.
Ghozland, S., Matthes, H.W., Simonin, F., Filliol, D., Kieffer, B.L., and Maldonado, R. (2002). Motivational effects of cannabinoids are mediated by mu-opioid and kappa- opioid receptors. J Neurosci 22, 1146-1154.
Ginosar, Y., Davidson, E.M., Meroz, Y., Blotnick, S., Shacham, M., and Caraco, Y. (2009). Mu-opioid receptor (A118G) single-nucleotide polymorphism affects alfentanil requirements for extracorporeal shock wave lithotripsy: a pharmacokinetic- pharmacodynamic study. Br J Anaesth 103, 420-427.
Glatt, S.J., Bousman, C., Wang, R.S., Murthy, K.K., Rana, B.K., Lasky-Su, J.A., Zhu, S.C., Zhang, R., Li, J., Zhang, B., et al. (2007). Evaluation of OPRM1 variants in heroin dependence by family-based association testing and meta-analysis. Drug Alcohol Depend 90, 159-165.
Gomes, I., Gupta, A., Filipovska, J., Szeto, H.H., Pintar, J.E., and Devi, L.A. (2004). A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. Proc Natl Acad Sci U S A 101, 5135-5139.
Gomes, I., Jordan, B.A., Gupta, A., Trapaidze, N., Nagy, V., and Devi, L.A. (2000). Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J Neurosci 20, RC110.
Griffiths, R.R. (1980). Common factors in human and infrahuman drug self- administration. Psychopharmacol Bull 16, 45-47.
Groer, C.E., Schmid, C.L., Jaeger, A.M., and Bohn, L.M. (2011). Agonist-directed interactions with specific beta-arrestins determine mu-opioid receptor trafficking, ubiquitination, and dephosphorylation. J Biol Chem 286, 31731-31741.
Guang, W., Wang, H., Su, T., Weinstein, I.B., and Wang, J.B. (2004). Role of mPKCI, a novel mu-opioid receptor interactive protein, in receptor desensitization, phosphorylation, and morphine-induced analgesia. Mol Pharmacol 66, 1285-1292.
Haberstock-Debic, H., Kim, K.A., Yu, Y.J., and von Zastrow, M. (2005). Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J Neurosci 25, 7847-7857.
149 Haberstock-Debic, H., Wein, M., Barrot, M., Colago, E.E., Rahman, Z., Neve, R.L., Pickel, V.M., Nestler, E.J., von Zastrow, M., and Svingos, A.L. (2003). Morphine acutely regulates opioid receptor trafficking selectively in dendrites of nucleus accumbens neurons. J Neurosci 23, 4324-4332.
Haefliger, J.A., Bruzzone, R., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., and Paul, D.L. (1992). Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping. J Biol Chem 267, 2057-2064.
Haklai-Topper, L., Mlechkovich, G., Savariego, D., Gokhman, I., and Yaron, A. (2010). Cis interaction between Semaphorin6A and Plexin-A4 modulates the repulsive response to Sema6A. EMBO J 29, 2635-2645.
Hamm, H.E., and Gilchrist, A. (1996). Heterotrimeric G proteins. Curr Opin Cell Biol 8, 189-196.
Harburg, G.C., Hall, F.S., Harrist, A.V., Sora, I., Uhl, G.R., and Eisch, A.J. (2007). Knockout of the mu opioid receptor enhances the survival of adult-generated hippocampal granule cell neurons. Neuroscience 144, 77-87.
Harrison, L.M., Kastin, A.J., and Zadina, J.E. (1998). Opiate tolerance and dependence: receptors, G-proteins, and antiopiates. Peptides 19, 1603-1630.
Hausmann, G., Banziger, C., and Basler, K. (2007). Helping Wingless take flight: how WNT proteins are secreted. Nat Rev Mol Cell Biol 8, 331-336.
He, L., Fong, J., von Zastrow, M., and Whistler, J.L. (2002). Regulation of opioid receptor trafficking and morphine tolerance by receptor oligomerization. Cell 108, 271- 282.
He, L., Kim, J.A., and Whistler, J.L. (2009). Biomarkers of morphine tolerance and dependence are prevented by morphine-induced endocytosis of a mutant mu-opioid receptor. FASEB J 23, 4327-4334.
Henry, A.G., White, I.J., Marsh, M., von Zastrow, M., and Hislop, J.N. (2011). The role of ubiquitination in lysosomal trafficking of delta-opioid receptors. Traffic 12, 170-184.
Hicke, L., and Dunn, R. (2003). Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu Rev Cell Dev Biol 19, 141-172.
Hill, S.J. (2006). G-protein-coupled receptors: past, present and future. Br J Pharmacol 147 Suppl 1, S27-37.
Hislop, J.N., Henry, A.G., Marchese, A., and von Zastrow, M. (2009). Ubiquitination regulates proteolytic processing of G protein-coupled receptors after their sorting to lysosomes. J Biol Chem 284, 19361-19370.
150 Hislop, J.N., Henry, A.G., and von Zastrow, M. (2011). Ubiquitination in the first cytoplasmic loop of mu-opioid receptors reveals a hierarchical mechanism of lysosomal down-regulation. J Biol Chem 286, 40193-40204.
Hislop, J.N., and von Zastrow, M. (2011). Role of ubiquitination in endocytic trafficking of G-protein-coupled receptors. Traffic 12, 137-148.
Hoehe, M.R., Kopke, K., Wendel, B., Rohde, K., Flachmeier, C., Kidd, K.K., Berrettini, W.H., and Church, G.M. (2000). Sequence variability and candidate gene analysis in complex disease: association of mu opioid receptor gene variation with substance dependence. Hum Mol Genet 9, 2895-2908.
House, C.M., Frew, I.J., Huang, H.L., Wiche, G., Traficante, N., Nice, E., Catimel, B., and Bowtell, D.D. (2003). A binding motif for Siah ubiquitin ligase. Proc Natl Acad Sci U S A 100, 3101-3106.
Hu, G., Chung, Y.L., Glover, T., Valentine, V., Look, A.T., and Fearon, E.R. (1997a). Characterization of human homologs of the Drosophila seven in absentia (sina) gene. Genomics 46, 103-111.
Hu, G., and Fearon, E.R. (1999). Siah-1 N-terminal RING domain is required for proteolysis function, and C-terminal sequences regulate oligomerization and binding to target proteins. Mol Cell Biol 19, 724-732.
Hu, G., Zhang, S., Vidal, M., Baer, J.L., Xu, T., and Fearon, E.R. (1997b). Mammalian homologs of seven in absentia regulate DCC via the ubiquitin-proteasome pathway. Genes Dev 11, 2701-2714.
Huang, C.J., Liu, H.F., Su, N.Y., Hsu, Y.W., Yang, C.H., Chen, C.C., and Tsai, P.S. (2008). Association between human opioid receptor genes polymorphisms and pressure pain sensitivity in females*. Anaesthesia 63, 1288-1295.
Huang, P., and Liu-Chen, L.Y. (2009). Detecting the mu opioid receptor in brain following SDS-PAGE with multiple approaches. Front Biosci (Elite Ed) 1, 220-227.
Hyman, S.E. (2007). The neurobiology of addiction: implications for voluntary control of behavior. Am J Bioeth 7, 8-11.
Ide, S., Han, W., Kasai, S., Hata, H., Sora, I., and Ikeda, K. (2005). Characterization of the 3' untranslated region of the human mu-opioid receptor (MOR-1) mRNA. Gene 364, 139-145.
Ikeda, K., Ichikawa, T., Kobayashi, T., Kumanishi, T., Oike, S., and Yano, R. (1999). Unique behavioural phenotypes of recombinant-inbred CXBK mice: partial deficiency of sensitivity to mu- and kappa-agonists. Neurosci Res 34, 149-155.
151 Ikeda, K., Kobayashi, T., Ichikawa, T., Kumanishi, T., Niki, H., and Yano, R. (2001). The untranslated region of (mu)-opioid receptor mRNA contributes to reduced opioid sensitivity in CXBK mice. J Neurosci 21, 1334-1339.
Imai, Y., Soda, M., Hatakeyama, S., Akagi, T., Hashikawa, T., Nakayama, K.I., and Takahashi, R. (2002). CHIP is associated with Parkin, a gene responsible for familial Parkinson's disease, and enhances its ubiquitin ligase activity. Mol Cell 10, 55-67.
Inturrisi, C.E. (2002). Clinical pharmacology of opioids for pain. Clin J Pain 18, S3-13.
Ishikawa, K., Nash, S.R., Nishimune, A., Neki, A., Kaneko, S., and Nakanishi, S. (1999). Competitive interaction of seven in absentia homolog-1A and Ca2+/calmodulin with the cytoplasmic tail of group 1 metabotropic glutamate receptors. Genes Cells 4, 381-390.
Iyer, K., Burkle, L., Auerbach, D., Thaminy, S., Dinkel, M., Engels, K., and Stagljar, I. (2005). Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins. Sci STKE 2005, pl3.
Jin, J., Kittanakom, S., Wong, V., Reyes, B.A., Van Bockstaele, E.J., Stagljar, I., Berrettini, W., and Levenson, R. (2010). Interaction of the mu-opioid receptor with GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence. BMC Neurosci 11, 33.
Johnson, E.E., Christie, M.J., and Connor, M. (2005). The role of opioid receptor phosphorylation and trafficking in adaptations to persistent opioid treatment. Neurosignals 14, 290-302.
Johnson, P.S., Wang, J.B., Wang, W.F., and Uhl, G.R. (1994). Expressed mu opiate receptor couples to adenylate cyclase and phosphatidyl inositol turnover. Neuroreport 5, 507-509.
Jordan, B.A., and Devi, L.A. (1999). G-protein-coupled receptor heterodimerization modulates receptor function. Nature 399, 697-700.
Jordan, B.A., Gomes, I., Rios, C., Filipovska, J., and Devi, L.A. (2003). Functional interactions between mu opioid and alpha 2A-adrenergic receptors. Mol Pharmacol 64, 1317-1324.
Kabbani, N., and Levenson, R. (2007). A proteomic approach to receptor signaling: molecular mechanisms and therapeutic implications derived from discovery of the dopamine D2 receptor signalplex. Eur J Pharmacol 572, 83-93.
Kabbani, N., Negyessy, L., Lin, R., Goldman-Rakic, P., and Levenson, R. (2002). Interaction with neuronal calcium sensor NCS-1 mediates desensitization of the D2 dopamine receptor. J Neurosci 22, 8476-8486.
152 Kapur, S., Sharad, S., Singh, R.A., and Gupta, A.K. (2007). A118g polymorphism in mu opioid receptor gene (oprm1): association with opiate addiction in subjects of Indian origin. J Integr Neurosci 6, 511-522.
Kasai, S., and Ikeda, K. (2011). Pharmacogenomics of the human micro-opioid receptor. Pharmacogenomics 12, 1305-1320.
Kato, A., Kawamata, N., Tamayose, K., Egashira, M., Miura, R., Fujimura, T., Murayama, K., and Oshimi, K. (2002). Ancient ubiquitous protein 1 binds to the conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3. J Biol Chem 277, 28934-28941.
Keith, D.E., Murray, S.R., Zaki, P.A., Chu, P.C., Lissin, D.V., Kang, L., Evans, C.J., and von Zastrow, M. (1996). Morphine activates opioid receptors without causing their rapid internalization. J Biol Chem 271, 19021-19024.
Kieffer, B.L., and Gaveriaux-Ruff, C. (2002). Exploring the opioid system by gene knockout. Prog Neurobiol 66, 285-306.
Kitanaka, N., Sora, I., Kinsey, S., Zeng, Z., and Uhl, G.R. (1998). No heroin or morphine 6beta-glucuronide analgesia in mu-opioid receptor knockout mice. Eur J Pharmacol 355, R1-3.
Kittanakom, S., Chuk, M., Wong, V., Snyder, J., Edmonds, D., Lydakis, A., Zhang, Z., Auerbach, D., and Stagljar, I. (2009). Analysis of membrane protein complexes using the split-ubiquitin membrane yeast two-hybrid (MYTH) system. Methods Mol Biol 548, 247- 271.
Knapp, R.J., Malatynska, E., Fang, L., Li, X., Babin, E., Nguyen, M., Santoro, G., Varga, E.V., Hruby, V.J., Roeske, W.R., et al. (1994). Identification of a human delta opioid receptor: cloning and expression. Life Sci 54, PL463-469.
Koch, T., Brandenburg, L.O., Schulz, S., Liang, Y., Klein, J., and Hollt, V. (2003). ADP- ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced mu-opioid receptor endocytosis. J Biol Chem 278, 9979-9985.
Koch, T., Kroslak, T., Averbeck, M., Mayer, P., Schroder, H., Raulf, E., and Hollt, V. (2000). Allelic variation S268P of the human mu-opioid receptor affects both desensitization and G protein coupling. Mol Pharmacol 58, 328-334.
Kong, H., Raynor, K., Yano, H., Takeda, J., Bell, G.I., and Reisine, T. (1994). Agonists and antagonists bind to different domains of the cloned kappa opioid receptor. Proc Natl Acad Sci U S A 91, 8042-8046.
153 Kostova, Z., Tsai, Y.C., and Weissman, A.M. (2007). Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation. Semin Cell Dev Biol 18, 770-779.
Kramer, J.W., Post, M.A., Brown, A.M., and Kirsch, G.E. (1998). Modulation of potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1 alpha-subunits. Am J Physiol 274, C1501-1510.
Kreek, M.J., Bart, G., Lilly, C., LaForge, K.S., and Nielsen, D.A. (2005). Pharmacogenetics and human molecular genetics of opiate and cocaine addictions and their treatments. Pharmacol Rev 57, 1-26.
Kroslak, T., Laforge, K.S., Gianotti, R.J., Ho, A., Nielsen, D.A., and Kreek, M.J. (2007). The single nucleotide polymorphism A118G alters functional properties of the human mu opioid receptor. J Neurochem 103, 77-87.
Lachman, H.M. (2006). An overview of the genetics of substance use disorders. Curr Psychiatry Rep 8, 133-143.
Lamprecht, R., and LeDoux, J. (2004). Structural plasticity and memory. Nat Rev Neurosci 5, 45-54.
Lasagna, L. (1954). Nalorphine (n-allylnormorphine); practical and theoretical considerations. AMA Arch Intern Med 94, 532-558.
Law, P.Y., Erickson, L.J., El-Kouhen, R., Dicker, L., Solberg, J., Wang, W., Miller, E., Burd, A.L., and Loh, H.H. (2000). Receptor density and recycling affect the rate of agonist-induced desensitization of mu-opioid receptor. Mol Pharmacol 58, 388-398.
Le Merrer, J., Becker, J.A., Befort, K., and Kieffer, B.L. (2009). Reward processing by the opioid system in the brain. Physiol Rev 89, 1379-1412.
Lentze, N., and Auerbach, D. (2008). Membrane-based yeast two-hybrid system to detect protein interactions. Curr Protoc Protein Sci Chapter 19, Unit 19 17.
Li, J.G., Haines, D.S., and Liu-Chen, L.Y. (2008). Agonist-promoted Lys63-linked polyubiquitination of the human kappa-opioid receptor is involved in receptor down- regulation. Mol Pharmacol 73, 1319-1330.
Li, Y., Chen, J., Bai, B., Du, H., Liu, Y., and Liu, H. (2012). Heterodimerization of human apelin and kappa opioid receptors: roles in signal transduction. Cell Signal 24, 991-1001.
Liang, Y.J., Wu, D.F., Yang, L.Q., Hollt, V., and Koch, T. (2007). Interaction of the mu- opioid receptor with synaptophysin influences receptor trafficking and signaling. Mol Pharmacol 71, 123-131.
154 Liao, D., Lin, H., Law, P.Y., and Loh, H.H. (2005). Mu-opioid receptors modulate the stability of dendritic spines. Proc Natl Acad Sci U S A 102, 1725-1730.
Lie, D.C., Colamarino, S.A., Song, H.J., Desire, L., Mira, H., Consiglio, A., Lein, E.S., Jessberger, S., Lansford, H., Dearie, A.R., et al. (2005). Wnt signalling regulates adult hippocampal neurogenesis. Nature 437, 1370-1375.
Lin, R., Karpa, K., Kabbani, N., Goldman-Rakic, P., and Levenson, R. (2001). Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A. Proc Natl Acad Sci U S A 98, 5258-5263.
Lin R, K.N., Binda A, Canfield V, Levenson R (2005). Use of the yeast two-hybrid system to detect receptor-protein interactions. In G-Protein-Coupled Receptor--Protein Interactions, G.S.a.O.D. BF, ed. (John Wiley & Sons, Inc.).
Liu, J., Stevens, J., Rote, C.A., Yost, H.J., Hu, Y., Neufeld, K.L., White, R.L., and Matsunami, N. (2001). Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein. Mol Cell 7, 927-936.
Liu, Y., Shah, S.V., Xiang, X., Wang, J., Deng, Z.B., Liu, C., Zhang, L., Wu, J., Edmonds, T., Jambor, C., et al. (2009). COP9-associated CSN5 regulates exosomal protein deubiquitination and sorting. Am J Pathol 174, 1415-1425.
Lotsch, J., Zimmermann, M., Darimont, J., Marx, C., Dudziak, R., Skarke, C., and Geisslinger, G. (2002). Does the A118G polymorphism at the mu-opioid receptor gene protect against morphine-6-glucuronide toxicity? Anesthesiology 97, 814-819.
Luo, W., Wang, Y., Hanck, T., Stricker, R., and Reiser, G. (2006). Jab1, a novel protease-activated receptor-2 (PAR-2)-interacting protein, is involved in PAR-2-induced activation of activator protein-1. J Biol Chem 281, 7927-7936.
Lynch, W.J., Nicholson, K.L., Dance, M.E., Morgan, R.W., and Foley, P.L. (2010). Animal models of substance abuse and addiction: implications for science, animal welfare, and society. Comp Med 60, 177-188.
Mague, S.D., and Blendy, J.A. (2010). OPRM1 SNP (A118G): involvement in disease development, treatment response, and animal models. Drug Alcohol Depend 108, 172- 182.
Majumdar, S., Grinnell, S., Le Rouzic, V., Burgman, M., Polikar, L., Ansonoff, M., Pintar, J., Pan, Y.X., and Pasternak, G.W. (2011). Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects. Proc Natl Acad Sci U S A 108, 19778-19783.
Marchese, A., Paing, M.M., Temple, B.R., and Trejo, J. (2008). G protein-coupled receptor sorting to endosomes and lysosomes. Annu Rev Pharmacol Toxicol 48, 601-629.
155 Marcol, W., Kotulska, K., Larysz-Brysz, M., and Kowalik, J.L. (2007). BDNF contributes to animal model neuropathic pain after peripheral nerve transection. Neurosurg Rev 30, 235-243; discussion 243.
Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E., and Gilbert, P.E. (1976). The effects of morphine- and nalorphine- like drugs in the nondependent and morphine- dependent chronic spinal dog. J Pharmacol Exp Ther 197, 517-532.
Martini, L., and Whistler, J.L. (2007). The role of mu opioid receptor desensitization and endocytosis in morphine tolerance and dependence. Curr Opin Neurobiol 17, 556-564.
Mathon, D.S., Lesscher, H.M., Gerrits, M.A., Kamal, A., Pintar, J.E., Schuller, A.G., Spruijt, B.M., Burbach, J.P., Smidt, M.P., van Ree, J.M., et al. (2005). Increased gabaergic input to ventral tegmental area dopaminergic neurons associated with decreased cocaine reinforcement in mu-opioid receptor knockout mice. Neuroscience 130, 359-367.
Matsuzawa, S., Takayama, S., Froesch, B.A., Zapata, J.M., and Reed, J.C. (1998). p53- inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: suppression by BAG-1. EMBO J 17, 2736-2747.
Matthes, H.W., Maldonado, R., Simonin, F., Valverde, O., Slowe, S., Kitchen, I., Befort, K., Dierich, A., Le Meur, M., Dolle, P., et al. (1996). Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid- receptor gene. Nature 383, 819-823.
Matthes, H.W., Smadja, C., Valverde, O., Vonesch, J.L., Foutz, A.S., Boudinot, E., Denavit-Saubie, M., Severini, C., Negri, L., Roques, B.P., et al. (1998). Activity of the delta-opioid receptor is partially reduced, whereas activity of the kappa-receptor is maintained in mice lacking the mu-receptor. J Neurosci 18, 7285-7295.
Mazarakou, G., and Georgoussi, Z. (2005). STAT5A interacts with and is phosphorylated upon activation of the mu-opioid receptor. J Neurochem 93, 918-931.
Mehnert, M., Sommer, T., and Jarosch, E. (2010). ERAD ubiquitin ligases: multifunctional tools for protein quality control and waste disposal in the endoplasmic reticulum. Bioessays 32, 905-913.
Miller, J., and Stagljar, I. (2004). Using the yeast two-hybrid system to identify interacting proteins. Methods Mol Biol 261, 247-262.
Milligan, G. (2005). Opioid receptors and their interacting proteins. Neuromolecular Med 7, 51-59.
Minami, M., and Satoh, M. (1995). Molecular biology of the opioid receptors: structures, functions and distributions. Neurosci Res 23, 121-145.
156 Moles, A., Kieffer, B.L., and D'Amato, F.R. (2004). Deficit in attachment behavior in mice lacking the mu-opioid receptor gene. Science 304, 1983-1986.
Molinari, P., Vezzi, V., Sbraccia, M., Gro, C., Riitano, D., Ambrosio, C., Casella, I., and Costa, T. (2010). Morphine-like opiates selectively antagonize receptor-arrestin interactions. J Biol Chem 285, 12522-12535.
Mollereau, C., Simons, M.J., Soularue, P., Liners, F., Vassart, G., Meunier, J.C., and Parmentier, M. (1996). Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene. Proc Natl Acad Sci U S A 93, 8666-8670.
Moriyoshi, K., Iijima, K., Fujii, H., Ito, H., Cho, Y., and Nakanishi, S. (2004). Seven in absentia homolog 1A mediates ubiquitination and degradation of group 1 metabotropic glutamate receptors. Proc Natl Acad Sci U S A 101, 8614-8619.
Moser, P., Wolinsky, T., Castagne, V., and Duxon, M. (2011). Current approaches and issues in non-clinical evaluation of abuse and dependence. J Pharmacol Toxicol Methods 63, 160-167.
Mueller, B., Klemm, E.J., Spooner, E., Claessen, J.H., and Ploegh, H.L. (2008). SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins. Proc Natl Acad Sci U S A 105, 12325-12330.
Myers, K.M., Bechtholt-Gompf, A.J., Coleman, B.R., and Carlezon, W.A., Jr. (2012). Extinction of conditioned opiate withdrawal in rats in a two-chambered place conditioning apparatus. Nat Protoc 7, 517-526.
Myung, J., Kim, K.B., and Crews, C.M. (2001). The ubiquitin-proteasome pathway and proteasome inhibitors. Med Res Rev 21, 245-273.
Neil, A. (1984). Affinities of some common opioid analgesics towards four binding sites in mouse brain. Naunyn Schmiedebergs Arch Pharmacol 328, 24-29.
Nemani, M., Linares-Cruz, G., Bruzzoni-Giovanelli, H., Roperch, J.P., Tuynder, M., Bougueleret, L., Cherif, D., Medhioub, M., Pasturaud, P., Alvaro, V., et al. (1996). Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor suppression. Proc Natl Acad Sci U S A 93, 9039-9042.
Nestler, E.J. (2001). Molecular neurobiology of addiction. Am J Addict 10, 201-217.
Nestler, E.J. (2004). Molecular mechanisms of drug addiction. Neuropharmacology 47 Suppl 1, 24-32.
Nugent, F.S., Penick, E.C., and Kauer, J.A. (2007). Opioids block long-term potentiation of inhibitory synapses. Nature 446, 1086-1090.
157 Obata, K., and Noguchi, K. (2006). BDNF in sensory neurons and chronic pain. Neurosci Res 55, 1-10.
Oldfield, S., Braksator, E., Rodriguez-Martin, I., Bailey, C.P., Donaldson, L.F., Henderson, G., and Kelly, E. (2008). C-terminal splice variants of the mu-opioid receptor: existence, distribution and functional characteristics. J Neurochem 104, 937- 945.
Oliver, P.L., Bitoun, E., Clark, J., Jones, E.L., and Davies, K.E. (2004). Mediation of Af4 protein function in the cerebellum by Siah proteins. Proc Natl Acad Sci U S A 101, 14901-14906.
Onogi, T., Minami, M., Katao, Y., Nakagawa, T., Aoki, Y., Toya, T., Katsumata, S., and Satoh, M. (1995). DAMGO, a mu-opioid receptor selective agonist, distinguishes between mu- and delta-opioid receptors around their first extracellular loops. FEBS Lett 357, 93-97.
Onoprishvili, I., Andria, M.L., Kramer, H.K., Ancevska-Taneva, N., Hiller, J.M., and Simon, E.J. (2003). Interaction between the mu opioid receptor and filamin A is involved in receptor regulation and trafficking. Mol Pharmacol 64, 1092-1100.
Pan, L., Xu, J., Yu, R., Xu, M.M., Pan, Y.X., and Pasternak, G.W. (2005). Identification and characterization of six new alternatively spliced variants of the human mu opioid receptor gene, Oprm. Neuroscience 133, 209-220.
Pan, Y.X. (2005). Diversity and complexity of the mu opioid receptor gene: alternative pre-mRNA splicing and promoters. DNA Cell Biol 24, 736-750.
Pan, Y.X., Xu, J., Mahurter, L., Bolan, E., Xu, M., and Pasternak, G.W. (2001). Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the Oprm gene. Proc Natl Acad Sci U S A 98, 14084-14089.
Pan, Y.X., Xu, J., Mahurter, L., Xu, M., Gilbert, A.K., and Pasternak, G.W. (2003). Identification and characterization of two new human mu opioid receptor splice variants, hMOR-1O and hMOR-1X. Biochem Biophys Res Commun 301, 1057-1061.
Pardon, M.C. (2010). Role of neurotrophic factors in behavioral processes: implications for the treatment of psychiatric and neurodegenerative disorders. Vitam Horm 82, 185- 200.
Parenty, G., Appelbe, S., and Milligan, G. (2008). CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer. Biochem J 412, 245-256.
Pasquier, C., Promponas, V.J., Palaios, G.A., Hamodrakas, J.S., and Hamodrakas, S.J. (1999). A novel method for predicting transmembrane segments in proteins based on a
158 statistical analysis of the SwissProt database: the PRED-TMR algorithm. Protein Eng 12, 381-385.
Pasternak, D.A., Pan, L., Xu, J., Yu, R., Xu, M.M., Pasternak, G.W., and Pan, Y.X. (2004). Identification of three new alternatively spliced variants of the rat mu opioid receptor gene: dissociation of affinity and efficacy. J Neurochem 91, 881-890.
Pasternak, G.W. (2004). Multiple opiate receptors: deja vu all over again. Neuropharmacology 47 Suppl 1, 312-323.
Pasternak, G.W. (2005). Molecular biology of opioid analgesia. J Pain Symptom Manage 29, S2-9.
Patel, M.B., Patel, C.N., Rajashekara, V., and Yoburn, B.C. (2002). Opioid agonists differentially regulate mu-opioid receptors and trafficking proteins in vivo. Mol Pharmacol 62, 1464-1470.
Peretti, D., Dahan, N., Shimoni, E., Hirschberg, K., and Lev, S. (2008). Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport. Mol Biol Cell 19, 3871-3884.
Petaja-Repo, U.E., Hogue, M., Laperriere, A., Bhalla, S., Walker, P., and Bouvier, M. (2001). Newly synthesized human delta opioid receptors retained in the endoplasmic reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and degraded by the proteasome. J Biol Chem 276, 4416-4423.
Petaja-Repo, U.E., Hogue, M., Laperriere, A., Walker, P., and Bouvier, M. (2000). Export from the endoplasmic reticulum represents the limiting step in the maturation and cell surface expression of the human delta opioid receptor. J Biol Chem 275, 13727- 13736.
Petruzzi, R., Ferraro, T.N., Kurschner, V.C., Golden, G.T., and Berrettini, W.H. (1997). The effects of repeated morphine exposure on mu opioid receptor number and affinity in C57BL/6J and DBA/2J mice. Life Sci 61, 2057-2064.
Pezet, S., and McMahon, S.B. (2006). Neurotrophins: mediators and modulators of pain. Annu Rev Neurosci 29, 507-538.
Pfeiffer, M., Kirscht, S., Stumm, R., Koch, T., Wu, D., Laugsch, M., Schroder, H., Hollt, V., and Schulz, S. (2003). Heterodimerization of substance P and mu-opioid receptors regulates receptor trafficking and resensitization. J Biol Chem 278, 51630-51637.
Pfeiffer, M., Koch, T., Schroder, H., Laugsch, M., Hollt, V., and Schulz, S. (2002). Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization. J Biol Chem 277, 19762-19772.
159 Picetti, R., Caccavo, J.A., Ho, A., and Kreek, M.J. (2012). Dose escalation and dose preference in extended-access heroin self-administration in Lewis and Fischer rats. Psychopharmacology (Berl) 220, 163-172.
Picker, M.J., Negus, S.S., and Powell, K.R. (1991). Differential cross-tolerance to mu and kappa opioid agonists in morphine-tolerant rats responding under a schedule of food presentation. Psychopharmacology (Berl) 103, 129-135.
Polekhina, G., House, C.M., Traficante, N., Mackay, J.P., Relaix, F., Sassoon, D.A., Parker, M.W., and Bowtell, D.D. (2002). Siah ubiquitin ligase is structurally related to TRAF and modulates TNF-alpha signaling. Nat Struct Biol 9, 68-75.
Prather, P.L., Tsai, A.W., and Law, P.Y. (1994). Mu and delta opioid receptor desensitization in undifferentiated human neuroblastoma SHSY5Y cells. J Pharmacol Exp Ther 270, 177-184.
Raehal, K.M., and Bohn, L.M. (2005). Mu opioid receptor regulation and opiate responsiveness. AAPS J 7, E587-591.
Ren, K., and Dubner, R. (2007). Pain facilitation and activity-dependent plasticity in pain modulatory circuitry: role of BDNF-TrkB signaling and NMDA receptors. Mol Neurobiol 35, 224-235.
Reyes-Gibby, C.C., Shete, S., Rakvag, T., Bhat, S.V., Skorpen, F., Bruera, E., Kaasa, S., and Klepstad, P. (2007). Exploring joint effects of genes and the clinical efficacy of morphine for cancer pain: OPRM1 and COMT gene. Pain 130, 25-30.
Ring, R.H., Alder, J., Fennell, M., Kouranova, E., Black, I.B., and Thakker-Varia, S. (2006). Transcriptional profiling of brain-derived-neurotrophic factor-induced neuronal plasticity: a novel role for nociceptin in hippocampal neurite outgrowth. J Neurobiol 66, 361-377.
Rios, C., Gomes, I., and Devi, L.A. (2006). mu opioid and CB1 cannabinoid receptor interactions: reciprocal inhibition of receptor signaling and neuritogenesis. Br J Pharmacol 148, 387-395.
Roberts, A.J., Gold, L.H., Polis, I., McDonald, J.S., Filliol, D., Kieffer, B.L., and Koob, G.F. (2001). Increased ethanol self-administration in delta-opioid receptor knockout mice. Alcohol Clin Exp Res 25, 1249-1256.
Roberts, A.J., McDonald, J.S., Heyser, C.J., Kieffer, B.L., Matthes, H.W., Koob, G.F., and Gold, L.H. (2000). mu-Opioid receptor knockout mice do not self-administer alcohol. J Pharmacol Exp Ther 293, 1002-1008.
Robinson, T.E., and Kolb, B. (1999). Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats. Synapse 33, 160-162.
160 Robinson, T.E., and Kolb, B. (2004). Structural plasticity associated with exposure to drugs of abuse. Neuropharmacology 47 Suppl 1, 33-46.
Rossi, G.C., Brown, G.P., Leventhal, L., Yang, K., and Pasternak, G.W. (1996). Novel receptor mechanisms for heroin and morphine-6 beta-glucuronide analgesia. Neurosci Lett 216, 1-4.
Roy, S., Liu, H.C., and Loh, H.H. (1998). mu-Opioid receptor-knockout mice: the role of mu-opioid receptor in gastrointestinal transit. Brain Res Mol Brain Res 56, 281-283.
Roy, S., Wang, J.H., Balasubramanian, S., Sumandeep, Charboneau, R., Barke, R., and Loh, H.H. (2001). Role of hypothalamic-pituitary axis in morphine-induced alteration in thymic cell distribution using mu-opioid receptor knockout mice. J Neuroimmunol 116, 147-155.
Rozenfeld, R., and Devi, L.A. (2007). Receptor heterodimerization leads to a switch in signaling: beta-arrestin2-mediated ERK activation by mu-delta opioid receptor heterodimers. FASEB J 21, 2455-2465.
Russo, S.J., Dietz, D.M., Dumitriu, D., Morrison, J.H., Malenka, R.C., and Nestler, E.J. (2010). The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus accumbens. Trends Neurosci 33, 267-276.
Salinas, P.C., and Zou, Y. (2008). Wnt signaling in neural circuit assembly. Annu Rev Neurosci 31, 339-358.
Schnell, S.A., and Wessendorf, M.W. (2004). Expression of MOR1C-like mu-opioid receptor mRNA in rats. J Comp Neurol 473, 213-232.
Schubert, U., Anton, L.C., Gibbs, J., Norbury, C.C., Yewdell, J.W., and Bennink, J.R. (2000). Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, 770-774.
Schulz, S., Mayer, D., Pfeiffer, M., Stumm, R., Koch, T., and Hollt, V. (2004). Morphine induces terminal micro-opioid receptor desensitization by sustained phosphorylation of serine-375. EMBO J 23, 3282-3289.
Shabalina, S.A., Zaykin, D.V., Gris, P., Ogurtsov, A.Y., Gauthier, J., Shibata, K., Tchivileva, I.E., Belfer, I., Mishra, B., Kiselycznyk, C., et al. (2009). Expansion of the human mu-opioid receptor gene architecture: novel functional variants. Hum Mol Genet 18, 1037-1051.
Sharma, S.K., Klee, W.A., and Nirenberg, M. (1975). Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc Natl Acad Sci U S A 72, 3092-3096.
161 Shi, J., Hui, L., Xu, Y., Wang, F., Huang, W., and Hu, G. (2002). Sequence variations in the mu-opioid receptor gene (OPRM1) associated with human addiction to heroin. Hum Mutat 19, 459-460.
Shi, L., Yue, J., You, Y., Yin, B., Gong, Y., Xu, C., Qiang, B., Yuan, J., Liu, Y., and Peng, X. (2006). Dok5 is substrate of TrkB and TrkC receptors and involved in neurotrophin induced MAPK activation. Cell Signal 18, 1995-2003.
Shmueli, A., Tsai, Y.C., Yang, M., Braun, M.A., and Weissman, A.M. (2009). Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: cross-talk between E3s in the endoplasmic reticulum. Biochem Biophys Res Commun 390, 758-762.
Sia, A.T., Lim, Y., Lim, E.C., Goh, R.W., Law, H.Y., Landau, R., Teo, Y.Y., and Tan, E.C. (2008). A118G single nucleotide polymorphism of human mu-opioid receptor gene influences pain perception and patient-controlled intravenous morphine consumption after intrathecal morphine for postcesarean analgesia. Anesthesiology 109, 520-526.
Simon, E.J., Hiller, J.M., and Edelman, I. (1973). Stereospecific binding of the potent narcotic analgesic (3H) Etorphine to rat-brain homogenate. Proc Natl Acad Sci U S A 70, 1947-1949.
Simonin, F., Valverde, O., Smadja, C., Slowe, S., Kitchen, I., Dierich, A., Le Meur, M., Roques, B.P., Maldonado, R., and Kieffer, B.L. (1998). Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal. EMBO J 17, 886-897.
Smith, M.H., Ploegh, H.L., and Weissman, J.S. (2011). Road to ruin: targeting proteins for degradation in the endoplasmic reticulum. Science 334, 1086-1090.
Snyder, S.H. (2004). Opiate receptors and beyond: 30 years of neural signaling research. Neuropharmacology 47 Suppl 1, 274-285.
Snyder, S.H., and Childers, S.R. (1979). Opiate receptors and opioid peptides. Annu Rev Neurosci 2, 35-64.
Sokolov, B.P., Jiang, L., Trivedi, N.S., and Aston, C. (2003). Transcription profiling reveals mitochondrial, ubiquitin and signaling systems abnormalities in postmortem brains from subjects with a history of alcohol abuse or dependence. J Neurosci Res 72, 756-767.
Somogyi, A.A., Barratt, D.T., and Coller, J.K. (2007). Pharmacogenetics of opioids. Clin Pharmacol Ther 81, 429-444.
162 Sora, I., Elmer, G., Funada, M., Pieper, J., Li, X.F., Hall, F.S., and Uhl, G.R. (2001). Mu opiate receptor gene dose effects on different morphine actions: evidence for differential in vivo mu receptor reserve. Neuropsychopharmacology 25, 41-54.
Sora, I., Takahashi, N., Funada, M., Ujike, H., Revay, R.S., Donovan, D.M., Miner, L.L., and Uhl, G.R. (1997). Opiate receptor knockout mice define mu receptor roles in endogenous nociceptive responses and morphine-induced analgesia. Proc Natl Acad Sci U S A 94, 1544-1549.
Stafford, K., Gomes, A.B., Shen, J., and Yoburn, B.C. (2001). mu-Opioid receptor downregulation contributes to opioid tolerance in vivo. Pharmacol Biochem Behav 69, 233-237.
Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. (1998). A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95, 5187-5192.
Studier, F.W. (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41, 207-234.
Suzuki, T., Otani, K., Koike, Y., and Misawa, M. (1988). Genetic differences in preferences for morphine and codeine in Lewis and Fischer 344 inbred rat strains. Jpn J Pharmacol 47, 425-431.
Szargel, R., Rott, R., Eyal, A., Haskin, J., Shani, V., Balan, L., Wolosker, H., and Engelender, S. (2009). Synphilin-1A inhibits seven in absentia homolog (SIAH) and modulates alpha-synuclein monoubiquitylation and inclusion formation. J Biol Chem 284, 11706-11716.
Szeto, C.Y., Tang, N.L., Lee, D.T., and Stadlin, A. (2001). Association between mu opioid receptor gene polymorphisms and Chinese heroin addicts. Neuroreport 12, 1103- 1106.
Talbot, J.N., Skifter, D.A., Bianchi, E., Monaghan, D.T., Toews, M.L., and Murrin, L.C. (2009). Regulation of mu opioid receptor internalization by the scaffold protein RanBPM. Neurosci Lett 466, 154-158.
Tan, E.C., Tan, C.H., Karupathivan, U., and Yap, E.P. (2003). Mu opioid receptor gene polymorphisms and heroin dependence in Asian populations. Neuroreport 14, 569-572.
Tang, X.C., McFarland, K., Cagle, S., and Kalivas, P.W. (2005). Cocaine-induced reinstatement requires endogenous stimulation of mu-opioid receptors in the ventral pallidum. J Neurosci 25, 4512-4520.
163 Tanowitz, M., and von Zastrow, M. (2003). A novel endocytic recycling signal that distinguishes the membrane trafficking of naturally occurring opioid receptors. J Biol Chem 278, 45978-45986.
Tegeder, I., and Geisslinger, G. (2004). Opioids as modulators of cell death and survival- -unraveling mechanisms and revealing new indications. Pharmacol Rev 56, 351-369.
Terenius, L. (1973). Stereospecific interaction between narcotic analgesics and a synaptic plasm a membrane fraction of rat cerebral cortex. Acta Pharmacol Toxicol (Copenh) 32, 317-320.
Trejo, J., Hammes, S.R., and Coughlin, S.R. (1998). Termination of signaling by protease-activated receptor-1 is linked to lysosomal sorting. Proc Natl Acad Sci U S A 95, 13698-13702.
Trescot, A.M., Datta, S., Lee, M., and Hansen, H. (2008). Opioid pharmacology. Pain Physician 11, S133-153.
Trigo, J.M., Martin-Garcia, E., Berrendero, F., Robledo, P., and Maldonado, R. (2010). The endogenous opioid system: a common substrate in drug addiction. Drug Alcohol Depend 108, 183-194.
Tsai, Y.C., and Weissman, A.M. (2011). Ubiquitylation in ERAD: reversing to go forward? PLoS Biol 9, e1001038.
Tuoc, T.C., and Stoykova, A. (2010). Roles of the ubiquitin-proteosome system in neurogenesis. Cell Cycle 9, 3174-3180.
Tzschentke, T.M. (1998). Measuring reward with the conditioned place preference paradigm: a comprehensive review of drug effects, recent progress and new issues. Prog Neurobiol 56, 613-672.
Tzschentke, T.M. (2007). Measuring reward with the conditioned place preference (CPP) paradigm: update of the last decade. Addict Biol 12, 227-462.
Uchida, M., Enomoto, A., Fukuda, T., Kurokawa, K., Maeda, K., Kodama, Y., Asai, N., Hasegawa, T., Shimono, Y., Jijiwa, M., et al. (2006). Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase. J Cell Sci 119, 3067-3077.
Ueda, H., Harada, H., Nozaki, M., Katada, T., Ui, M., Satoh, M., and Takagi, H. (1988). Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go. Proc Natl Acad Sci U S A 85, 7013-7017. van Ree, J.M., and de Wied, D. (1980). Involvement of neurohypophyseal peptides in drug-mediated adaptive responses. Pharmacol Biochem Behav 13 Suppl 1, 257-263.
164 Volkow, N.D., and Li, T.K. (2005). Drugs and alcohol: treating and preventing abuse, addiction and their medical consequences. Pharmacol Ther 108, 3-17. von Zastrow, M., Svingos, A., Haberstock-Debic, H., and Evans, C. (2003). Regulated endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological adaptation to opiate drugs. Curr Opin Neurobiol 13, 348-353.
Waldhoer, M., Bartlett, S.E., and Whistler, J.L. (2004). Opioid receptors. Annu Rev Biochem 73, 953-990.
Wang, D., Quillan, J.M., Winans, K., Lucas, J.L., and Sadee, W. (2001). Single nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein coupling and calmodulin binding. J Biol Chem 276, 34624-34630.
Wang, D., Sadee, W., and Quillan, J.M. (1999). Calmodulin binding to G protein- coupling domain of opioid receptors. J Biol Chem 274, 22081-22088.
Wang, H.L., Hsu, C.Y., Huang, P.C., Kuo, Y.L., Li, A.H., Yeh, T.H., Tso, A.S., and Chen, Y.L. (2005). Heterodimerization of opioid receptor-like 1 and mu-opioid receptors impairs the potency of micro receptor agonist. J Neurochem 92, 1285-1294.
Wang, J.B., Johnson, P.S., Persico, A.M., Hawkins, A.L., Griffin, C.A., and Uhl, G.R. (1994). Human mu opiate receptor. cDNA and genomic clones, pharmacologic characterization and chromosomal assignment. FEBS Lett 338, 217-222.
Wheeler, T.C., Chin, L.S., Li, Y., Roudabush, F.L., and Li, L. (2002). Regulation of synaptophysin degradation by mammalian homologues of seven in absentia. J Biol Chem 277, 10273-10282.
Whistler, J.L., Chuang, H.H., Chu, P., Jan, L.Y., and von Zastrow, M. (1999). Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction. Neuron 23, 737-746.
Williams, J.T., Christie, M.J., and Manzoni, O. (2001). Cellular and synaptic adaptations mediating opioid dependence. Physiol Rev 81, 299-343.
Wong, Y.H., Demoliou-Mason, C.D., and Barnard, E.A. (1988). ADP-ribosylation with pertussis toxin modulates the GTP-sensitive opioid ligand binding in digitonin-soluble extracts of rat brain membranes. J Neurochem 51, 114-121.
Wu, D.F., Koch, T., Liang, Y.J., Stumm, R., Schulz, S., Schroder, H., and Hollt, V. (2007). Membrane glycoprotein M6a interacts with the micro-opioid receptor and facilitates receptor endocytosis and recycling. J Biol Chem 282, 22239-22247.
165 Wyles, J.P., McMaster, C.R., and Ridgway, N.D. (2002). Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum. J Biol Chem 277, 29908-29918.
Xu, J., Xu, M., Hurd, Y.L., Pasternak, G.W., and Pan, Y.X. (2009). Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene. J Neurochem 108, 962-972.
Xue, J.C., Chen, C., Zhu, J., Kunapuli, S., DeRiel, J.K., Yu, L., and Liu-Chen, L.Y. (1994). Differential binding domains of peptide and non-peptide ligands in the cloned rat kappa opioid receptor. J Biol Chem 269, 30195-30199.
Yang, L., Wang, S., Lim, G., Sung, B., Zeng, Q., and Mao, J. (2008). Inhibition of the ubiquitin-proteasome activity prevents glutamate transporter degradation and morphine tolerance. Pain 140, 472-478.
Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y., Matsuoka, K., Yoshida, M., Tanaka, K., et al. (2002). E3 ubiquitin ligase that recognizes sugar chains. Nature 418, 438-442.
Yun, S., Moller, A., Chae, S.K., Hong, W.P., Bae, Y.J., Bowtell, D.D., Ryu, S.H., and Suh, P.G. (2008). Siah proteins induce the epidermal growth factor-dependent degradation of phospholipase Cepsilon. J Biol Chem 283, 1034-1042.
Zangen, A., Ikemoto, S., Zadina, J.E., and Wise, R.A. (2002). Rewarding and psychomotor stimulant effects of endomorphin-1: anteroposterior differences within the ventral tegmental area and lack of effect in nucleus accumbens. J Neurosci 22, 7225- 7233.
Zhu, J., Chen, C., Xue, J.C., Kunapuli, S., DeRiel, J.K., and Liu-Chen, L.Y. (1995). Cloning of a human kappa opioid receptor from the brain. Life Sci 56, PL201-207.
Zhu, Y., King, M.A., Schuller, A.G., Nitsche, J.F., Reidl, M., Elde, R.P., Unterwald, E., Pasternak, G.W., and Pintar, J.E. (1999). Retention of supraspinal delta-like analgesia and loss of morphine tolerance in delta opioid receptor knockout mice. Neuron 24, 243- 252.
VITA Stephanie Justice-Bitner
EDUCATIONAL BACKGROUND
Master of Science in Veterinary Science, 2004 University of Nebraska, Lincoln, NE Non-thesis, emphasis in immunology
Bachelor of Science in Microbiology, 1997 Colorado State University, Fort Collins, CO
PROFESSIONAL EXPERIENCE
Visiting Assistant Professor of Biology, Department of Biology 8/11-present King’s College, Wilkes-Barre, PA
Adjunct Professor of Biology, Department of Biology 8/09-5-09 Elizabethtown College, Elizabethtown, PA
Temporary Faculty- Biology, Kulpmont/Berwick 8/04-12/05 Luzerne County Community College, Nanticoke, PA
Research Tech III, Veterinary Diagnostic Center 8/03-1/04 University of Nebraska, Lincoln, NE
PUBLICATIONS
Justice-Bitner S, Petko, J, Jin J, Wong V, Kittanakom S, Ferraro T, Stagljar I, and Levenson R, "MOR is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two- Hybrid Screens" (Submitted)
ABSTRACTS
“Discovery of Novel mu-Opioid Interacting Proteins Involved in the Ubiquitin Pathway.” Ubiquitin Drug Discovery and Diagnostics Conference, Hyatt at the Bellevue, Philadelphia, PA, October 2009.