Agonist-selective regulation of the mu by βarrestins

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of

Philosophy in the Graduate School of The Ohio State University

By

Chad Edward Groer

Graduate Program in Integrated Biomedical Science Program

The Ohio State University

2010

Dissertation Committee:

Professor Laura M. Bohn. Co-Advisor

Professor Wolfgang Sadée, Co-Advisor

Professor John Oberdick

Professor Lane Wallace

Copyright by

Chad Edward Groer

2010

ABSTRACT

Morphine and other mediate their effects through activation of the mu (MOR). Activation of the MOR results in recruitment of regulatory proteins, , that can regulate how this receptor signals. In vivo studies suggest that disruption of βarrestin-mediated MOR regulation may enhance -induced antinociception and reduce tolerance and certain unwanted side effects. Therefore, by understanding the cellular mechanisms by which this receptor is regulated, the development of which preserve the beneficial effects of opiates while eliminating unwanted side effects may be possible. In this dissertation we test the hypothesis that MOR can bias

MOR- interactions, and that arrestin recruitment profiles, in turn, may determine cellular responses evoked by these agonists.

In the first data portion of this dissertation, we characterize several novel

MOR agonists that are unable to promote βarrestin recruitment. Herkinorin is a moderately selective at the MOR, based on the structure of a natural product, . We find that herkinorin promotes very little MOR phosphorylation, does not recruit βarrestins, and does not induce receptor internalization in transfected cells. Herkinorin is unable to induce βarrestin recruitment or MOR internalization under conditions that facilitate receptor

ii phosphorylation and subsequent arrestin recruitment with other agonists. We also evaluated several derivatives of herkinorin with similar βarrestin recruitment and MOR internalization profiles. Therefore, herkinorin and its derivatives may be a promising step toward recapitulating ’s effects in βarr2-KO mice, which have been used to demonstrate that MOR activation without recruiting

βarrestin2 may be therapeutically useful, by producing analgesia with reduced side effects.

In the second data portion of this dissertation, we evaluate the interaction and functional consequences of MOR regulation by βarrestin1 and βarrestin2, in response to the classical agonists, DAMGO and morphine. Using both qualitative (microscopy) and quantitative (cell surface biotinylation and BRET) approaches, we have confirmed that DAMGO can induce robust interactions between the MOR and both βarrestins. Morphine, however, selectively promotes interactions with βarrestin2. Additionally, the agonist specific βarrestin interactions are required for internalization of the MOR. Finally, we show that

βarrestin1 is required for agonist-induced MOR ubiquitination, such that only

DAMGO, and not morphine, is able to promote MOR ubiquitination.

Taken together, these data suggest that MOR regulation is highly dependent on the complement of proteins available to interact with the MOR, and that the nature of the ligand can determine how the MOR is regulated by the available proteins. Therefore, the development of biased ligands for the MOR should focus activation of the MOR, but circumventing βarrestin-mediated

iii regulation. These concepts may be critical to consider in the development of opiate compounds designed to retain efficacy, while reducing the occurrence of unwanted side effects.

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DEDICATION

This dissertation is dedicated to:

my parents, Ed and Lana Groer and my brother, sister-in-law, and niece, Eric, Trish, and Autumn Groer.

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ACKNOWLEDGMENTS

First of all, I would like to thank my advisor, Laura M. Bohn, Ph.D., for her guidance, resources, and patience during my graduate studies. In addition to providing all resources that needed to perform my dissertation research, she provided invaluable advice, guidance, and encouragement regarding my project, without which, I would have not succeeded.

Next, I would like to thank all members of our lab, both past and present.

Kirsten Raehal (formerly grad student, now post-doc) and Cullen Schmid (grad student) have been with me in Laura’s lab since the beginning. Both of these women have helped me countless times with technical and writing issues and have helped me think critically about my project. Both Kirsten and Cullen are great colleagues and great friends. John Streicher (post-doc) helped with technical issues and dissertation writing. Alex Jaeger (undergrad) helped develop the BRET cell lines. Bob Moyer, Ph.D., Lori Hudson, and Sarah Teich are former members of the lab who have also helped me tremendously, as well.

I would like to thank my committee members for their time and for their suggestions concerning my dissertation work: Wolfgang Sadee, Dr.rer.nat (co-

vi advisor), John Oberdick, Ph.D., and Lane Wallace, Ph.D. I would also like to thank Gopi Tejwani, Ph.D. for serving on my candidacy exam committee with only days notice to replace one member who could not serve due to health problems.

I would like to thank Tom Prisinzano, Ph.D. at the University of Kansas

(formerly Univ. of Iowa) and his lab members Kevin Tidgewell, PhD. and Kim

Lovell for synthesizing herkinorin and the derivatives.

Thanks to Robert Lefkowitz, M.D. at Duke University for providing all four

MEF lines.

I would like to thank Dr. Allen Yates, Dr. Virginia Sanders, and Dr. Tom

Boyd for their work in directing the IBGP program.

I would like to thank all administrative assistants that have made my life much less hectic, including Christine Kerr, Sherry Ring, Gina Pace, Amy

Lahmers, Kelly Dillon, Elaine Wakely, Mary Krosky, and Lynn Wylie.

This work was funded by NIDA: DA14600 (LMB), DA18860 (LMB),

DA18151 (TEP).

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VITA

March 28, 1982 . . . ………………...... Born – Pittsburgh, PA

June 2000....…………..…………….Graduated from Quigley Catholic High School Baden, PA

2002-2004...... Undergraduate Research Assistant Dept. of Molecular and Cellular Biochemistry College of Medicine The Ohio State University Columbus, OH

June 2004. …...... ……………………...... B.S. in Pharmaceutical Sciences Magna Cum Laude, Graduated with Distinction and Honors College of Pharmacy The Ohio State University Columbus, OH

June 2004-Present……………...……………….…………..……….Ph.D. Candidate Integrated Biomedical Science Graduate Program College of Medicine The Ohio State University Columbus, OH

March 2009-Present………………………..…………… External Graduate Student Scripps Florida Jupiter, FL

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Publications

Tidgewell K, Groer CE, Harding WW, Lozama A, Schmidt M, Marquam A, Hiemstra J, Partilla JS, Dersch CM, Rothman RB, Bohn LM, and Prisinzano TE. Herkinorin Analogues with Differential β-Arrestin-2 Interactions. J. Med. Chem. 2008 Apr 24;51(8):2421-31.

Groer CE, Tidgewell K, Moyer RA, Harding WW, Rothman RB, Prisinzano TE, and Bohn LM. An opioid agonist that does not induce mu-opioid receptor-- arrestin interactions or receptor internalization. Mol Pharmacol. 2007 Feb;71(2):549-57.

Fields of Study

Major Field: Integrated Biomedical Science Program

Emphasis in Molecular Pharmacology, Pharmacogenomics, and Therapeutics

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Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments ...... vi

Vita ...... viii

List of Tables ...... xiii

List of Figures ...... xiv

CHAPTER 1 Introduction ...... 1

1.1 Clinical Aspects of ...... 1

1.2 Opioid Receptors ...... 2

1.3 Classical Regulation of -Coupled Receptors ...... 3

1.4 Functional Selectivity...... 7

1.5 Agonist-Directed Regulation of the MOR- In Vitro Evidence ...... 8

1.5.1 MOR Phosphorylation ...... 9

1.5.2 βarrestin Recruitment ...... 10

1.5.3 Internalization ...... 13

1.5.4 Desensitization ...... 14 x

1.5.5 Agonist-Directed MOR Signaling ...... 16

1.6 Functional Selectivity at the Mu Opioid Receptor- In Vivo Relevance ...... 17

1.7 Hypothesis and Overview of Chapters 2 and 3 ...... 19

1.8 Figures ...... 22

CHAPTER 2 Charcterization of Novel Mu Opioid Receptor Agonists that are Unable to Promote βarrestin Recruitment ...... 24 2.1 Introduction ...... 24

2.2 Materials and Methods ...... 28

2.3 Results ...... 37

2.5 Discussion ...... 45

2.6 Tables and Figures ...... 52

CHAPTER 3 Agonist Directed βarrestin Interactions Determine Mu Opioid Receptor Regulation ...... 66 3.1 Introduction ...... 66

3.2 Materials and Methods ...... 69

3.3 Results ...... 75

3.5 Discussion ...... 80

3.6 Tables and Figures ...... 86

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CHAPTER 4 Conclusions ...... 99

References ...... 107

xii

List of Tables

Table 2.1. Binding affinities at opioid receptors using [125I]OXY as radioligand...... 53

Table 2.2. [35S]-GTPγS coupling in CHO cells stably expressing µ opioid receptors...... 54

Table 3.1. Specific binding parameters of [3H]- to HA-MOR WT and βarr1/2-KO MEFs...... 87

Table 3.2. BRET analysis of interactions between the MOR and βarrestins- Potency and Efficacy...... 92

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List of Figures

Figure 1.1 Schematic representation of classical regulation of G protein-coupled receptors by βarrestins...... 22

Figure 1.2 Functional selectivity for the mu opioid receptor...... 23

Figure 2.1. Chemical structures of salvinorin A, herkinorin and four herkinorin derivatives...... 52

Figure 2.2. Herkinorin promotes MOR-mediated ERK1/2 phosphorylation...... 55

Figure 2.3. Agonist induced MOR phosphorylation at Ser375...... 57

Figure 2.4. Agonist-induced βarrestin2 interactions with the MOR...... 58

Figure 2.5. Agonist-induced internalization of the MOR...... 60

Figure 2.6. Agonist-induced MOR internalization under conditions in which morphine induces MOR internalization...... 62

Figure 2.7. Herkinorin and four derivatives induce MOR-mediated ERK1/2 phosphorylation...... 63

Figure 2.8. Agonist-induced arrestin2-GFP translocation...... 64

Figure 2.9. Agonist-induced MOR-YFP internalization...... 65

Figure 3.1. Cellular localization and saturation binding of HA-MOR stably expressing WT and arr1/2 KO MEFs...... 86

Figure 3.2. Agonist-induced HA-MOR phosphorylation at Ser375...... 88 xiv

Figure 3.3. Agonist-induced βarrestin-GFP translocation...... 89

Figure 3.4. BRET analysis of dose dependent agonist-induced increase in interactions between the MOR and βarrestins...... 90

Figure 3.5. Agonist-induced MOR trafficking in WT and arr1/2 KO MEFs...... 93

Figure 3.6. βarrestin rescue of HA-MOR internalization in βarr1/2 KO MEFs....95

Figure 3.7 Time course of agonist-induced MOR ubiquitination in WT and βarr1/2-KO MEFs...... 97

Figure 3.8 Agonist-induced MOR ubiquitination in WT and βarrestin-KO MEFs...... 98

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CHAPTER 1

INTRODUCTION

1.1 Clinical Aspects of Opioids

Opioids are drugs that are effective and widely used analgesics for treatment of moderate to severe pain states [1]. While opioids are effective in controlling pain in many patients, as much as 23% of patients opt to discontinue opioid therapy due to the adverse side effects, which include nausea/vomiting, constipation, sedation, cognitive dysfunction, respiratory suppression, addiction, and physical dependence [2]. While tolerance develops to many of these side effects during opioid treatment, it rarely develops to constipation, which is the most common side effect and is a major factor in patient noncompliance and the discontinuation of opioid therapy [2-3]. Furthermore, the threat of developing dependence and addiction, as well as adverse side effects, makes physicians hesitant to prescribe and patients hesitant to use opiates to manage pain.

Hence, though opiates are some of the best analgesics available, the occurrence of side effects can limit their clinical utility. Therefore, by understanding the cellular mechanisms of opioid actions, development of analgesics which preserve 1 the beneficial effects of opiates while eliminating unwanted side effects may be possible.

1.2 Opioid Receptors

Opioids produce the majority of their physiological effects by activating opioid receptors, which are G protein-coupled receptors (GPCRs). Radioligand binding and behavioral studies have suggested the existence of at least three types of opioid receptors, termed the mu (MOR), delta (DOR), and kappa (KOR) opioid receptors [4-8]. Subsequently, the genes of the three receptors were cloned, confirming the existence of three distinct opioid receptors [9-21]. Each opioid receptor exhibits unique expression patterns in the central nervous system

(CNS) [15,22-24], as well as the enteric nervous system and immune cells [25].

Other GPCRs show sequence and structural homology to this family, including the sigma receptors and the opioid-like/nociception receptor (ORL-1). These two receptors, however, exhibit very low binding affinities for most traditional and clinically used opioid ligands and will not be discussed further [26].

Activation of the MOR and DOR has been predominantly associated with the analgesic effects of opioids [22]. The prototypical and most widely used opiate, morphine, binds the MOR preferentially, but has modest affinity at the

DOR and KOR as well [22,27-28]. Morphine’s physiological effects include antinociception, respiratory suppression, constipation, and, in mice, increased locomotor activity. Additionally, mice show preference for morphine and other 2 opiates in experiments designed to test for drug reinforcement, and they become physically dependent on opiates following chronic treatment, as assessed by antagonist precipitated withdrawal. Though morphine binds to all three opioid receptor types (MOR, DOR, and KOR), pharmacological inhibitor and knockout mouse studies show that these effects are predominately mediated by the MOR.

Either selective antagonism [29] or genetic deletion of the MOR abolishes all of the above mentioned morphine-induced behaviors [22,24,27,30-33]. Therefore, the MOR is a major target for pharmacotherapy for pain management.

1.3 Classical Regulation of G Protein-Coupled Receptors

The mu opioid receptor (MOR) is a GPCR, and like most GPCRs, the activated MOR is subject to classical regulatory mechanisms including phosphorylation, arrestin binding, desensitization, and internalization (Figure 1.1)

[34-36]. In this classical model, the agonist-activated GPCR is rapidly phosphorylated by G protein receptor kinases (GRKs). The GRK family contains

7 members, which can be divided into three categories (GRKs 1 and 7, GRKs 2 and 3, GRKs 4, 5, and 6) based on sequence homology, function, and sub- cellular localization [37-42]. GRKs 1 and 7 are expressed exclusively in the ocular system. GRKs 2, 3, 5, 6, and to a lesser extent 4, are widely expressed.

While GRKs 2 and 3 are located predominantly in the cytosol and must be recruited to the membrane by GPCR-activated Gβγ protein subunits, GRKs 4, 5, and 6 are associated with the membrane by polybasic domains or palmitoylation 3

[43]. GRK-mediated phosphorylation is important for both desensitization and internalization of many GPCRs [44]. For example, over-expression of GRKs was able to promote agonist-induced phosphorylation and internalization of a β2AR mutant that was otherwise resistant to trafficking [45-46]. Similarly, over- expression of GRK2 or a dominant negative mutant of GRK2 can augment or attenuate trafficking of m2 muscarinic receptors, respectively [47].

The phosphorylated GPCR can serve as a substrate for arrestin binding

[48-50]. Arrestin protein binding is most robust when GPCRs are both phosphorylated and activated. Arrestins terminate further G protein-mediated signaling by interfering with the coupling of GPCRs to G proteins, in a process called desensitization. Currently, 4 arrestin proteins are known [51]. Two are expressed almost exclusively in the visual system (arrestin1 and arrestin4), and two are ubiquitously expressed (arrestin2 and arrestin3; also called βarrestin1 and βarrestin2, respectively) and may regulate most GPCRs. βarrestin1 and 2 have been tagged with green fluorescent protein (βarr1 or 2-GFP) and used to monitor in real time activation of and βarrestin recruitment to activated GPCRs

[48]. Using this approach, Oakley et al. [48,52] observed that while some

GPCRs recruit both βarrestins with equal affinity upon activation (i.e. angiotensin

II type 1A and V2 receptors; termed Class B), some receptors recruit

βarrestin2 with higher affinity than βarrestin1 (i.e. β2 (β2AR) and MOR; termed Class A).

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In addition to their roles in desensitization, GRKs and arrestins promote internalization of GPCRs [37,49-50,53-60]. Internalization causes receptors to be removed from the cell surface and thereby not available for signaling. In some instances, however, signaling pathways are activated by internalized receptors [61]. Furthermore, internalization of GPCRs can promote either degradation of the receptors or their resensitization and recycling back to the membrane. GRKs promote GPCR internalization by facilitating arrestin binding.

Arrestins, in turn, bind directly to AP-2 and clathrin and tether receptors to clathrin-coated pits for internalization [62-63]. Over-expression of βarrestins can augment internalization of both wildtype and an internalization deficient mutant of the β2 adrenergic receptor (β2AR). In contrast, dominant-negative βarrestin mutants or the absence of βarrestins drastically attenuates β2AR or angiotensin II type 1A receptor internalization, respectively [49,64]. Finally, disruption of the interactions between βarrestins and either clathrin or AP-2 also attenuates β2AR internalization [54-59].

Once internalized, the receptor can be either resensitized and returned to the cell surface for another signaling event or degraded in the lysosome in a process called downregulation [65]. βArrestins are important in this process as they have been shown to promote ubiquitination of some GPCRs. This was first demonstrated for the β2AR, which requires βarrestin2 for agonist-induced ubiquitination and down-regulation [66]. Removal of βarrestins or the use of a lysine deficient β2AR mutant prevented agonist-induced downregulation of this

5 receptor. βarrestin involvement for ubiquitination and/or downregulation of several other GPCRs, including the CXCR4, IGFR-1R, KOR, M1 and M2 mACHR receptors, have also been demonstrated [67-70]. In some cases, however,

βarrestins seem to promote recycling rather than degradation of receptors. Vines el al. [71] showed that recycling of the N-formyl receptor was attenuated in the βarrestin deficient cells. Collectively, these studies demonstrate that

βarrestins play important roles GPCR trafficking, recycling, and downregulation.

While βarrestins have classically been defined for their roles in regulating

GPCRs in the processes described above, βarrestins can also act as positive mediators of many GPCRs [72-74]. Using cells lacking expression of βarrestins or by siRNA knockdown of βarrestins, Azzi et al. [75] and Shenoy et al. [76] showed β2AR-meidated activation of ERK1/2 required βarrestin expression when certain agonists were used. Similarly, βarrestin2 was shown to complex with components of the ERK1/2 signaling cascade in response to angiotensisn II type

2A stimulation [77-79]. For these and other GPCRs, such as the V2 vasopressin receptor [77-81], parathyroid [82], CC 7

[83], and the serotonin 2A receptor [84], βarrestin-mediated signaling is dependent on the particular agonist used to activate each receptor. Certain agonists promote βarrestin-mediated ERK1/2 activation while other agonists at the same receptor may promote ERK1/2 activation through G protein mediated pathways. Further, some agonists may utilize both the βarrestin-mediated and G protein-mediated pathway simultaneously, though not necessarily with the same

6 potency or efficacy. This phenomenon is referred to as functional selectivity, and is discussed below.

1.4 Functional Selectivity

Functional selectivity (also referred to as ligand bias or collateral efficacy) is a phenomenon wherein a particular ligand will stabilize a receptor in a conformation that may preferentially activate one signaling pathway over another

[85]. Different ligands at the same receptor may stabilize different receptor conformations, which could in turn promote activation of different signaling or regulatory pathways (Figure 1.2). GPCRs mediate many of their biological responses by coupling to G proteins, which in turn modulate a variety of downstream effectors, such as , ion channels, and transcription factors. As discussed above, activation of GPCRs leads to their phosphorylation,

βarrestin association, and trafficking. In addition to their regulatory roles,

βarrestins can also serve to mediate signaling cascades that are independent of

G protein activation. Therefore, βarrestins can act to dampen G protein mediated signaling and also promote other signaling cascades. Activation of a single receptor can lead to modulation of many different downstream effectors some of which are dampened by, and others which are promoted by, βarrestins.

Interestingly, downstream effectors are not always modulated to the same extent or even in the same direction.

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Many GPCRs have been shown to exhibit functional selectivity, including the MOR [86-88], CCR7 [83], β2AR [76], and serotonin 2A receptor (5HT2AR)

[84]. For the 5HT2AR-mediated head twitch response, serotonin requires

βarrestin to mediate this behavior, while another 5HT2AR agonist, DOI does not.

For the MOR, several ligands were shown to promote two phases of a calcium response with very different potencies [88]. These examples illustrate that different ligands acting at the same receptor can elicit different biological effects, such that each distinct ligand for a receptor may activate a unique subset of all possible signaling cascades associated with the receptor.

1.5 Agonist-Directed Regulation of the MOR- In Vitro Evidence

The MOR is a GPCR and primarily couples to Gi/o proteins, leading to the inhibition of adenylyl cyclase. Furthermore, MOR activation leads to the activation of inwardly rectifying potassium channels, inhibition of voltage-gated calcium channels, and activation of a variety of second messenger systems can be mediated through the MOR, including PKC, CamKII, PLC and ERK1/2. Upon activation, the MOR is subject to classical regulatory mechanisms including phosphorylation, βarrestin recruitment, and internalization. This classical regulation, however, has been demonstrated to be dependent on the ligand used to activate the receptor.

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1.5.1 MOR Phosphorylation

Upon stimulation, the MOR is phosphorylated. Agonists such as and DAMGO induce robust phosphorylation of the receptor within seconds of exposure. Morphine and a novel full MOR agonist, herkinorin, however, induce much less robust phosphorylation [35,89-92]. These data suggest that DAMGO and etorphine stabilize the MOR in a conformation that permits robust interactions with and activation of GRKs. In contrast, morphine and herkinorin promote conformations of the MOR that seem to be less suitable substrates for intracellular kinases. Importantly, over-expression of GRK2 can dramatically augment morphine-induced MOR phosphorylation, suggesting that the expression levels of kinases within the cell can also influence MOR phosphorylation. In addition, there are at least 12 serine and threonine residues in the C-terminal tail of the MOR that may be phosphorylated. Several studies have shown that the specific pattern of phosphorylation of these residues depends on the agonist used [59,89,92-94]. Importantly, morphine induced MOR phosphorylation is abolished in the S375A MOR mutant, while DAMGO phosphorylation is reduced, suggesting that while DAMGO induces MOR phosphorylation at several residues, morphine-induced phosphorylation requires

Ser375, either for direct phosphorylation or as an essential residue for the phosphorylation of other sites [89,92-93]. It has been proposed that these differences in phosphorylation are in part due to activation of different kinases.

Different agonist-induced receptor conformations may promote interactions with

9 different intracellular kinases, and result in unique phosphorylation patterns.

Indeed, over expression of GRK2 can increase MOR phosphorylation by etorphine and morphine [35], while PKC blockade may affect only morphine- induced MOR phosphorylation [94]. Therefore, cell specific expression of kinases may influence how the MOR is regulated by distinct agonists.

1.5.2 βarrestin Recruitment

Once activated by ligand and phosphorylated by kinases, the affinity between GPCRs and βarrestins increases [49-50]. Therefore, since the agonist can influence the phosphorylation state of the receptor and phosphorylation can influence βarrestin binding, agonists may differ in their ability to cause interactions between the receptor and βarrestin recruitment profiles. Indeed,

Zhang et al. [35] observed a striking difference in agonist induced βarrestin2-

GFP recruitment to the MOR. While etorphine induced robust βarr2-GFP recruitment to the MOR, morphine was unable to induce detectable βarrestin2-

GFP recruitment. When GRK2 or GRK6 is over-expressed, however, morphine can induce detectable recruitment of βarr2-GFP [34-35,90,95]. In addition, we have observed that GRKs 3, 4, and 5 also allow visualization of morphine- induced βarr2-eGFP recruitment. These results further support the hypothesis that a ligand’s ability to induce receptor phosphorylation can directly influence

βarrestin binding. Further, it suggests that GRK protein levels in the cell can determine whether morphine can promote MOR phosphorylation and hence

βarrestin recruitment. Additionally, PKC, PKA, CamKII, and ERK1/2 have been 10 shown to regulate the morphine-bound MOR [94,96-100], and may affect morphine-induced MOR phosphorylation and thus βarrestin interactions.

Therefore, the morphine-activated MOR could be regulated differently in different cell types, based on the intracellular complement of kinases in each cell type.

In addition to the intracellular complement of kinases, the two ubiquitously expressed βarrestins can also interact with the MOR, and agonist-dependent differences have been demonstrated for βarrestin1 recruitment to the MOR.

Using cells that lack expression of both βarrestins [64] to avoid competition with endogenous βarrestins, Bohn et al. [34] showed that while etorphine robustly recruited both βarrestins, morphine preferentially induced recruitment of βarr2-

GFP. These results are consistent with observations by Oakley et al. [52], who showed that the MOR recruits βarrestin2 with higher affinity than βarrestin1.

While etorphine can promote robust phosphorylation of the MOR and promote robust recruitment of both βarrestins, morphine-induced phosphorylation is very weak. Therefore, morphine induces very weak interactions with the higher affinity, βarrestin2, which is only detectable upon over-expression of GRKs or in the absence of endogenous βarrestins. In contrast, these cellular manipulations cannot overcome the inability of morphine to promote recruitment of the lower affinity βarrestin1, suggesting that the criteria for βarrestin1 recruitment to the

MOR is more stringent than for βarestin2.

Since morphine has been shown as a partial agonist in some G protein coupling studies, an attractive hypothesis is that the efficacy of a ligand to induce

11 receptor activation will determine βarrestin recruitment. Contrary to this hypothesis, however, a class of novel compounds, including Herkinorin based on the natural product Salvinorin A, have been identified that do not recruit either

βarrestin but maintain maximal efficacy at promoting G protein coupling, adenylyl cyclase inhibition, and ERK1/2 activation [90,101-103]. Further, a study by

Molinari et al. [104] directly compared G protein coupling efficacy and arrestin recruitment by a panel of MOR agonist. Indeed they found that a linear relationship did not exist. Some agonists assessed, including , were shown to be partial agonists with respect to G protein coupling, while having no efficacy for βarrestin2 recruitment. Morphine, on the other hand, shows partial G protein coupling efficacy but also displayed partial efficacy at

βarrestin2 recruitment. Interestingly, displayed approximately equal efficacy as morphine for βarrestin2 recruitment, but displayed full efficacy for G protein coupling. Collectively, these data suggest that the structure of the ligand can directly influence the βarrestin recruitment profile to the MOR and that receptor activation is necessary, but not sufficient, for βarrestin recruitment.

Finally, the extent of coupling to G proteins promoted by a ligand does not predict the extent of βarrestin interaction. Gurevich and Gurevich [51] suggested that

βarrestin binding and G protein coupling differentially involve the intracellular loops and the C-terminal tail of the MOR. It is likely that ligands induce different conformations of these intracellular domains of the MOR, which in turn accounts for the variety of βarrestin and G protein coupling efficacies observed [104].

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1.5.3 Internalization

Agonist-induced phosphorylation and arrestin binding can determine the trafficking pattern of GPCRs, including the MOR [105]. Further, MOR phosphorylation patterns and βarrestin recruitment are dependent on the specific ligand (see above). Therefore, it follows that ligand-specific internalization should occur for the MOR. Indeed, Arden et al. [86] first demonstrated that while

DAMGO promotes robust internalization of the MOR in transfected cells, morphine treatment does not, even though both agonists showed similar efficacy for promoting coupling to G proteins [106]. These results have been subsequently repeated and confirmed in numerous other studies [35-

36,87,90,92,107-110].

For the MOR, internalization profiles for different agonists mirror the agonists’ abilities to promote phosphorylation and βarrestin binding. Agonists, such as etorphine and DAMGO, that promote robust receptor phosphorylation and βarrestin recruitment cause extensive MOR internalization. In stark contrast, the morphine-bound receptor induces weak phosphorylation and βarrestin2 recruitment and therefore is internalized poorly. Morphine-induced internalization can be augmented by over-expression of GRK2 [35,90,92] suggesting that an agonists’ ability to promote MOR internalization is dependent on its ability to promote receptor phosphorylation and βarrestin binding. Consistent with this hypothesis, herkinorin, which does not promote βarrestin recruitment even in the presence of GRK2 over-expression, does not induce MOR internalization

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[90,101]. These data strongly suggest that while the cellular complement of kinases is important to determine phosphorylation, βarrestin binding, and internalization, the ligand must promote a receptor conformation that is amenable to interaction with these intracellular partners.

The agonist-dependent difference in MOR internalization has also been shown in vivo using guinea pig ileum [111], rat locus coeruleus [112], and rat cortical neurons [92]. Further, as cell culture studies suggest that the cellular complement of proteins can influence how the agonist-bound MOR traffics, the same seems to be true in vivo. Using rat nucleus accumbens neurons,

Haberstock-Debic et al. [113] demonstrated “compartment-selective” internalization. Morphine did not induce MOR internalization in the cell bodies, but did induce receptor internalization in the dendrites of the neurons. They also observed morphine-induced MOR internalization in striatal neurons [114]. These results suggest that the interactions between the receptor and intracellular regulatory proteins may differ between cell types and even between compartments within the same cell, and hence result in different agonist-induced responses.

1.5.4 Desensitization

Upon activation, the MOR becomes desensitized, such that it can no longer respond to agonist stimulation. Phosphorylation and βarrestin binding are important regulatory events that contribute directly to desensitization of the MOR.

Mutation of Thr394 or Ser375 to alanine dramatically reduces DAMGO-induced 14 phosphorylation and desensitization of the MOR [89,91-92]. Further, over- expression of βarrestins in cell systems can augment agonist-induced desensitization of GTP coupling, inhibition of adenylyl cyclase, or induction of potassium current [36,107,115-116], while expression of dominant negative

βarrestin mutants prevents agonist-induced desensitization [107].

Since different agonists promote different MOR phosphorylation patterns and βarrestin recruitment profiles, and MOR phosphorylation and βarrestin recruitment directly mediate MOR desensitization, it follows that MOR desensitization is dependent on the agonist used. Indeed, agonist-dependent

MOR desensitization has been observed for the MOR using various endpoints, including GTP coupling, inhibition of adenylyl cyclase, changes in electrophysiological membrane potential, and calcium release [36,92,94,117-

120]. DAMGO-induced MOR desensitization has been shown to be dependent on GRK2 activity and βarrestin expression. Dominant negative mutants of GRK2 greatly attenuate DAMGO-induced desensitization [94,121-122], and genetic deletion of βarrestin2 ablates DAMGO-induced desensitization of a calcium response [120].

Morphine, however, seems to engage different mechanisms of MOR desensitization that is highly dependent on the system in which the MOR is being examined. Morphine can promote GRK2-mediated MOR regulation when expression levels are adequate [34-35,114]. In other studies, dominant negative mutants of GRK2 had no effect on morphine-induced desensitization [94,121-

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122], while PKC inhibition or use of PKC knockout tissues greatly attenuated morphine-induced MOR desensitization [94,121,123-124]. In some systems, morphine did not induce desensitization of inhibition of adenylyl cyclase or -promoted potassium current [36,125]. The diversity of these reports exemplifies the role of the cellular environment in MOR regulation, as all of these studies were conducted in different cell systems and used different endpoints to assess desensitization of the MOR. Therefore, when morphine is the agonist,

MOR desensitization is greatly influenced by the complement of proteins expressed in the immediate vicinity of the MOR. On the other hand, DAMGO may stabilize the MOR in a conformation that is subject exclusively to GRK and

βarrestin-mediated regulation.

1.5.5 Agonist-Directed MOR Signaling

In addition to agonist-specific regulation of the MOR, different ligands have also been shown to lead to distinct induction of downstream signaling cascades. This was first demonstrated by Quillan et al. [88] showing that morphine and etorphine differed in their relative abilities to modulate each of two phases of calcium release.

More recently, studies have examined the agonist-selective activation of

ERK1/2, a downstream effecter of the MOR. MOR-mediated activation of this pathway can occur through the traditional G protein mediated pathway or a

βarrestin dependent pathway [126-127] in a ligand dependent manner. The specific mechanism of ERK activation seems to have consequences on ERK 16 localization and gene transcription [126,128-130]. In these two distinct mechanisms of ERK activation, the ligand can actually determine whether

βarrestin assumes its traditional role to desensitize signaling or βarrestin acts to promote signaling.

1.6 Functional Selectivity at the Mu Opioid Receptor- In Vivo Relevance

Consistent with the agonist specificity of MOR-βarrestin interactions seen in vitro, in vivo studies using mice that lack βarr2 (βarr2-KO) reveal agonist- dependent behavioral differences. While acute antinociceptive profiles are similar between WT and βarr2-KO mice in response to and , presumably due to compensation by βarr1, morphine-induced antinociception is dramatically different between the genotypes [34,123,131-133]. Compared to wildtype (WT) mice, the βarr2 KO mice display enhanced and prolonged morphine-induced antinociception. Antinociceptive profiles of arr1 KO mice, however, do not differ from WT mice in response to morphine.

In addition to dramatically altered acute responses to morphine in βarr2-

KO mice, chronic morphine is unable to induce tolerance in these mice in the hotplate test [131], which is a measure of supra-spinal pain perception [134], while wildtype mice develop substantial tolerance with the same treatment.

Furthermore, after chronic morphine treatment, the MORs in the periaqueductal grey (PAG) and brainstem ( regions involved in pain perception) of wildtype mice are desensitized, while MORs from these same brain regions in βarr2-KO 17 mice retain the ability to induce G protein coupling [131]. The βarr2-KO mouse studies are consistent with previous studies in rats showing that chronic morphine induces MOR desensitization specifically in brain regions associated with pain perception, while little or no desensitization of MORs was observed in brain regions not thought to play a role in pain perception [135-138]. For antinociceptive responses in mice, βarrestin2 negatively regulates the MOR.

Thus, in the absence of βarrestin2, morphine analgesic responses are augmented [34,123,131,133].

In addition to analgesia, opiates can also produce some undesired effects, such as constipation and respiratory suppression [139]. In stark contrast to antinociceptive responses, however, both morphine-induced constipation and respiratory suppression were reduced, rather than enhanced, in the βarr2-KO mice compared to WT [139]. Furthermore, the authors show that the difference between genotypes is maintained using a peripherally restricted opiate. These data suggest that βarrestin2 regulation of the MOR may be very different in the colon than in the CNS, such that βarrestin may be mediating MOR signaling in this tissue. Removal of this βarrestin-mediated signaling would result in the observed attenuation of the response. Indeed, βarrestin have been show to promote signaling for other GPCRs [73], and more recently for the MOR.

Alternatively, however, disruption of other GPCRs may be involved in these effects.

18

1.7 Hypothesis and Overview of Chapters 2 and 3

This dissertation presents work that tests the hypothesis that MOR agonists can bias MOR-arrestin interactions, and that arrestin recruitment profiles, in turn, may determine cellular responses evoked by these agonists.

In Chapter 2, we characterize several novel MOR agonists that are unable to promote βarrestin recruitment. Herkinorin is a moderately selective agonist at the MOR, based on the structure of a natural product, Salvinorin A, which is a highly selective and potent KOR agonist. Therefore we evaluated herkinorin for its ability to induce classical regulation of the MOR. We find that herkinorin promotes very little MOR phosphorylation, does not recruit βarrestins, and does not induce MOR internalization in HEK293 cells. Though morphine induces a similar pattern for MOR regulation, over-expression of GRK2 is sufficient to promote morphine induced βarr2 recruitment and MOR internalization. Herkinorin, however, is still unable to induce βarrestin recruitment or MOR internalization under these conditions. We also evaluated several derivatives of herkinorin with similar βarrestin recruitment and MOR internalization profiles. These data provide evidence that while morphine- induced regulation of the MOR can be influenced by kinase levels, herkinorin and its derivatives stabilize receptor conformations that are not appropriate for

βarrestin recruitment or internalization. Further, given that herkinorin and its derivatives are fully efficacious MOR agonists, these data suggest that MOR activation and βarrestin mediated regulation are separable events, such that

19 conformational requirements for G protein coupling are distinct from those involved in βarrestin regulation. Therefore, herkinorin and its derivatives may be a promising step toward recapitulating morphine’s effects in βarr2-KO mice.

MOR activation that can circumvent βarrestin regulation may be therapeutically useful, by producing analgesia with reduced side effects (Figure 1.2).

In Chapter 3, we evaluate the interaction and functional consequences of

MOR regulation by βarrestin1 and βarrestin2, in response to the classical agonists, DAMGO and morphine. We have stably expressed the MOR in WT,

βarr1-KO, βarr2-KO, and βarr1/2-KO mouse embryonic fibroblasts (MEFs) to evaluate the role of βarrestins in classical MOR regulation. Using both qualitative

(microscopy) and quantitative (cell surface biotinylation and BRET) approaches, we have confirmed that DAMGO can induce robust interactions between the

MOR and both βarrestins. Morphine, however, selectively promotes interactions with βarrestin2. Additionally, the agonist specific βarrestin interactions are required for trafficking of the MOR. Finally, we show that βarrestin1 is required for agonist-induced MOR ubiquitination, such that only DAMGO, and not morphine, is able to promote MOR ubiquitination.

The studies presented in this dissertation show that MOR signaling and regulation can be affected differently, in a ligand-dependent manner, further indicating functional selectivity at the MOR. We demonstrate that while all ligands tested are full agonists at the MOR, they induce βarrestin regulation in three different ways. DAMGO can induce robust interaction with both βarrestins

20 while morphine specifically recruits βarrestin2. Finally, herkinorin does not promote βarrestin recruitment. The βarrestin interactions with the MOR result in functional differences in MOR trafficking and ubiquitination. Furthermore, in the case of the morphine-bound MOR, regulation can be manipulated by altering cellular kinase levels. Taken together, these data suggest that MOR regulation is highly dependent on the complement of proteins available to interact with the

MOR, and that the nature of the ligand can determine how the MOR is regulated by the available proteins. Therefore, the development of biased ligands for the

MOR should focus activation of the MOR, but circumventing βarrestin-mediated regulation. These concepts will be critical to consider in the development of opiate compounds designed to retain analgesic efficacy, while reducing the occurrence of unwanted side effects.

21

1.8 Figures

Agonist

Desensitization

G P P arrestin1&2 G G GRK G

Internalization

Signaling Resensitization Ubiquitination Lysosomal degradation

Figure 1.1 Schematic representation of classical regulation of G protein-coupled receptors by βarrestins. Rapidly following agonist activation and G protein signaling, GRKs phosphorylate the GPCR and facilitate the recruitment of βarrestins. Association of βarrestins prevents further G protein coupling and signaling and facilitates internalization by scaffolding internalization machinery to the receptor. Following internalization, the receptor may be resensitized and recycled to the cell surface or degraded in the lysosome. (Adapted from Bohn et al., Neuromolecular Medicine, 2004) [140]

22

Antinociception Fentanyl No change in Etorphine MOR βarr 1/2 βarr2 KO mice Methadone

Increased in Morphine MOR βarr 2 βarr2 KO mice

Novel Increased in MOR No βarr Agonists WT mice?

Figure 1.2 Functional selectivity for the mu opioid receptor. Functional selectivity is a phenomenon wherein different ligands acting at the same receptor can differentially activate signaling or regulatory pathways. For the mu opioid receptor (MOR), the three groups of ligands shown all activate the MOR with respect to G protein coupling, but promote differential recruitment of βarrestins. Fentanyl, etorphine, and methadone promote robust interactions between the MOR and both βarrestins. Morphine, however, selectively promotes βarrestin2 recruitment. Novel agonists, such as herkinorin, do not promote detectable interactions between the MOR and either βarrestin. The βarrestin recruitment profiles have important implications in vivo. In βarr2-KO mice, morphine- induced antinociception is dramatically augmented, while the antinociceptive profiles to fentanyl, etorphine, and methadone are not different from WT mice, presumably due to βarrestin1 compensation in the absence of βarrestin2. Novel MOR agonists, such as herkinorin, that do not promote βarrestin interactions with the MOR may recapitulate in WT mice the effects of morphine in βarr2-KO mice. These experiments are ongoing.

23

CHAPTER 2

CHARACTERIZATION OF NOVEL MU OPIOID RECEPTOR AGONISTS THAT

ARE UNABLE TO PROMOTE βARRESTIN RECRUITMENT

2.1 Introduction

The mu opioid receptor (MOR) is a G protein-coupled receptor (GPCR) and as such, when activated it is subject to classical regulatory mechanisms including G protein receptor kinase (GRK)-mediated phosphorylation, βarrestin recruitment, and internalization [49,58,60]. In vitro studies show that while the

MOR is regulated by these classical mechanisms after stimulation with many agonists, such as DAMGO and fentanyl, which promote robust MOR phosphorylation, βarrestin recruitment, and MOR internalization, morphine fails to induce robust phosphorylation of the MOR, recruitment of βarrestins, or receptor internalization in HEK-293 cells [34-36,86].

The inability of morphine to induce these regulatory mechanisms, however, can be overcome by GRK2 over-expression in cell culture. These data suggest that the agonist activating the MOR can promote unique receptor conformations that determine MOR regulation in a GRK-dependent manner, as

24 over-expression of GRK2 in HEK cells augments morphine-induced MOR phosphorylation, which then facilitates βarrestin2 recruitment and receptor internalization [34-35]. The increase in MOR phosphorylation is likely responsible for the robust βarrestin2 recruitment when GRK2 is over-expressed.

Since βarrestins can act as scaffolds for internalization machinery, the increased

βarrestin2 recruitment, in turn, enhances MOR internalization [34-35,141-142].

Therefore, by over-expressing GRK2, the cellular environment is biased toward

βarrestin-mediated MOR regulation.

Two ubiquitously expressed βarrestins, βarrestin1 and βarrestin2, are involved in the classical regulation of most GCPRs [51,60,143]. Though both

βarrestins can be recruited to the MOR by a number of opiate agonists, such as methadone and fentanyl, cell culture studies suggest that βarrestin2 is preferentially important in morphine-induced MOR regulation. By eliminating competition with endogenous βarrestins through the use of mouse embryonic fibroblasts that lack expression of both βarrestins (βarr1/2-KO MEFs), morphine- induced βarr2-GFP, but not βarr1-GFP, translocation can be observed [34].

These data are consistent with the classification of the MOR as a “class B”

GPCR [52], which promotes βarrestin2 recruitment with higher affinity than

βarrestin1. Taken together, these data demonstrate that the MOR can interact with either βarrestin1 or βarrestin2, but that this interaction is influenced by the ligand and the phosphorylation state of the receptor.

25

In addition to cell culture studies, genetic ablation of βarrestin2 in mice

(βarr2-KO mice) also demonstrates a morphine bias for βarrestin2 regulation of the MOR, as several morphine-induced behavioral responses are altered in these mice. βArr2-KO mice display enhanced and prolonged morphine-induced antinociception and attenuated tolerance to morphine compared to wildtype (WT) mice, while deletion of βarrestin1 does not impact morphine induced antinociception [34,131,133]. Interestingly, acute antinociceptive profiles and the development of tolerance are similar between WT and βarr2-KO mice in response to fentanyl and methadone [131,133,144], which may be due to compensation by βarrestin1, as these agonists promote recruitment of βarrestin1 in HEK cells. Though the deletion of βarrestin2 promotes enhanced morphine- induced antinociception, these animals display significantly attenuated acute morphine-induced constipation and naloxone-induced withdrawal signs following chronic morphine treatment [139,144]. These data suggest that the interaction of the MOR with βarrestin2 may be a critical point in MOR regulation in vivo as well as in vitro. Further, the interactions between the MOR and βarrestins may have implications in the development of pharmacotherapies, as a MOR agonist that does not promote βarrestin interactions may be a means to maintain analgesic efficacy while reducing the occurrence of unwanted side effects.

To this end, we have evaluated several novel opioids based on the structure of a natural product, Salvinorin A, which is purified from . This compound binds selectively to kappa opioid receptors (KOR)

26 and is a potent and fully efficacious KOR agonist, as assessed by GTP coupling and adenylyl cyclase inhibition [145-146]. Interestingly, however, salvinorin A was shown to induce significantly attenuated KOR internalization compared to the classical KOR agonist, U50,488H [147-149], suggesting that βarrestin regulation of this receptor may be altered. In an effort to engineer novel analgesic drugs, the chemical structure of salvinorin A was modified to alter the receptor selectivity of the compound. One particular compound derived in this series, termed herkinorin, showed selective binding to and agonist activity at the

MOR, as assessed by competition binding and GTP coupling studies, respectively [103](Harding et al., 2005). Given the altered KOR internalization by the parent compound, salvinorin A, we assessed whether herkinorin may promote attenuated βarrestin regulation of the MOR.

In the studies presented in this chapter, we characterize βarrestin- mediated regulation of the MOR by herkinorin. We show that herkinorin is an agonist at the MOR in HEK-293 cells, as assessed by phosphorylation of the downstream kinase, ERK1/2. Moreover, herkinorin induces weak MOR phosphorylation that is comparable to morphine, yet herkinorin is unable to induce βarrestin2 recruitment or internalization of the MOR, even under conditions in which morphine can do so. Further, MOR regulation was assessed in response to four herkinorin derivatives that showed improved selectivity for the

MOR compared to herkinorin. Three of the four derivatives tested do not recruit

βarrestin2 or promote MOR internalization. Therefore, these compounds

27 represent a class of MOR ligands that do not induce βarrestin-mediated regulation and, considering the improved properties of morphine in βarr2-KO mice, may be a promising step toward the development of analgesics with reduced side effects.

2.2 Materials and Methods

Drugs. DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin; Tocris, Ellisville, MO), morphine sulfate pentahydrate (Sigma, St. Louis, MO), and naloxone hydrochloride (Tocris, Ellisville, MO) were dissolved in distilled water to 10mM.

Herkinorin was dissolved in DMSO to 10mM. Since herkinorin is prone to degradation, powder was produced in small quantities, protected from light, and dissolved immediately before each experiment. 10mM stocks were diluted with

Minimal essential medium (MEM) to working concentrations for experiments. For imaging experiments, MEM did not contain phenol red.

Constructs. mouse mu opioid receptor ((gi1055230) is HA tagged on the N- terminus (HA-MOR); rat βarrestin1 (gi949985) and βarrestin2 (gi949986) are tagged on the C-terminus with enhanced green florescent protein (βarr2-eGFP and βarr1-eGFP), mouse MOR is tagged on the C-terminus with yellow florescent protein (MOR-YFP), mouse MOR1D is tagged on the C-terminus with

GFP (MOR1D-GFP)

28

Stable Expression of HA-MOR in HEK-293 cells. HEK-293 cells were transduced with HA-MOR using Murine Stem Cell Virus (MSCV). Transfected cells were selected in the presence of (1ug/mL, Calbiochem). A

FACS Aria flow cytometer was used to select for high expressing cells (top

~25%) using an anti-HA AlexaFluor 488 conjugate antibody (1:200; Invitrogen,

Eugene, Oregon). Cells were sub-cloned 3 times to select for clones that showed predominantly membrane expression of HA-MOR, as assessed using an anti-HA AlexaFluor488 conjugate antibody and visualized by confocal microscopy.

ERK Phosphorylation. HEK-293 cells stably expressing an HA-tagged mouse mu opioid receptor (HA-MOR HEK; ~2 pmol receptor/ mg membrane protein) were serum-starved for 30 minutes at 37oC. Naloxone (10µM) was added during serum starvation where indicated. Cells were incubated with drug at the concentrations and times indicated in the figures and figure legends. Cells were washed twice with phosphate buffered saline (PBS) on ice and collected in lysis buffer (20 mM Tris HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1%

NP40, 0.25% deoxycholate, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM

NaF, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN)).

Lysates were homogenized by passing through a 28.5 gauge needle twice and spun at 20,000 x g for 30 minutes at 4oC. Protein were quantitated (Dc Protein

29

Assay kit, Biorad, Hercules, CA) and normalized with 4X sample buffer (Biorad,

Hercules, CA) (62.5mM Tris-HCl, pH6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue) containing 5% β-mercaptoethanol. 20µg of proteins were run on 10% Bis-Tris gels (Biorad or Invitrogen, Carlsbad, CA) and transferred to

PVDF membranes (Immobilon-P, Millipore, Bedford, MA). Blots were blocked with 5% non-fat milk in Tris buffered saline with 0.1% Tween-20 (TBST) and immuno-blotted for total ERK1/2 (p44/42 MAP kinase antibody, 1:1000 in 5%

BSA in TBST, Cell Signaling Technology, Danvers, MA). Blots were washed in

TBST and incubated with HRP-conjugated donkey anti-rabbit secondary antibody

(GE Healthcare, Buckinghamshire, UK) for 1 hour at room temperature.

Chemiluminescence was detected using the Kodak 2000R imaging system

(Eastman Kodak Company, Rochester, NY). Blots were stripped and re-probed for phospho-ERK1/2 (p-ERK (E-4), sc-7383, Santa Cruz Biotechnology, Inc.,

Santa Cruz, CA). Densitometry was assessed using the Kodak imaging software. Phospho-ERK1/2 levels were normalized to the total ERK1/2 levels

(normalized values equal P-ERK divided by T-ERK). Stimulation over vehicle treated was determined for each blot using GraphPad Prism software (GraphPad

Software, Inc., San Diego, CA). Wildtype mouse embryonic fibroblasts (WT

MEF) and MEFs lacking endogenous βarrestin 1 and 2 (βarr1/2-KO MEF)

(Kohout et al., 2001; Bohn et al., 2004b) were stably transduced with HA-MOR using MSCV retroviral expression system. ERK1/2 phosphorylation was

30 measured in MEFs as described above. ERK1/2 activation studies were performed in parallel with imaging experiments to ensure activity of all agonists.

MOR Phosphorylation. HEK cells stably expressing the HA-MOR were serum starved for 15 minutes, followed by a 10 minute treatment with saline, DAMGO

(1μM), morphine (10μM), or herkinorin (10μM). Cells transfected with empty vector (mock) and treated with DAMGO (1μM) are included as a control. Cells were washed twice with PBS on ice and collected in lysis buffer (same as ERK lysis buffer). Lysates were homogenized on a rotator for 1 hour at 4oC and cleared at 20,000 x g for 30 minutes at 4oC. Equal amounts of protein (700-

1000µg) were added to 70µl of anti-HA agarose conjugate (Sigma, St. Louis,

MO) and incubated overnight on a rotator at 4oC. Buffer without protein was added to HA agarose for no protein control. Precipitate was washed and proteins were eluted in 30µl 4X XT sample buffer (Biorad) at 95oC for 4 minutes.

Proteins were resolved on 10% Bis-Tris XT Precast Gels (Biorad) and transferred to PVDF membranes. Blots were blocked with 5% milk in TBST, washed with

TBST, and incubated with an antibody specific for the MOR phosphorylated at

Ser375 ((Phospho-MOR (Ser375), 1:500 in 5% BSA in TBST, Cell Signaling

Technology, Danvers, MA) overnight at 4oC. Blots were washed in TBST and incubated with HRP-conjugated donkey anti-rabbit secondary antibody (GE

Healthcare, Buckinghamshire, UK) for 1 hour at room temperature.

Chemiluminescence was detected using the Kodak 2000R imaging system

31

(Eastman Kodak Company, Rochester, NY). Blots were stripped and re-probed with an antibody against the C-terminus of the MOR (1:1000 in 5% milk in TBST,

Sigma, St. Louis, MO). Densitometry was assessed using the Kodak imaging software. Phospho-MOR levels were normalized to the total receptor per lane

(normalized values equal P-MOR divided by T-MOR). Stimulation over vehicle treated was determined for each blot using GraphPad Prism software (GraphPad

Software, Inc., San Diego, CA).

Cross-linking and Co-Immunoprecipitation. These studies were based on methods described by Shenoy et al. [76] and Gesty-Palmer et al. [82] with some modification. HEK stably expressing the HA-MOR were washed with PBS + 10 mM HEPES and then incubated in PBS-HEPES buffer for 20 minutes at 37°C.

Cells were then treated with vehicle (0.1% DMSO), DAMGO (1µM), or herkinorin

(10μM) for 5 minutes. The membrane permeable cross-linking reagent, dithiobis[succinimidylpropionate] (DSP, Pierce, Rockford, IL) was prepared in

DMSO and administered drop-wise to the plates (2mM final DSP concentration, at <10% DMSO). Plates were incubated at room temperature with constant agitation for 20 minutes. The cross-linking reaction was stopped by the addition of 1 M Tris-HCl pH 7.4 to give a final concentration of 50 mM Tris. Cells generally came off of the plates, so they were collected and centrifuged 2000 rpm and then washed 4X in TBS. Cells were resuspended in lysis buffer (50mM

Tris HCl pH 7.4, 150mM NaCl, 5mM EDTA, 1% NP40, 1mM sodium

32 orthovanadate, 1mM PMSF, 1mM NaF, and protease inhibitor cocktail (Roche) and solubilized overnight at 4°C with rotation. Lysates were cleared centrifugation at 12000 rpm and incubated with anti-HA agarose conjugate

(Sigma) for 2 hours at 4°C with rotation. Precipitate was washed and proteins were eluted with 30µl 4X XT sample buffer at 100oC for 10 minutes. Proteins were resolved on 10% Bis-Tris XT Precast Gels (Biorad) and transferred to

PVDF membranes. Blots were blocked with 5% milk in TBST, washed with

TBST, and incubated with an antibody against βarrestins (A1CT, 1:1000 in 5% milk in TBST) (Shenoy et al. 2006, Gesty-Palmer et al. 2006) overnight at 4oC.

A1CT was generously provided by Dr. Robert Lefkowitz (Duke University). Blots were washed in TBST and incubated with HRP-conjugated donkey anti-rabbit secondary antibody (GE Healthcare, Buckinghamshire, UK) for 1 hour at room temperature. Chemiluminescence was detected using the Kodak 2000R imaging system (Eastman Kodak Company, Rochester, NY). Blots were stripped and re- probed with an antibody against the C-terminus of the MOR (1:1000 in 5% milk in

TBST, Sigma, St. Louis, MO). Densitometry was assessed using the Kodak imaging software. βArrestin levels were normalized to the total receptor per lane.

Stimulation over vehicle treated was determined for each blot using GraphPad

Prism software (GraphPad Software, Inc., San Diego, CA). WT and βarr1/2-KO

MEF lysates are included as controls for A1CT antibody (Kohout et al. 2001).

33

βArrestin Translocation. HEK-293 cells were transiently transfected with HA-

MOR (10µg) and βarr2-GFP (2µg) using electroporation. For some experiments,

HA-MOR HEK stable lines were transiently transfected with βarr2-GFP, but no differences in responses were observed from transiently transfected cells. Cells were serum-starved for 10-20 minutes with MEM without phenol red. Basal images were taken using an Olympus confocal microscope. Drugs were added and GFP localization was monitored for up to 30 minutes. Translocation of

βarr2-GFP to the plasma membrane was first observed at 60 to 90 seconds after drug and was maximally robust by 5 minutes. Cells were observed to 30 minutes to make sure that drugs which did not promote translocation did not simply have a delayed response. Several cells were imaged from each plate, and the at least four transfections were performed for each condition. Where indicated, GRK2

(5µg) was included in the transfection.

MOR-YFP Trafficking. HEK-293 cells were transiently transfected with MOR-

YFP (2µg) using electroporation. Cells were serum-starved for 30 minutes with

MEM without phenol red. Basal images were taken using an Olympus confocal microscope. Drugs were added and cells were incubated at 37oC for two hours.

Images were taken every 30 minutes during the two hours drug treatment.

Several cells were imaged from each plate, and at least four transfections were performed for each condition. Where indicated, GRK2 (5µg) was included in the

34 transfection. Where indicated, mouse MOR-1D-GFP (2µg) was used instead of

MOR-YFP.

Immunofluorescence of Surface MORs. HA-MOR HEK stable line was plated into a collagen coated 96-well optical plate. Cells were serum-starved for 30 minutes and treated with DMAGO (1µM) or herkinorin (10µM) for up to 2 hours.

Cells were washed 2X with MEM, fixed with 4% paraformaldehyde (PFA) at room temperature for 20 minutes, and blocked in MEM plus 5% goat serum. Cells were then incubated with an anti-HA antibody (monoclonal 12CA5 clone, 1:500,

Roche) in blocking buffer overnight at 4oC. Cells were washed 3X in blocking buffer and incubated with a goat anti-mouse IgG AlexaFluor488 conjugate secondary antibody (1:1000, Molecular Probes/Invitrogen) in blocking buffer for 1 hour at room temperature. Cells were washed 3X in PBS, and fluorescence was measured using a Fusion plate reader (Perkin Elmer Life and Analytical

Sciences, Boston, MA). Data are normalized to cells in which no agonists were added (0 minutes time point). Nonspecific signal (wells without primary antibody) was subtracted from each data point. Cells were viewed and imaged with a 10X objective after each experiment to ensure that cells had not washed off of the plate (Figure 2.5.C). If cells did wash off, that particular well measurement was not used in analysis. Cells were also treated in parallel on confocal imaging plates, and imaged with a 40X objective. All images were taken with an Olympus confocal microscope.

35

Biotinylated MOR internalization. HA-MOR HEK cells were serum starved

(30min., 37oC) and surface proteins were labeled with suflo-NHS-SS-biotin

(600µg/µl, Pierce, Rockford, IL) for 30 minutes at 4oC. Cells came off plates and were collected, combined, and pelletted by centrifugation at 2000rpm for 2 minutes. Cells were washed with TBS (50mM Tris HCl pH7.4, 150mM NaCl) to quench un-reacted biotin and spun as above. Cells were then divided equally into tubes for drug treatment. Cells were resuspended in MEM containing vehicle (0.1% DMSO), DAMGO (1M), or herkinorin (10µM) and incubated for 1 hour in a 37oC water bath. Remaining surface biotin was then stripped with glutathione stripping buffer (50mM L-glutathione, 75mM NaCl, 75mM NaOH,

10% FBS, in H2O) for 15 minutes on ice. Glutathione was then quenched with iodoacetamide buffer (50mM iodoacetamide, 1% BSA in PBS) for 15 minutes on ice. Cells were washed 2X in PBS, resuspended in extraction buffer (10mM Tris

HCl pH7.4), 120mM NaCl , 25mM KCl, 1mM PMSF, protease inhibitor cocktail

(Roche), 1 mg/mL iodoacetamide, 0.5% Triton X-100), and solubilized for 2 hours at 4oC with rotation. Lysates were cleared at 12,000 x g for 30 minutes at 4oC and equal amounts of protein (700-1000µg) were added to 40µl of Avidin agarose beads (Pierce) overnight at 4oC with rotation to precipitate biotinylated proteins. Precipitate was washed 3X and proteins were eluted as in co- immunoprecipitation experiments. Proteins were resolved and transferred to

PVDF membranes (as above). Blots were blocked with 5% milk in TBST and

36 incubated with an antibody against the C-terminus of the MOR (1:1000 in 5% milk in TBST, Neuromics, Edina, MN) overnight at 4oC. Blots were washed in

TBST and incubated with HRP-conjugated donkey anti-rabbit secondary antibody

(GE Healthcare, Buckinghamshire, UK) for 1 hour at room temperature.

Chemiluminescence was detected using the Kodak 2000R imaging system

(Eastman Kodak Company, Rochester, NY). Densitometry was assessed using the Kodak imaging software. Stimulation over vehicle was determined for each blot using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA).

Several controls are included: “100%”-biotinylated cells without treatment or stripping, “Strip”-biotinylated cells stripped directly after un-reacted biotin was quenched, “Mock”-HEK cells transfected with empty vector and treated with

DAMGO, “No Prot”-buffer only was added to Avidin beads.

2.3 Results

The structure of herkinorin is based on the natural product, salvinorin A

(Figure 2.1). Our collaborators have shown that, in contrast to salvinorin A, herkinorin has greater affinity at the MOR than the KOR and very low affinity at the DOR [103] (Table 2.1). Further, herkinorin is fully efficacious at promoting

GTP coupling to the MOR [103] (Table 2.2) and inhibiting forskolin-stimulated cAMP accumulation [102]. Finally, herkinorin is almost 3 fold more potent at promoting GTP coupling to the MOR than the KOR [103]. Collectively, these

37 data indicate that herkinorin selectively binds to and is a fully efficacious agonist at the MOR.

Activation of the MOR induces phosphorylation of ERK1/2 downstream of

G protein activation in vitro [90,126-127,150]. Therefore, to further assess herkinorin agonist activity in parallel with the receptor regulation studies, we evaluated the phosphorylation state of ERK1/2 as an indicator of receptor activation. Similar to the prototypical MOR agonist DAMGO, herkinorin promotes significant ERK1/2 phosphorylation by 2 minutes that lasts to at least 30 minutes post-treatment in HA-MOR HEK cells (Figure 2.2B). Further, herkinorin dose- dependently induces ERK1/2 phosphorylation (Figure 2.2A), which can be blocked by pretreatment with the , naloxone (Figure 2.).

Many GPCRs have been shown to utilize βarrestins for the activation of ERK1/2

[73,80,82,84]. To test whether DAMGO, morphine, and herkinorin can induce

ERK1/2 phosphorylation in the absence of βarrestins, we used mouse embryonic fibroblasts that lack expression of both endogenous βarrestins (βarr1/2-KO

MEFs). DAMGO, morphine, and herkinorin were able to activate ERK1/2 in the

WT MEFs as well as in the βarr1/2-KO MEFs, suggesting that MOR-mediated

ERK1/2 activation with these agonists does not require βarrestins (Figure 2.2D).

Agonist activation of the MOR can induce phosphorylation of the receptor, which can affect its interactions with βarrestins. Phosphorylation of the MOR at

Ser375 is of particular interest, because it has been shown to be important for

MOR trafficking [93] and desensitization [92] in an agonist dependent manner.

38

Therefore, we assessed the ability of herkinorin to promote MOR phosphorylation at Ser375. DAMGO, morphine, and herkinorin all induce MOR phosphorylation at Ser375 (Figure 2.3). Morphine and herkinorin, however, induce significantly less phosphorylation than DAMGO at this particular residue.

Agonist-induced phosphorylation of the MOR increases its affinity for

βarrestins [35,90]. Therefore, since herkinorin induces weak MOR phosphorylation, we asked whether herkinorin could also promote βarrestin2 recruitment. Agonist-induced βarrestin2 recruitment was assessed by confocal microscopy in HEK-293 cells transiently transfected with the HA-MOR and βarr2-

GFP [48]. In the basal state, βarr2-GFP is localized to the cytosol of the cells

(Figure 2.4A and 2.8A). DAMGO treatment results in the accumulation of green puncta around the plasma membrane, indicative of the recruitment of βarr2-GFP to activated MORs. This effect can be observed as early as 30 to 60 seconds after DAMGO treatment, peaks at about 5 minutes, and can be observed to at least 20 minutes post-treatment. In contrast, both morphine and herkinorin are unable to induce detectable βarr2-GFP translocation to the plasma membrane.

To rule out the possibility that the time course was changed for morphine and herkinorin, cells were monitored every 30 seconds for the first ten minutes after treatment and approximately every 15 minutes for up to 2 hours. Translocation was not observed following treatment with morphine or herkinorin at any time point, which for morphine is in agreement with previous reports [34-35]. In addition, herkinorin failed to promote βarrrestin1 recruitment (data not shown).

39

To test whether herkinorin could promote interactions with endogenous

βarrestins and to ensure that the GFP tag was not interfering with βarrestin recruitment to the MOR, HA-MOR complexes were stabilized with a membrane permeable cross-linking reagent (DSP), after a 5 minute drug treatment.

Complexes were then immunoprecipitated and βarrestin interactions were determined by western blot analysis. Consistent with confocal studies, DAMGO treatment leads to an increase in the amount βarrestin co-immunoprecipitated with the MOR compared to vehicle (Figure 2.4B). In contrast, herkinorin treatment did not increase the amount of βarrestin co-immunoprecipitated over vehicle treated cells.

Phosphorylation of the MOR increases its affinity for βarrestins. Since herkinorin induced weak MOR phosphorylation and over-expression of GRK2 in

HEK cells has been shown to increase morphine-induced MOR phosphorylation

[92] and morphine-induced βarr2-GFP translocation [34-35], we asked whether over-expression of GRK2 could facilitate herkinorin-induced βarr2-GFP recruitment. HEK-293 cells were transiently transfected with HA-MOR, βarr2-

GFP, and GRK2. Similar to what has been observed by others [35], morphine promotes βarr2-GFP translocation in HEK cells only when GRK2 is over- expressed (Figure 2.8A). Even with over-expression of this receptor kinase, however, herkinorin was unable to promote detectable βarr2-GFP, even at doses as high as 100µM (Figure 2.4C and 2.8A). Morphine treatment promoted βarr2-

GFP recruitment in the same cells that failed to do so with herkinorin, suggesting

40 that the cells were over-expressing GRK2, since morphine does not promote detectable translocation in this cell line otherwise (Figure 2.4A). Further, the combination of morphine and herkinorin in cells not over-expressing GRK2 does not induce translocation (data not shown).

βArrestins promote receptor internalization through the endocytic pathway.

Therefore, we tested whether the inability of herkinorin to recruit βarrestins would lead to impaired receptor trafficking. HEK-293 cells were transiently transfected with MOR-YFP, and agonist-induced receptor trafficking in live cells was observed every 30 minutes for 2 hours using confocal microscopy (Figure 2.5A).

In the absence of agonist, MOR-YFP was localized to the plasma membrane of the cells. DAMGO treatment causes a decrease in the membrane fluorescence and the appearance of intracellular vesicles, which is indicative of receptor internalization [49]. DAMGO-induced internalization begins about 20 minutes post-treatment and persists for the 2 hours in which the cells were observed. In contrast, neither morphine nor herkinorin are able to promote MOR-YFP internalization in these cells. At all time points examined after morphine or herkinorin treatment, the MOR-YFP remained localized to the plasma membrane.

In addition to confocal microscopy, we also used two quantitative approaches to measure agonist-induced internalization of the MOR. First, we utilized immunocytochemistry to quantitate the amount of receptors remaining on the membranes of cells following drug treatment. HA-MOR HEK cells were treated with DAMGO or herkinorin for various times, followed by fixation without

41 permeabilization, and surface receptors were labeled with the anti-HA

AlexaFluor488 conjugate. Quantification of immunofluorescence and corresponding confocal images show that DAMGO treatment leads to a time dependent decrease in surface fluorescence, indicating that MORs have been internalized (Figure 2.5B and C). In contrast, herkinorin treatment does not cause a decrease in surface fluorescence over the 2 hour treatment period, suggesting that herkinorin does not promote MOR internalization.

In addition to immunocytochemistry, we also used a cell surface biotinylation assay to quantitate agonist-induced internalization (Figure 2.5D).

Surface proteins of HA-MOR HEK cells were labeled with biotin prior to drug treatment. After 1 hour in the presence of vehicle or agonist, the remaining surface biotin was stripped away. The remaining biotinylated proteins were precipitated with avidin and internalized MORs were analyzed by western blot.

DAMGO treatment results in a significant increase in internalized receptors. In contrast, herkinorin treatment does not result in receptor internalization compared to vehicle.

In addition to augmenting morphine-induced MOR phosphorylation and

βarrestin recruitment, over-expression of GRK2 promotes morphine-induced

MOR-YFP internalization [92] (Figure 2.6A). In contrast to morphine, however, over-expression of GRK2 is not sufficient to promote herkinorin-induced MOR internalization.

42

The C-terminal tail of GPCRs is important for agonist-induced phosphorylation and internalization [151-153]. A naturally occurring splice variant of the MOR, termed MOR-1D, differs from the wildtype MOR only in the number of potential phosphorylation sites within the C-terminal tail. This variant has been shown to promote robust MOR phosphorylation and internalization in response to morphine treatment in HEK-293 cells in the absence of GRK2 over- expression [152]. We also demonstrate that this variant can internalize with morphine treatment in transiently transfected HEK cells (Figure 2.6B). Unlike morphine, however, herkinorin is still unable to promote internalization of this

MOR variant. These data suggest that even in systems that are biased toward

βarrestin2 recruitment and MOR internalization, the herkinorin-bound receptor is still resistant to βarrestin2 mediated regulation of the MOR.

In an effort to improve the solubility and stability of herkinorin, several derivatives of herkinorin were generated (Figure 2.1) and evaluated for opioid receptor binding and activation by our collaborators (Table 2.1 and 2.2) [101].

Three of these derivatives have a substituent added to the 3-position of the benzene ring of herkinorin. The fourth derivative, herkamide, has an amide linkage, instead of the ester linkage of herkinorin. All four derivatives show affinity and selectivity for the MOR (Table 2.1) [101]. The methoxy derivative shows less affinity at the MOR than herkinorin, but maintains MOR selectivity.

The bromo derivative and herkamide show increased affinity and selectivity at the MOR compared to herkinorin, while the nitro derivative shows less affinity

43 and selectivity at the MOR. Functional studies in MOR-expressing CHO cells demonstrate that the methoxy, bromo, and nitro derivatives are less potent and less efficacious than herkinorin (Table 2.2). Both the methoxy and bromo derivatives, however, are full agonists compared to DAMGO, while the nitro derivative is a partial agonist. Finally, herkamide is more potent than herkinorin and maintains full efficacy. These data demonstrate that all four derivatives are selective MOR agonists.

Similar to the studies previously described, we evaluated the phosphorylation state of ERK1/2 in parallel with translocation and trafficking studies as an indicator of MOR activation. We show that all four herkinorin derivatives promote a dose-dependent increase in ERK1/2 phosphorylation

(Figure 2.7A). In all cases, agonist-induced ERK1/2 phosphorylation can be blocked by pretreatment with the opioid receptor antagonist, naloxone, and is absent in mock transfected cells, suggesting that ERK1/2 phosphorylation is indeed mediated by the MOR. Direct comparison shows that all derivatives can promote ERK1/2 phosphorylation to the same extent as DAMGO and herkinorin

(Figure 2.7B).

We then asked whether the herkinorin derivatives retained herkinorin’s inability to recruit βarr2-GFP to the MOR. HEK-293 cells were transiently transfected with HA-MOR and βarr2-GFP, and agonist-induced recruitment was assessed by confocal microscopy. Consistent with our previous studies,

DAMGO induces robust βarr2-GFP translocation to the plasma membrane in

44

HEK-293 cells transiently transfected with HA-MOR (Figure 2.8A), while morphine promotes detectable βarr2-GFP translocation only in the presence of

GRK2 over-expression. Again, herkinorin is unable to promote detectable translocation even with GRK2 over-expression. Like herkinorin, the methoxy, bromo, and nitro derivatives are unable to promote detectable βarr2-GFP translocation in the GRK2 over-expressing cells (Figure 2.8B). Somewhat surprisingly, herkamide promotes robust βarr2-GFP translocation in the presence or absence of GRK2 over-expression.

We then assessed whether the βarrestin recruitment profile of the herkinorin derivatives would correlate with each agonist’s ability to promote MOR trafficking using HEK-293 cells transiently transfected with MOR-YFP. Controls are shown in Figure 2.9A, and internalization profiles correlate with βarrestin recruitment profiles. Like herkinorin, the methoxy, bromo, and nitro derivatives are unable to promote MOR-YFP internalization, even with GRK2 over- expression (Figure 2.9B). In correlation with its ability to promote robust

βarrestin2 translocation, herkamide promotes robust MOR-YFP internalization under both conditions.

2.5 Discussion

The main finding in this chapter is that herkinorin and several of its derivatives are MOR agonists, yet they do not promote βarrestin interactions or

45

MOR trafficking, even in the presence of GRK2 over-expression. Therefore, these compounds represent a novel class of MOR biased agonists, because they promote activation of certain signaling pathways (G protein coupling and ERK1/2 activation), while they do not promote other pathways (βarrestin recruitment and internalization). Genetic deletion or knockdown of βarrestin2 in vivo results in enhanced antinociceptive responses and attenuated antinociceptive tolerance and other side effects induced by morphine [131,139,154]. Therefore, pharmacological disruption of MOR/βarrestin interactions by using agonists such as herkinorin and three of its derivatives, which can activate the MOR without promoting MOR/βarrestin interactions, may be therapeutically beneficial.

Phosphorylation of the MOR has been shown to play a very important role in βarrestin recruitment, receptor trafficking, and desensitization of G protein signaling. Moreover, the phosphorylation state of the MOR can dictate agonist- specific regulation [59,89,92-94,107,116]. Specifically, phosphorylation at

Ser375 is of particular interest, because it has been shown to be important for several parameters of MOR regulation. First, mutation of Ser375 to alanine completely abolishes morphine-induced MOR internalization [92-93]. Second, phosphorylation at this residue has been shown to be required for morphine induced MOR desensitization [92]. Interestingly, DAMGO has been shown to promote phosphorylation of additional residues of the MOR, and Ser375 is only partially responsible for DAMGO-induced internalization and desensitization of the MOR. These studies demonstrate that while Ser375 phosphorylation is

46 required for morphine-induced MOR regulation, additional phosphorylation sites can also promote agonist-induced MOR regulation [89,93-94]. Further, our studies suggest that phosphorylation at Ser375 is not sufficient to promote MOR internalization, as herkinorin promotes phosphorylation of this residue but does not promote MOR internalization (Figure 2.3, 2.5, and 2.6).

In HEK-293 cells stably expressing the MOR, both morphine and herkinorin promote MOR phosphorylation at Ser375 over vehicle, but much less robust than DAMGO (Figure 2.3). This suggests that DAMGO stabilizes the

MOR in a conformation that is highly amenable to phosphorylation by kinases, such as GRKs. In contrast, morphine and herkinorin may stabilize different conformations of the MOR that are poor substrates for GRKs or other kinases.

GRK2 over-expression, however, increases morphine-induced phosphorylation of Ser375 [92] and promotes robust interactions with arr2 [34-35], and thus receptor trafficking is also enhanced. In contrast, over-expression of GRK2 does not promote herkinorin-induced βarrestin2 translocation or enhance MOR trafficking. One possible explanation is that, unlike morphine, over-expression of

GRK2 does not enhance the ability of herkinorin to promote MOR phosphorylation; however, we did not test this directly. Therefore, the potential of an agonist to promote phosphorylation at this residue may have important implications on MOR regulation, in that agonists that do not promote robust phosphorylation at this residue may elude arr2-mediated regulation.

47

In addition to GRK2 other kinases (GRKs, CamKII, PKC, PKA, and

ERK1/2) have been shown to regulate the MOR [35,92,94-100]. Moreover, at least 12 serines and threonines in the C-terminal tail of the MOR may be subject to phosphorylation and pattern of phosphorylation may be influenced by the agonist used [89,92-93]. However, we have not evaluated whether other residues are phosphorylated following herkinorin treatment or whether kinases other than GRK2 may be involved.

We have used ERK1/2 phosphorylation as an indicator of MOR activation in parallel with trafficking studies. We observed that herkinorin promotes a dose- and time dependent increase in phosphorylated ERK1/2 that can be blocked by naloxone (Figure 2.2A, B, and C). These data are consistent with the results of

GTP coupling studies (Table 2.2) [101-103], suggesting herkinorin and its four derivatives are agonists at the MOR. We also demonstrate that βarrestins are not required to mediate ERK1/2 phosphorylation, since DAMGO, morphine, and herkinorin promote this effect in cells lacking expression of both βarrestins

(Figure 2.2D). These data suggest that MOR-mediated ERK1/2 activation by these compounds is mediated through the G protein pathway.

Herkinorin is a full agonist at the MOR as assessed by GTP coupling [101-

103], adenylyl cyclase inhibition [102], and ERK1/2 activation [90], yet it does not recruit βarrestins or internalize the MOR. A fully efficacious MOR agonist that does not promote βarrestin interactions, such as herkinorin, may have unique therapeutic benefits. For instance, βarr2-KO mice show enhanced morphine-

48 induced antinociception with significantly attenuated tolerance, physical dependence, constipation and respiratory suppression [123,131,133,139,144].

Therefore, MOR agonists that do not promote βarrestin-mediated regulation may promote desired effects (i.e. analgesia), while reducing unwanted side effects

(i.e. constipation and respiratory suppression) in wildtype mice. Importantly, however, βarr2-KO mice display enhanced morphine-induced reward [132], suggesting that an agonist that does not induce βarrestin2 regulation of the MOR may also be more prone to abuse.

While the unique properties of herkinorin may be promising in development of novel analgesics, the results presented here show the effects of herkinorin in cultured cells over-expressing the HA-MOR and must be interpreted with caution. The cellular environment in which the MOR is expressed dictates how an agonist promotes regulation and signaling. For example, in HEK cells, we can modulate morphine regulation of the MOR simply by over-expressing

GRK2. Further, morphine has been shown to promote different MOR trafficking patterns in different neuronal populations, as well as in different regions within the same neuron [113-114]. Therefore, herkinorin-induced regulation of the

MOR may differ in endogenous tissues. For instance, different cellular kinases

(i.e. other GRKs, PKC, PKA) may be more abundant and/or active in physiologically relevant tissues and may interact with the herkinorin-bound MOR.

Therefore, herkinorin-induced activation and regulation of the MOR should be evaluated in vivo. Studies are ongoing in our lab to measure herkinorin-induced

49 analgesia in WT and βarr2-KO mice. Since herkinorin does not promote interactions between the MOR and βarrestin2, we would expect to observe no difference in antinociceptive responses between the genotypes. Further, we would expect herkinorin to promote less constipation, respiratory suppression, and physical dependence than morphine in WT mice, due to its inability to promote βarr2/MOR interactions.

Despite the promising characteristics of herkinorin, several factors limit its potential as a therapeutically useful compound. Though the parent compound salvinorin A is thought to be the most potent, naturally occurring known, it is very short acting [145]. In fact the hallucinogenic effects can wear off in as little as a few minutes. Therefore, herkinorin activity in vivo may also be very brief. We have observed that, while herkinorin can produce antinociception in mice when administered locally, no activity is observed following systemic injection. This may be due to rapid metabolism or degradation by serum esterases. Furthermore, herkinorin is very insoluble in aqueous vehicles, which also confounds systemic administration. Finally, although herkinorin is selective for the MOR, it retains modest affinity (Table 2.1) and full efficacy (Table 2.2) at the KOR. Therefore, herkinorin may produce unwanted KOR-mediated effects, such as hallucinations.

In order to improve upon the selectivity, stability, and solubility of herkinorin, several derivatives of herkinorin were synthesized (Figure 2.1). The four derivatives studied in this dissertation all were MOR agonists that display

50 different selectivity at the MOR compared to herkinorin (Table 2.1 and Table 2.2,

Figure 2.7). These data suggest that substituents at the 3-position of the benzene ring on herkinorin can influence MOR binding and functional potency and efficacy. Modifications at this position, however, do not seem to interfere with the unique MOR regulation induced by herkinorin. Interestingly, herkamide promotes very robust βarrestin recruitment and MOR trafficking. The amide linkage in herkamide may result in a different orientation of the benzene ring compared to herkinorin, such that the MOR assumes a conformation that is amenable to βarrestin mediated regulation.

In conclusion, we have characterized MOR agonists that do not induce

βarrestin recruitment or MOR trafficking. Taken together with our previous in vivo studies, MOR agonists, such as herkinorin, that do not promote βarrestin mediated regulation of the MOR, may prove to be useful analgesics with reduced occurrence of unwanted side effects. Further synthesis of ligands based on the structure of herkinorin and its derivatives is needed to improve upon the physical limitations of these compounds. More globally, we have shown that one can selectively activate certain pathways while not promoting activation of other pathways at the MOR, demonstrating functional selectivity at this receptor.

51

2.6 Tables and Figures

Herkinorin R = H R Methoxy R = OCH3 Bromo R = Br Nitro R = NO2 Herkamide O = N; R=H

Herkinorin R = H R Methoxy R = OCH3 Bromo R = Br Nitro R = NO2 Herkamide O = N; R=H

Figure 2.1. Chemical structures of salvinorin A, herkinorin and four herkinorin derivatives. A benzene ring was added to salvinorin A to make herkinorin. A Methoxy-, Bromo-, or Nitro- group has been added to the para- position of the benzoyl group of herkinorin. For herkamide, the ester linkage (box) of herkinorin has been replaced with an amide linkage. Adapted from Groer et al. Mol Pharm. 2007 and Tidgewell, Groer et al. J Med Chem. 2008 [90,101].

52

Ki ± SD, nM Selectivity µ   µ/ Salvinorin A >1000 5790 ± 980 1.9 ± 0.2 >525 Herkinorin 12 ± 1 1170 ± 60 90 ± 2 0.13 Herkinorin derivatives Methoxy- 70 ± 4 1860 ± 140 540 ± 40 0.13 Bromo- 10 ± 1 1410 ± 80 740 ± 40 0.01 Nitro- 260 ± 210 >10000 570 ± 40 0.46 Herkamide 3.1 ± 0.4 810 ± 30 7430 ± 880 0.0004

Table 2.1. Binding affinities at opioid receptors using [125I]OXY as radioligand. Herkinorin and four derivatives show selectivity for the µ opioid receptor. Harding et al. J Med Chem. 2005; Tidgewell, Groer et al. J Med Chem. 2008 [101,103]. These studies were performed by the Rothman laboratory and are presented here for reference.

53

EMAX EC50 ± SD, nM (% stimulation) DAMGO 40 ± 4 100 ± 4 Herkinorin 500 ± 140 130 ± 4 Herkinorin derivatives Methoxy- 830 ± 100 94 ± 3* Bromo- 4890 ± 980 108 ± 8* Nitro- 1370 ± 230 46 ± 2* Herkamide- 360 ± 60 134 ± 5

Table 2.2. [35S]-GTPγS coupling in CHO cells stably expressing µ opioid receptors. Emax is percent stimulation compared to DAMGO. * p<0.05 versus herkinorin. Tidgewell, Groer et al. J Med Chem. 2008 [101]. These studies were performed by the Rothman laboratory and are presented here for reference.

54

A. Herkinorin P-ERK T-ERK

DAMGO P-ERK T-ERK 6 ** DAMGO * C. Herkinorin 4

2 P-ERK OverControl T-ERK

Fold PhosphorylationFold 0 0 10 100 500 1000 10000 [Drug], nM

B. Herkinorin P-ERK T-ERK DAMGO D. P-ERK T-ERK

7 DAMGO 6 P-ERK Herkinorin

5 * WT T-ERK

4 -

3 P-ERK KO

2 arr1/2 T-ERK  Fold IncreaseFold of 1 P-ERK1/2 Over Basal P-ERK1/2 0 0 10 20 30 Time (min)

Figure 2.2. Herkinorin promotes MOR-mediated ERK1/2 phosphorylation. HEK- 293 cells stably expressing HA-tagged MOR were serum starved and treated with drug. Cell lysates were analyzed by western blot. Phospho-ERK1/2 bands were normalized to total ERK bands. A. Cells were treated with DAMGO or herkinorin for 10 minutes at the concentrations indicated. Densitometric analysis reveals significant differences between the dose-response of DAMGO and herkinorin (n=2-6) (two-way ANOVA: drug p<0.01, dose p<0.0001, interaction p<0.01; * p< 0.05, ** p<0.01vs. DAMGO Bonferroni post-hoc analysis; n=3-6). Mean ± S.E.M. is shown. B. Cells were treated with DAMGO (1µM) or herkinorin (10µM) for the indicated times. Representative immunoblots are shown. (continued) 55

(Figure 2.2 continued) Densitometric analysis reveals a difference between maximally efficacious doses of DAMGO and herkinorin (n=3-5) (two-way ANOVA: drug p<0.0001, time p<0.0001, interaction p>0.05; * p<0.05 vs. DAMGO at 10 minutes Bonferroni post-hoc analysis; n=3-6) as assessed by two-way ANOVA ( p < 0.05) and a significant difference between the compounds at the 10-min point (* p < 0.01 Student's t test). Mean ± S.E.M. is shown. C. Pre-treatment with 10µM naloxone during serum starve prevents 1µM herkinorin-induced ERK1/2 activation. D. WT and βarr1/2-KO MEFs stably expressing HA-MOR were treated with drug for 10 minutes. DAMGO, morphine, and herkinorin (all 1µM) activate ERK1/2 in both cell lines. Adapted from Groer et al. Mol Pharm. 2007 [90].

56

P-MOR 75

T-MOR 75 8 IP: HA-MOR ** 6

4 ## # *** *

2 (Ser375)over Vehicle

Fold IncreaseFold P-MOR of 0

Vehicle DAMGO Morphine Herkinorin

Figure 2.3. Agonist induced MOR phosphorylation at Ser375. HEK-293 cells stably expressing HA-MOR were treated with saline, DAMGO (1 µM), morphine (10 µM), or herkinorin (10 µM) for 10 min. The receptor was immunoprecipitated from cell lysates using an anti-HA antibody-agarose bead complex. Precipitated proteins were analyzed by western blot. Phospho-MOR (Ser375) bands were normalized to total MOR bands. Representative immunoblots are shown. Densitometric analyses of three experiments performed in duplicate or triplicate are shown as mean ± S.E.M. All drugs induce MOR phosphorylation at Ser375 (*** p<0.001, ** p<0.01, * p<0.05 vs saline; student’s t test). Morphine and herkinorin induce less MOR phosphorylation than DAMGO (## p<0.01, # p<0.05 vs DAMGO; Student’s t test, n=4-6). Adapted from Groer et al. Mol Pharm. 2007 [90].

57

A. Basal DAMGO Morphine Herkinorin

Antibody IP: HA Controls ** B. 3

2

50 arr

 37 1

75 Vehicle of Fold

MOR 50 0 HEK-MOR lysate Veh DAM Herk Herkinorin C. Basal 2µM 100µM + Morphine

Figure 2.4. Agonist-induced βarrestin2 interactions with the MOR. A. HEK-293 cells were transiently transfected with the HA-MOR and βarr2-GFP. βarr2-GFP shows cytosolic localization in untreated cells. DAMGO (1µM) induces βarr2-GFP translocation to the membrane within 5 minutes (white arrow; puncta at membrane). In contrast, neither morphine (10µM, 10min) nor herkinorin (2µM, 10min) induces detectable βarr2- GFP translocation to the plasma membrane. B. HEK-293 cells stably expressing the HA-MOR were treated with vehicle (0.1%DMSO), DAMGO (1µM), or herkinorin (10µM) for 5 minutes. Proteins were cross-linked using a membrane-permeable cross-linking reagent (DSP), and the HA-MOR and βarrestins were co-immunoprecipitated using an (continued)

58

(Figure 2.4 continued) anti-HA antibody. Precipitates were analyzed by western blot. Representative blots are shown. “Mock” control refers to HEK cells transfected with empty vector, and “No Prot” control contained no lysates during immunoprecipitation. βArrestin antibody (A1CT) controls show lysates from WT and βarr1/2 KO MEFs, and MOR antibody control shows whole cell lysate from HEK cells stably expressing the HA-MOR. Densitometric analysis of two experiments and mean ± S.E.M. are shown. DAMGO treatment results in significant pulldown of βarrestin, while herkinorin does not (** p<0.01, student’s t test, n=3-4). C. HEK-293 cells were transiently transfected with HA-MOR, βarr2-GFP, and GRK2. Under these conditions, herkinorin (2µM at 10min or 100µM at 30min) does not induce βarr2-GFP translocation. In contrast, the same cells treated with morphine (10µM, 10min) display translocation, demonstrating that the cells do express the HA- MOR and GRK2. Adapted from Groer et al. Mol Pharm. 2007 [90].

59

A. Basal DAMGO Morphine Herkinorin

B. C. Basal DAMGO Herkinorin

100

75

(% Control) (% 10X 50 10 M Herkinorin

1 M DAMGO 40X Immunofluorescence 0 30 60 90 120 Time (min)

D.

2.0 IP: Avadin *** IB: MOR 1.5

1.0

0.5

Internalization, Fold OverFold Control 0.0

Vehicle DAMGO Herkinorin

Figure 2.5. Agonist-induced internalization of the MOR. A. HEK-293 cells were transiently transfected with the MOR tagged with YFP on the C-terminus (MOR-YFP). In untreated cells, MOR-YFP is primarily localized to the plasma membrane. (continued) 60

(Figure 2.5 continued) DAMGO (1µM, 30min) promotes MOR-YFP internalization, shown by the formation of intracellular vesicles (arrow) and the disappearance of membrane localization. Neither morphine (5µM, 60min) nor herkinorin (10µM, 120min) induce detectable MOR-YFP internalization. B. HEK-293 cells stably expressing the HA-MOR were treated with either DAMGO (1µM) or herkinorin (10µM) for the times indicated. Cells were fixed but not permeabilized. Surface expression was measured using a fluorescently labeled anti- HA antibody. Graph shows mean ± S.E.M. and reveals a difference between the two drugs (two-way ANOVA: drug p<0.0001, time p<0.001, interaction p<0.0001; Bonferroni post-hoc analysis: 15min p<0.01, all other time points p<0.001, n=12-14). C. Immunocytochemistry using fluorescently labeled anti-HA antibody shows surface HA- MOR expression before (basal) and after (60min) DAMGO (1µM) or herkinorin (10µM). Top, images were taken with a 10X objective from 96-well plates assayed in figure 2.5.B. Bottom, 40X images from parallel dishes. D. Agonist-induced HA-MOR internalization was quantified in HEK-293 cells stably expressing HA-MOR cell surface biotinylation assays. Surface proteins were labeled with biotin, followed by a 1 hour treatment with vehicle (0.1% DMSO), DAMGO (1µM), or herkinorin (10µM). Biotin remaining on the surface was stripped. Intracellular biotin was precipitated using avidin. Precipitate was analyzed by western for HA-MOR. Controls: protein biotinylation (“100%”), glutathione stripping (“Strip”), HEK with empty vector (“Mock”), and no lysate during avidin- precipitation (“No Prot”). Representative immunoblot is shown. Densitometric analysis of three experiments done in duplicate shows the mean  S.E.M. of internalized HA- MOR. DAMGO induces internalization of the HA-MOR over vehicle, but herkinorin does not (*** p<0.001 vs vehicle; Student’s t-test, n=5-6). Adapted from Groer et al. Mol Pharm. 2007 [90].

61

A. MOR-YFP + GRK2

Basal 30 min 60 min

Morphine Herkinorin

B. MOR1D-GFP

Basal 30 min 60 min Morphine

Basal 60 min 120 min Herkinorin

Figure 2.6. Agonist-induced MOR internalization under conditions in which morphine induces MOR internalization. A. HEK-393 cells were transiently transfected with MOR-YFP and GRK2. Under these conditions, morphine induces internalization of MOR-YFP (arrows, vesicles). In contrast, herkinorin is unable to induce MOR-YFP internalization. B. HEK-393 cells were transiently transfected with MOR1D-GFP. Morphine induces internalization of this MOR variant (arrows, vesicles), but herkinorin does not. Adapted from Groer et al. Mol Pharm. 2007 [90].

62

A. Herkinorin P-ERK T-ERK Methoxy P-ERK T-ERK P-ERK Bromo T-ERK P-ERK Nitro T-ERK Amide P-ERK T-ERK 0 0.01 0.1 0.5 1 10 10 10 [Drug] (µM) + - 10 µM Naloxone B. - + CHO cells w/o MOR

P-ERK

T-ERK 100 nM 8 1000 nM

6

4 *

OverVehicle 2

Fold PhosphorylationFold 0

Figure 2.7. Herkinorin and four derivatives induce MOR-mediated ERK1/2 phosphorylation. CHO cells stably expressing the human MOR were treated with the indicated drugs for 10 minutes. Cell lysates were analyzed by western blot. A. Shown are representative dose-response blots of at least three experiments. B. TOP: Representative immunoblots are shown. BOTTOM: Densitometric analysis of two experiments done in triplicate shows mean ± S.E.M. All treatments induce ERK1/2 phosphorylation (p<0.0001 vs vehicle for all treatments; student’s t test, n=5-13). The nitro derivative is less potent than all other treatments (* p<0.05 vs all other treatments; student’s t test, n=5-13). Adapted from Tidgewell, Groer et al. J Med Chem. 2008 [101].

63

A. Basal DAMGO Morphine Herkinorin + GRK 2 GRK +

B. Herkinorin Derivatives

Methoxy Bromo Nitro Amide + GRK 2 GRK +

Figure 2.8. Agonist-induced arrestin2-GFP translocation. HEK-293 cells were transiently transfected with HA-MOR and βarr2-GFP and with or without GRK2 over- expression. Cells were serum starved and treated with DAMGO (1µM) or other compounds (10µM). Live cells were monitored for up to 20 minutes. Representative images from at least three experiments are shown. A. DAMGO induces robust translocation of βarr2-GFP (arrows, green puncta) to the plasma membrane. Morphine, however, can only induce translocation when GRK2 is over-expressed. Herkinorin is unable to induce detectable βarr2-GFP translocation even in the presence of GRK2 over-expression. B. Three herkinorin derivatives (Methoxy, Bromo, and Nitro) are unable to induce detectable βarr2-GFP translocation even in the presence of GRK2 over-expression. Herkamide induces robust βarr2-GFP translocation to the plasma membrane under both conditions. Adapted from Tidgewell, Groer et al. J Med Chem. 2008 [101]. 64

A. Basal DAMGO Morphine Herkinorin + GRK 2 GRK +

B. Herkinorin Derivatives

Methoxy Bromo Nitro Amide + GRK 2 GRK +

Figure 2.9. Agonist-induced MOR-YFP internalization. HEK-293 cells were transiently transfected with MOR-YFP with or without GRK2 over-expression. Cells were serum starved and treated with DAMGO (1µM) or other compounds (10µM). Live cells were monitored for up to 2 hours. Representative images from at least three experiments are shown. A. DAMGO induces robust internalization of MOR-YFP (arrows, vesicles). Morphine, however, can only induce MOR-YFP internalization when GRK2 is over-expressed. Herkinorin is unable to induce detectable internalization even in the presence of GRK2 over-expression. B. Three herkinorin derivatives (Methoxy, Bromo, and Nitro) are unable to induce detectable MOR-YFP internalization even in the presence of GRK2 over-expression. Herkamide induces robust internalization under both conditions. Adapted from Tidgewell, Groer et al. J Med Chem. 2008 [101].

65

CHAPTER 3

AGONIST DIRECTED βARRESTIN INTERACTIONS DETERMINE

MU OPIOID RECEPTOR REGULATION

3.1 Introduction

Morphine and other opiates are among the most clinically useful analgesics, and their actions are mediated largely through the mu opioid receptor

(MOR). As a G protein-coupled receptor (GPCR), the MOR is subject to regulation paradigms that include GRK-mediated phosphorylation, βarrestin recruitment, and internalization [34-36]. Further, βarrestins have been shown to scaffold ubiquitination machinery such as E3 ligases to GPCRs, including the β2 adrenergic receptor (B2AR) and the chemokine receptor (CXCR4) [66-67], but this has not been demonstrated for the MOR.

βArrestin-mediated MOR regulation is determined by the agonist bound to the receptor. This was first recognized when Arden et al. [86] observed that while DAMGO promoted robust internalization of the MOR in HEK-293 cells, morphine treatment failed to do so. Subsequent studies demonstrated that the attenuated morphine-induced MOR internalization is a consequence of the

66 morphine-bound receptor being a poor substrate for phosphorylation and

βarrestin recruitment. In contrast, DAMGO induces robust MOR phosphorylation and βarrestin recruitment. These agonist-directed differences are an example of functional selectivity at the MOR, wherein both agonists promote MOR activation, but they differ in their abilities to promote βarrestin regulation of the MOR [34-

36,87,90,107-110,118,125].

The means by which an agonist can direct signaling and regulation, however, also depends on the complement of proteins expressed in residence with the receptor. A clear example of the cellular environment influencing MOR regulation is that GRK2 over-expression augments MOR phosphorylation,

βarrestin2 recruitment, and internalization in HEK-293 cells [35,90,92]. This is illustrated further in a study wherein morphine-induced MOR trafficking was observed in the dendrites of nucleus accumbens neurons, but not in the soma of the same neurons [113]. Since the relative expression of proteins may differ between these regions, it is attractive to postulate that the expression of accessory proteins (perhaps GRKs) may be sufficient to promote morphine- mediated MOR internalization.

Furthermore, substantial evidence suggests that βarrestins play a pivotal role in regulation of the MOR in vivo. In vivo studies using mice that lack

βarrestin2 (βarr2-KO) reveal a myriad of behavioral differences in response to morphine compared to their WT littermates, including enhanced thermal antinociception, reduced tolerance, reduced constipation, reduced signs of

67 withdrawal, enhanced dopamine release, and enhanced reward profiles

[34,123,131,133,139,144]. Therefore, it is highly likely that βarrestin2 plays a very important role in regulating the MOR in vivo. Interestingly, while morphine’s effects are dramatically altered in the βarr2-KO mice, several other agonists, such as fentanyl and methadone, promote similar antinociceptive profiles in both the WT and βarr2-KO mice [34,123,131,133,139,144]. The lack of differences between the WT and βarr2-KO mice in response to fentanyl and methadone may be a consequence of their ability to promote robust MOR phosphorylation, and thus promote compensatory MOR regulation by βarrestin1. It has been suggested that the morphine-bound MOR cannot recruit βarrestin1, and thus this compensatory regulation cannot occur. This is supported by the observation that morphine antinociception is not altered in βarr1-KO mice [34].

Therefore, we sought to determine whether a fundamental difference exists between βarrestin1 and βarrestin2 mediated regulation of the MOR. We utilized cells lacking expression of βarrestins (βarr1/2-KO, βarr1-KO, and βarr2-

KO MEFs) to assess the ability of two classic MOR ligands, DAMGO and morphine, to promote classical GPCR regulation of the MOR as a function of

βarrestin expression. We show that DAMGO induces recruitment of both

βarrestin1 and βarrestin2 to the MOR and that either βarrestin is sufficient to promote DAMGO-induced internalization, while βarrestin1 is critical to promote

DAMGO-induced MOR ubiquitination. Morphine, however, promotes interactions

68 between the MOR and βarrestin2 only. This interaction is sufficient to promote

MOR internalization, but not MOR ubiquitination.

3.2 Materials and Methods

Drugs. DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin; Tocris, Ellisville, MO) and morphine (morphine sulfate pentahydrate; Sigma, St. Louis, MO) were dissolved in distilled water to 10mM. 10mM stock solutions were diluted in PBS to working concentrations.

Constructs. mouse mu opioid receptor ((gi1055230) is HA tagged on the N- terminus (HA-MOR); rat βarrestin1 (gi949985) and βarrestin2 (gi949986) are tagged on the C-terminus with enhanced green florescent protein (βarr2-eGFP and βarr1-eGFP), rat βarrestin1 (gi949985) and βarrestin2 (gi949986) are Myc- tagged on the N-terminus (Myc-βarr1 and Myc-βarr2), mouse MOR is tagged on the C-terminus with yellow florescent protein (MOR-YFP)

Stable Expression of HA-MOR in WT and arr1/2 KO MEFs. MEFs were transduced with HA-MOR using Murine Stem Cell Virus (MSCV). Transfected cells were selected in the presence of puromycin (1ug/mL, Calbiochem). A

FACS Aria flow cytometer was used to select for high expressing cells (top

~25%) using an anti-HA AlexaFluor 488 conjugate antibody (1:200; Invitrogen,

69

Eugene, Oregon). Cells were sub-cloned 3 times to select for WT and arr1/2

KO MEFs that showed similar amounts of radioligand binding.

Immunofluorescence. HA-MOR WT and βarr1/2 KO MEFs were serum-starved

(30min), fixed (4%PFA, 20min, 4oC), and permeabilized in PBS+ (PBS with 5% goat serum, 0.3% TritonX-100, and 0.02% Sodium Azide, 30min, RT). HA-MOR was labeled using an anti-HA AlexaFluor 488 conjugate antibody (1:200).

Fluorescence was visualized with a 100X objective on an Olympus Fluoview 300 confocal microscope and Olympus Fluoview imaging software version 4.3.

Radioligand Binding. Membranes of HA-MOR WT and arr1/2 KO MEFs were prepared using Teflon on glass dounce homogenization and centrifugation at

20,000 x g at 4oC for 30 minutes. Membranes were re-suspended in 50mM Tris pH7.4 using dounce homogenization and protein content was quantified. Total binding was assessed in 200l reactions containing 20g membrane protein and the indicated concentration of [3H]-naloxone (Perkin Elmer, Boston, MA). Non- specific binding was determined in the presence of 40M 6-naltrexol. Reactions were incubated at room temperature for 1 hour and were terminated by rapid filtration through GF/B filter and washed with ice cold water. Radioactivity was determined using liquid scintillation counting. Data are reported as fmol receptor per mg membrane protein.

70

MOR Phosphorylation. HA-MOR WT and arr1/2 KO MEFs were serum starved for 15 minutes, followed by a 10 minute treatment with vehicle (PBS), DAMGO

(1μM), or morphine (10μM). The HA-MOR was immunoprecipitated from cell lysates using an anti-HA agarose conjugate as in Chapter 2 methods section.

Precipitate was analyzed by western blot using antibodies against the MOR phosphorylated at Serine 375 (Cell Signaling, Danvers, MA; 1:500) and the C- terminus of the MOR (Neuromics, Edina, MN; 1:1000), as in Chapter 2.

Phosphorylated MORs were normalized to the amount of total MOR precipitated in each sample.

βarrestin Translocation- Microscopy. arr1/2 KO MEFs were transiently transfected with HA-MOR (5µg) and either βarr1-GFP or βarr2-GFP (2.5µg) using electroporation and plated on collagen-coated glass cover slips. After incubation at 37 ºC for 36 hours, cells incubated with an anti-HA AlexaFluor 594 conjugate antibody in serum-free media for 30 minutes at 37oC. Cells were washed with serum-free media and basal images were obtained, followed by drug treatment. Cells were monitored each minute throughout the 30 minute drug treatment. Representative cells at 10-20 minutes are shown. Images were taken as described in Immunofluorescence section.

Bioluminescence Resonance Energy Transfer (BRET)- HEK-293 cells stably over-expressing GRK2 were virally transduced with HA-MOR-Rluc. Cells

71 expressing high levels of MOR-Rluc (top 30% of positive cells) were selected by flow cytometry. Cells were then transiently transfected with either βarr1-GFP2 or

βarr2-GFP2 by electroporation. Two days post transfection, cells were collected in BRET buffer (PBS supplemented with 0.1 g/L CaCl2, 0.1 g/L MgCl2, and 1 g/L

Glucose) and 100,000 cells (in 40µl buffer) were added to each well of a white

96-well optiplate (Perkin Elmer). Cells were treated with 5 µl of varying concentrations of drug and allowed to incubate at 37 ºC for 5 minutes, followed by the addition of 5 µl of coelenterazine 400a (final concentration of 5 µM).

Luminescence readings were taken at 515 nm and 395 nm using an Envision plate reader. BRET ratio equals 510nm/395nm. Background signal (cells not expressing βarr1/2-GFP2) was subtracted from all ratios. Ratios were then normalized to vehicle responses. Agonist-induced interactions between MOR and βarrestins were detected as an increase in normalized BRET ratio.

MOR-YFP Trafficking. WT and arr1/2 KO MEFs were transiently transfected with MOR-YFP (2µg) by electroporation. Internalization was assessed as described in Chapter 2 methods section.

Immunofluorescence of Internalized MOR. Internalization was assessed in HA-

MOR WT and βarr1/2-KO MEFs. Cells were serum starved for 30 minutes followed by drug treatment for 2 hours. Immunofluorescence was performed as described above.

72

Biotinylated MOR Internalization. HA-MOR WT and arr1/2 KO MEFs were serum starved and surface proteins were labeled with biotin. Cells were washed with TBS followed by treatment with vehicle, DAMGO (1M), or morphine (10µM) for 2hrs at 37oC. Remaining surface biotin was then stripped with glutathione stripping buffer and then washed with iodoacetamide buffer. Biotinylated proteins were precipitated from cell lysates with Avidin beads and analyzed by western blot using an antibody against the C-terminus of the MOR (Neuromics,

Edina, MN; 1:1000). Control: “100%”- cells were not incubated in stripping buffer, “Strip”- Cells were biotinylated, stripped and lysed without drug treatment.

A more detailed protocol is in Chapter 2 methods section.

βarrestin Rescue of MOR Internalization- Confocal Imaging. HA-MOR arr1/2-

KO MEFs were transiently transfected with Myc-βarr1 (2.5μg) or Myc-βarr2

(2.5µg) using electroporation. After incubation at 37 ºC for 36 hours, cells were serum-starved for 30 minutes at 37oC followed by treatment with vehicle,

DAMGO (1M), or morphine (10µM) for 2hrs at 37oC. Cells were fixed and permeabilized as above. Cells were incubated with three antibodies in PBS+ in the following order, with several PBS+ washes after each antibody: anti-Myc

(Clontech, Mountainview, CA; 1:100, 4oC, overnight), goat anti-mouse Alexa

Fluor 568 conjugate (Invitrogen, Eugene, Oregon; 1:2000, RT, 2hrs), and anti-HA

73

Alexa Fluor 488 conjugate (Invitrogen, Eugene, Oregon; 1:100, 4oC, overnight).

Images were obtained as described above.

βArrestin Rescue of MOR Internalization- Biotinylation. HA-MOR arr1/2-KO

MEFs were transiently transfected with Myc-βarr1 (2.5μg) or Myc-βarr2 (10µg) using electroporation. One day post transfection, cells that were plated on collagen-coated coverslips were fixed, permeabilized, and stained using an antibody against the Myc tag. If approximately 20% transfection efficiency was obtained, biotinylation experiment was performed as described above two days post transfection.

MOR Ubiquitination- HA-MOR WT and arr1/2 KO MEFs were treated with drug for the times indicated. Cells were washed twice with PBS on ice and collected in lysis buffer (50 mM Tris HCl pH8.0, 150 mM NaCl, 5 mM EDTA, 0.1% SDS,

10% glycerol, 1% NP40, 0.5% deoxycholate, 10 mM sodium orthovanadate, 10 mM NaF, protease inhibitor cocktail (Roche), and 10mM N-ethylmaleimide

(NEM). Lysates were homogenized on a rotator for at least 20 minutes 4oC and cleared at 12,000 x g for 30 minutes at 4oC. Equal amounts of protein (700-

1000µg) were added to 25µl of anti-HA agarose conjugate and incubated overnight on a rotator at 4oC. Precipitate was washed 4X in PBS and proteins were eluted in 35µl 1X XT sample buffer with 1.25% β-mercaptoethanol at 55oC for 30 minutes. 30µl of supernatant was removed and boiled at 100oC for 3

74 minutes. 25µl of precipitated proteins were resolved on 4-12% Bis-Tris NuPAGE gels (Invitrogen) and transferred to PVDF membranes. Blots were blocked with

5% milk in TBST and incubated simultaneously with an antibody against ubiquitin

(P4D1, Santa Cruz Biotech., Santa Cruz, CA, 1:500) and an antibody against C- terminus of the MOR (Neuromics, Edina, MN; 1:4500) overnight at 4oC. Blots were washed in TBST and incubated simultaneously with a goat anti-rabbit

IRDye 680 conjugate (Licor, Lincoln, NE, 1:15000) and goat anti-mouse IRDye

800 conjugate (Licor, 1:15000) in 5% milk for 1 hour at room temperature protected from light. Signal was detected using the Licor Odyssey imager. Band intensity was determined using Odyssey software version 1.2. Ubiquitin signal was normalized to the total receptor per lane. Stimulation over vehicle treated was determined for each blot using GraphPad Prism software (GraphPad

Software, Inc., San Diego, CA).

3.3 Results

WT and βarr1/2 KO MEFs stably expressing HA-MOR were generated using a murine stem cell virus (MSCV) transduction system. Radioligand binding was used to isolate cell lines of each genotype that express similar amounts of naloxone binding sites. Saturation binding using [3H]-naloxone shows that both the WT and βarr1/2-KO MEFs express approximately 1.3 pmol receptor per mg protein (Figure 3.1 and Table 3.1) and that the affinity of naloxone for the HA-

75

MOR does not significantly differ between genotypes (Table 3.1). Furthermore, clones were selected based on uniform cell surface expression of the HA-MOR under basal conditions, as assessed by confocal microscopy (Figure 3.1).

Since βarrestin association with the MOR can be facilitated by receptor phosphorylation, we assessed the phosphorylation state of the MOR at Ser375 following agonist treatment, as phosphorylation of this residue is associated with

MOR internalization and desensitization [92-93]. In both the WT and βarr1/2 KO

MEFs, DAMGO and morphine induce phosphorylation at Ser375 (Figure 3.2).

Further, morphine induces significantly less robust phosphorylation than DAMGO in both cell lines, consistent with previous observations [35].

Following agonist activation and subsequent receptor phosphorylation, the

MOR has increased affinity for βarrestins. To determine agonist specificity in

βarrestin recruitment, βarr1/2 KO MEFs were transiently transfected with HA-

MOR and either βarr1-eGFP or βarr2-eGFP. In the absence of agonist, βarr1- eGFP expression is localized in the cytosol and nucleus, while βarr2-eGFP expression is primarily confined to the cytosol (Figure 3.3). Cells were then treated with agonist, and arrestin translocation was assessed only in HA-MOR positive cells using confocal microscopy. DAMGO induces robust translocation of both βarr2-eGFP and βarr1-eGFP. In contrast, morphine promotes detectable translocation of only βarr2-GFP. This is consistent with a previous report in transiently transfected βarr1/2-KO MEFs by Bohn et al. [34]. The authors showed that etorphine, which induces robust MOR phosphorylation similar to

76

DAMGO, promotes translocation of both βarrestins, while morphine selectively promotes recruitment of βarrestin2.

To obtain a more quantitative assessment of the βarrestin preference induced by DAMGO and morphine, we utilized a Bioluminescence Resonance

Energy Transfer (BRET) assay. HEK-293 cells stably over-expressing GRK2 and HA-MOR-Rluc were transiently transfected with either βarr1-GFP2 or βarr2-

GFP2. Agonist-induced interactions between MOR and βarrestins were detected as an increase in GFP2 fluorescence over Rluc luminescence. DAMGO and morphine promote robust interactions between the MOR and βarrestin2, with similar potencies (Figure 3.4A and Table 3.2). Morphine, however, is significantly less efficacious at promoting βarrestin2 interactions when compared to DAMGO (two-way ANOVA: DAMGO vs. morphine; p<0.0001 for drug, p<0.0001 for concentration, p=0.6075 for the interaction; Bonferroni post-test

*p<0.05 for 1µM and 10µM doses) (Figure 3.4A). Therefore, morphine can be classified as a partial agonist for recruitment of βarrestin2. Interestingly, while

DAMGO can promote βarrestin1 interactions with the MOR, morphine is unable to induce this interaction (two-way ANOVA: DAMGO vs. morphine; p<0.01 for drug, p<0.05 for concentration, p=0.0636 for the interaction; Bonferroni post-test

*p<0.05 for 10µM dose) (Figure 3.4B and Table 3.2), which is consistent with microscopy observations.

Given that βarrestins can promote GPCR internalization by scaffolding the receptor to internalization machinery [42,49,53,55-57], we assessed the role of

77

βarrestins in agonist-induced MOR internalization using WT and βarr1/2-KO

MEFs. These experiments were performed in MEFs either transiently transfected with MOR-YFP or stably expressing HA-MOR and MOR trafficking was visualized by confocal microscopy (Figure 3.5A). In the absence of agonist or in the presence of vehicle alone, the MOR is primarily localized to the plasma membrane. The WT MEFs internalize the MOR in response to DAMGO, and to a lesser extent in response to morphine. In contrast, neither drug is able to promote MOR internalization in the absence of βarrestins.

Further, these observations were quantified using a cell surface biotinylation assay. Both DAMGO and morphine increase the amount of biotinylated HA-MOR protected from glutathione stripping in the WT MEFs, due to MOR internalization (Figure 3.5B and C). Morphine exposure, however, leads to significantly less MOR internalization than DAMGO. In the arrestin null

MEFs, on the other hand, MOR internalization was not observed with either agonist.

To determine the role of each βarrestin in agonist-induced MOR internalization, we assessed the ability of Myc-tagged βarrestins to rescue MOR internalization in the βarr1/2-KO MEFs. βarr1/2-KO MEFs stably expressing the

HA-MOR were transiently transfected with either Myc-βarr1 or Myc-βarr2, and internalization was assessed by confocal microscopy only in Myc positive cells

(Figure 3.6A). Consistent with our arrestin recruitment studies which demonstrate that both DAMGO and morphine promote MOR interactions with

78

βarrestin2 (Figure 3.3 and 3.4A), Myc-βarr2 transfection was able to rescue

MOR internalization induced by both DAMGO and morphine (Figure 3.6).

Transfection with Myc-βarr1, however, only restores MOR internalization in response to DAMGO, which also consistent with agonist-specific βarrestin1 recruitment (Figure 3.3 and 3.4B). The effect of agonist-specific βarrestin rescue of MOR internalization can also be seen using cell surface biotinylation assays (Figure 3.6B and C), and recapitulate the effects observed with microscopy.

Trafficking of GPCRs can serve as a means to compartmentalize signaling scaffolds, and the association of βarrestins can determine the inclusion of certain proteins within the receptor complex [73,105]. By acting to scaffold E3 ubiquitin ligases, βarrestins have been shown to play a critical role in agonist-induced ubiquitination of several GPCRs [65]. Therefore, we asked whether the MOR was ubiquitinated in response to agonist treatment and whether MOR ubiquitination was a function of βarrestin expression. WT and βarr1/2-KO MEFs were treated with DAMGO or morphine for the times indicated (Figure 3.7).

DAMGO promotes MOR ubiquitination in a time dependent manner in WT MEFs that peaks after 1 hour of treatment and persists to at least 2 hours. In the absence of βarrestins, DAMGO-induced MOR ubiquitination was not observed.

Interestingly, morphine was unable to promote MOR ubiquitination in the WT

MEFs.

79

Since DAMGO promotes βarrestin1 recruitment to the MOR and

βarrestin1 is able to rescue MOR internalization in the βarr1/2 MEFs, we asked whether either βarrestin alone could promote agonist-induced MOR ubiquitination. Using MEFs that lack expression of each individual βarrestin

(βarr1-KO, βarr2-KO), we assessed the role of each βarrestin in agonist-induced

MOR ubiquitination (Figure 3.8). Consistent with our time course analysis, we observed that DAMGO, but not morphine, promotes MOR ubiquitination in the

WT MEFs, while neither drug promotes MOR ubiquitination in the βarr1/2-KO

MEFs. In the absence of βarrestin1, agonist-induced MOR ubiquitination was not observed. The deletion of βarrestin2, however, had no significant impact on the response profile observed in the WT MEFs. Taken together, these findings suggest that βarrestin1, and not βarrestin2, plays a critical role in ubiquitination of the MOR.

3.5 Discussion

In this chapter, we assessed canonical regulation of the MOR in response to the classical MOR agonists, DAMGO and morphine, as a function of agonist- directed βarrestin interactions with the MOR. Our first objective in these studies was to validate and quantitate the agonist-directed βarrestin recruitment profiles of DAMGO and morphine. Agonists that promote robust MOR phosphorylation, such as DAMGO, have previously been shown to promote robust βarrestin2

80 recruitment [34-35,52,90,104]. Morphine-induced βarrestin2 recruitment has been much more difficult to assess, and can only be visualized with GRK2 over expression in HEK-293 cells or in the absence of endogenous βarrestins in cells.

Recently, Molinari et al. [104] were able to quantitate morphine-induced

βarrestin2 interactions with the MOR using BRET.

βArrestin1 interactions with MOR have received much less attention.

Bohn et al. [34] assessed the ability of both etorphine and morphine to promote

βarr1-GFP recruitment in cells that lack endogenous βarrestin expression.

These qualitative data suggest that agonists that promote robust MOR phosphorylation, such as etorphine, also promote βarrestin1 recruitment. In contrast, βarr1-GFP recruitment was not detectable when morphine was used.

Therefore, we used both confocal microscopy (Figure 3.3) and BRET (Figure

3.4) to assess βarrestin interactions in response to DAMGO and morphine.

Using these two approaches we were able to confirm that DAMGO induces interactions between the MOR and both βarrestins. Further, we were able to quantitate the interaction between the morphine-bound MOR and βarrestin2. We find that while morphine does promote βarrestin2 interactions with the MOR, the interactions are much less robust than that promoted by DAMGO, which may account for the difficulty in observing this interaction using only microscopy.

Importantly, our BRET system utilizes HEK-293 cells that over-express GRK2, which has been shown to promote βarrestin interactions with the MOR. Even in this system that is biased toward βarrestin interactions, morphine is unable to

81 promote any detectable BRET signal between βarrestin1 and the MOR. These data suggest that the morphine-bound MOR is highly resistant to regulation by

βarrestin1. Collectively, these data support the hypothesis that βarrestin1 cannot regulate the MOR in response to morphine in the βarr2-KO mice. Hence, the dramatic behavioral differences between the WT and βarr2-KO mice are observed. In contrast, agonists that promote robust MOR phosphorylation, such as fentanyl and methadone, can promote βarrestin1-mediated MOR regulation in the βarr2-KO mice, which may be why antinociceptive profiles do not differ from

WT mice.

We then sought to determine whether the agonist-selective recruitment of

βarrestins would have functional consequences with respect to MOR internalization and ubiquitination, as βarrestins have been shown to act as scaffolds for both internalization and ubiquitination machinery [50,65]. We have observed a striking difference in the capabilities of βarrestins to promote MOR internalization and ubiquitination. We show that while βarrestins are required for agonist-induced MOR internalization (Figure 3.5), either βarrestin1 or βarrestin2 is sufficient to mediate DAMGO-induced MOR trafficking (Figure 3.6).

Consistent with morphine promoting selective recruitment of βarrestin2 to the

MOR, however, only βarrestin2 is able to rescue morphine induced MOR internalization. In contrast to the reciprocity of βarrestins in promoting MOR internalization, our data suggest that only βarrestin1 can promote agonist- induced MOR ubiquitination (Figure 3.7 and 3.8). As a consequence of their

82

βarrestin recruitment profiles, in turn, only DAMGO, and not morphine, can promote MOR ubiquitination. Therefore, agonists can differentially recruit

βarrestins, which in turn determine MOR regulation with regards to internalization and ubiquitination.

Morphine-induced MOR regulation seems to be highly dependent on the intracellular environment in which the MOR is expressed. Many cell culture studies have manipulated kinase or βarrestin expression to alter morphine- induced MOR trafficking. In vivo, Haberstock-Debic et al. [113-114] showed that morphine-induced MOR trafficking differs between neurons, and even within different regions of the same neurons. It is likely that these differences in morphine-induced trafficking are due to differential expression of proteins available to regulate the morphine-bound MOR. Indeed, the most robust example of the sensitivity of the morphine-bound MOR to expression of regulatory proteins is the dramatically altered morphine-induced behavioral profile in the βarr2-KO mice [123,131,133,139,144]. Other opioids, which promote robust MOR phosphorylation, such as DAMGO and methadone, may promote a receptor conformation that has high affinity for GRKs and both

βarrestins. Therefore, such opioids may not be as sensitive to the specific complement of intracellular proteins.

In contrast to MOR trafficking, however, our data suggests that only

βarrstin1 can mediate agonist-induced MOR ubiquitination. The preference for a particular βarrestin in mediating agonist-induced GPCR regulation has been

83 observed previously. The β2AR and the V2 vasopressin receptors utilize

βarrestin2 to promote receptor ubiquitination [66,155], while the -like growth factor-1 receptor (IGF-1R) and the chemokine receptor CXCR4, preferentially utilize βarrestin1 to promote receptor ubiquitination [67,156].

Though ubiquitination is thought mediate trafficking of receptors to the lysosome for degradation [65,157], ubiquitination-independent trafficking to the lysosome has been reported for the delta opioid receptor (DOR) [158-159].

Several proteins (Vps4 and Hrs), which are involved in lysosomal sorting of ubiquitinated proteins, are also utilized to traffic non-ubiquitinated receptors to lysosomes. Several studies have observed that chronic treatment with morphine does not cause MOR down-regulation in whole mouse brain or brainstem

[131,160-162]. This may be due to morphine’s inability to promote MOR ubiquitination.

Overall, our results demonstrate that MOR regulation is highly dependent on the agonist, such that an agonist’s ability to promote MOR/βarrestin interactions dictates MOR internalization and ubiquitination. DAMGO exposure results in the classical model of GPCR regulation in which the MOR is robustly phosphorylated, recruits both βarrestins, internalizes, and is ubiquitinated. On the other hand, morphine, which can be considered functionally selective at the

MOR, promotes weak phosphorylation, preferentially recruits βarrestin2, and does not promote MOR ubiquitination. These results demonstrate that βarrestin1 and βarrestin2, have differential roles in MOR regulation. While both βarrestins

84 can promote MOR trafficking, only βarrestin1 is sufficient to promote ubiquitination of the MOR.

85

3.6 Tables and Figures

A. WT βarr1/2 KO

B. 1000 WT arr1/2 KO 750

500

250

(fmol/mg protein) (fmol/mg Receptor numberReceptor 0 0 1 2 3 4 [3H]-Naloxone (nM)

Figure 3.1. Cellular localization and saturation binding of HA-MOR stably expressing WT and arr1/2 KO MEFs. A. HA-MOR expressing cells are labeled using an anti-HA AlexaFluor 488 conjugate antibody and display primarily membrane expression. Representative images are shown from three independent experiments. B. Specific binding was determined with increasing concentrations of [3H]-naloxone, using 40M 6-naltrexol to determine non-specific binding. Specific binding of the radioligand was fit to a non-linear regression one site binding curve using GraphPad Prism software (n=3). Binding parameters and statistical analyses are shown in Table 3.1.

86

B Genotype K (nM) MAX D (fmol per mg)

WT 1.526 ± 0.1891 1232 ± 60.80

βarr1/2 KO 2.158 ± 0.448 1356 ± 103.5

Table 3.1. Specific binding parameters of [3H]-Naloxone to HA-MOR WT and

βarr1/2 KO MEFs. Mean ± SEM are shown. Neither KD nor BMAX differ significantly between genotypes (p>0.05, Student’s t-test, n=3-4).

87

A.

150 P-MOR 75

150 T-MOR 75

WT arr1/2 KO

B. 20 Vehicle DAMGO *** 15 Morphine ## *** *** 10 # ***

5

Fold of Vehicle of Fold P-MOR (Ser375) P-MOR

0 WT arr1/2 KO

Figure 3.2. Agonist-induced HA-MOR phosphorylation at Ser375. The HA-MOR was immunoprecipitated from cell lysates of HA-MOR WT and βarr1/2 KO MEFs that were treated with vehicle, DAMGO (1µM), or morphine (10µM) for 10min. The extent of MOR phosphorylation was determined by western blotting. A. Representative immunoblots are shown. B. Densitometric analysis of seven experiments shows mean  S.E.M. of phospho-MOR normalized to total MOR. DAMGO and morphine induce phosphorylation at Ser375 in both genotypes (*** p<0.001 for agonist treatment vs vehicle of same genotype, Student’s t-test, n=8-12). Morphine induces less robust phosphorylation than DAMGO in both genotypes (# p<0.05, ## p<0.01 vs DAMGO of same genotype; Student’s t-test, n=8-9).

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βarr1/2 KO MEF + HA-MOR

βarr1-eGFP βarr2-eGFP

Basal

DAMGO Morphine

Figure 3.3. Agonist-induced βarrestin-GFP translocation. βarr1/2 KO MEFs were transiently transfected with HA-MOR and either βarr1-GFP or βarr2-GFP. HA-MOR was labeled with anti-HA AlexaFluor 594 conjugate antibody (red) followed by treatment with DAMGO (1µM) or morphine (10µM). Representative confocal images of βarrestin-GFP translocation are shown from at least three experiments per drug. DAMGO induces βarr1-GFP and βarr2-GFP translocation to the plasma membrane (white arrows, puncta). Morphine induces detectable translocation of βarr2-GFP only.

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A. 0.020 DAMGO Morphine 0.015 * * 0.010

0.005

arr2Ratio BRET  0.000 -10 -9 -8 -7 -6 -5 Log [Agonist], M B. DAMGO 0.008 Morphine 0.006

0.004 *

0.002

arr1Ratio BRET  0.000 -10 -9 -8 -7 -6 -5 Log [Agonist], M

Figure 3.4. BRET analysis of dose dependent agonist-induced increase in interactions between the MOR and βarrestins. HEK-293 cells stably expressing MOR-Rluc and GRK2 were transiently transfected with either βarr2-GFP2 (A.) or βarr1- GFP2 (B.). Five minutes following drug treatment, coelenterazine 400a was added and dual luminescence readings were taken at 510 nm and 395 nm. BRET ratio equals 510nm/395nm. Background signal (cells not expressing βarr1/2-GFP2) was subtracted from all ratios. Ratios were then normalized to vehicle responses. Graphs show mean ± S.E.M. of 8 experiments (n=5-8 for each data point); data were fit to a non-linear regression curve using GraphPad Prism software. Calculated potencies and efficacies are reported in Table 3.2. A. Both DAMGO and morphine induce a dose-dependent increase βarr2 interactions with the MOR, but morphine is less efficacious, as the curves (continued) 90

(Figure 3.4 continued) differ significantly (two-way ANOVA: DAMGO vs. morphine; p<0.0001 for drug, p<0.0001 for concentration, p=0.6075 for the interaction; Bonferroni post-test *p<0.05). B. DAMGO induces a dose-dependent increase βarr1 interactions with the MOR, but morphine does not (two-way ANOVA: DAMGO vs. morphine; p<0.01 for drug, p<0.05 for concentration, p=0.0636 for the interaction; Bonferroni post-test *p<0.05).

91

E EC (nM) MAX 50 (BRET ratio) (95% C.I.) (95% C.I.) βarr2-GFP2 19.2 0.01719 DAMGO (5.8-63.4) (0.01516-0.01922) 46.1 0.01017** Morphine (6.8-313.4) (0.00847-0.01308) βarr1-GFP2 23.2 0.00677 DAMGO (5.2-103.0) (0.00567-0.00786)

Morphine not converged not converged

Table 3.2. BRET analysis of interactions between the MOR and βarrestins- Potency and Efficacy. Data from 8 experiments (n=5-8 for each data point) were fit to a non-linear regression curve using GraphPad Prism software. DAMGO and morphine promote MOR interactions with βarr2-GFP2 with similar potencies (p>0.05, Student’s t- test), but DAMGO is significantly more efficacious (** p<0.01 vs DAMGO-βarr2-GFP2, Student’s t-test). DAMGO promotes interactions between the MOR and βarr1-GFP2, while morphine does not.

92

A. MOR-YFP HA-MOR

WT arr1/2 KO WT arr1/2 KO

Basal

DAMGO Morphine

B. C. WT arr1/2 KO *** 2.5 150 ## 2.0 WT *** 75 1.5 150

βarr1/2 Internalization, 1.0 Fold OverFold Vehicle KO 75

Vehicle DAMGO Morphine

Figure 3.5. Agonist-induced MOR trafficking in WT and arr1/2 KO MEFs. A. Left two panels. Transiently transfected MOR-YFP is primarily localized to the plasma membrane in both cell lines under basal conditions. DAMGO (1M, 2hrs) and (continued)

93

(Figure 3.5 continued) morphine (10M, 2hrs) induce internalization in WT MEFs. In arr1/2 KO MEFs, however, MOR-YFP remains on the cell surface after treatment with either drug. Right two panels. WT and βarr1/2 KO MEFs stably expressing the HA-MOR were treated as above, followed by fixation, permeabilization, and labeling of the HA-MOR by an anti-HA AlexaFluor 488 conjugate. DAMGO and morphine lead to an increase in intracellular receptor staining only in the WT MEFs. Representative images are shown from at least three experiments. B. Agonist-induced HA-MOR internalization was quantified in HA- MOR WT and arr1/2 KO MEFs using cell surface biotinylation assays. Representative immunoblots are shown. Controls for protein biotinylation (“100%”) and glutathione stripping (“Strip”) are included. C. Densitometric analysis of seven biotinylation experiments done in duplicate shows the mean  S.E.M. of internalized HA-MOR. DAMGO and morphine induce internalization in WT MEFs (*** p<0.001 vs vehicle; Student’s t-test, n=8-9), but not in the arr1/2 KO MEFs (p>0.05; Student’s t-test, n=10- 14). Morphine induces significantly less MOR internalization than DAMGO in the WT MEFs (## p<0.01; Student’s t-test, n=7-8).

94

A. βarr1/2 KO MEF + HA-MOR

Myc-βarr1 Myc-βarr2

Basal

DAMGO Morphine

B. C. Myc-arr1 Myc-arr2 + Myc- *** + Myc- 2.00 βarr1 βarr2 1.75 **

1.50 ***

1.25

DAMGO DAMGO

Morphine Morphine

Vehicle Vehicle 1.00

150 Internalization, Fold OverFold Vehicle 0.75

IB: MOR IB: 75

Vehicle DAMGO Morphine

Figure 3.6. βarrestin rescue of HA-MOR internalization in βarr1/2 KO MEFs. A. βarr1/2-KO MEFs were transiently transfected with either Myc-βarr1 or Myc-βarr2. Following DAMGO (1µM) or morphine (10µM) treatment, cells were fixed and (continued)

95

(Figure 3.6 continued) permeabilized. Myc-βarrestins are labeled with an anti-Myc antibody, followed by an anti-mouse secondary Alexa Fluor 568 conjugate (red). HA-MOR is labeled using anti- HA AlexaFluor 488 conjugate (green). Representative images are shown from at least three experiments. Expression of Myc-βarr2 rescues both DAMGO and morphine induced HA-MOR internalization. In contrast, expression of Myc-βarr1 rescues HA-MOR internalization only by DAMGO, and not morphine. B. Agonist-induced HA-MOR internalization was quantified in arr1/2 KO MEFs transiently transfected with either Myc-βarr1 or Myc-βarr2 using cell surface biotinylation assays. Representative immunoblots are shown. C. Densitometric analysis of three experiments done in duplicate or triplicate shows the mean  S.E.M. of internalized HA-MOR. Myc-βarr2 transfection rescues both DAMGO and morphine induced internalization (*** p<0.001, ** p<0.01 vs vehicle; Student’s t-test, n=4-5). Myc-βarr1 transfection rescues only DAMGO-induced HA-MOR internalization (*** p<0.001; Student’s t-test, n=8).

96

A. WT MEF Time DAMGO Morphine

Minutes 0

 0

30 60 30 60

120 120 150

100 IB: UB IB:

MOR 75 -

150

100 IP: HA IP:

75 IB: MOR IB:

B. arr1/2-KO MEF C. WT DAMGO WT Morphine Time DAMGO 150 Minutes KO DAMGO ***

 140 ***

0

15 30 60 120 150 130 ### * ###

100 120 IB: UB IB: MOR 75 ## - 110 150 100 100

IP: HA IP: 90 % Ubiquitination Over Basal % Ubiquitination IB: MOR IB: 75 0 15 30 60 120 Time (minutes)

Figure 3.7 Time course of agonist-induced MOR ubiquitination in WT and βarr1/2- KO MEFs. The HA-MOR was immunoprecipitated from cell lysates of WT and βarr1/2- KO MEFs stably expressing the HA-MOR that were treated with DAMGO (1µM) or morphine (10µM) for the times indicated. The extent of MOR ubiquitination was determined by western blotting. Representative immunoblots are shown for WT (A.) and βarr1/2-KO (B.) MEFs. C. Densitometric analysis of eight experiments shows mean  S.E.M. of ubiquitinated MOR normalized to total MOR (n=3-29 for each data point). In WT MEFs, DAMGO induces a time-dependent increase in ubiquitinated MOR, while morphine does not (two-way ANOVA WT DAMGO vs WT morphine; p<0.01 for time, p<0.001 for drug, p<0.01 for the interaction; Bonferroni post-test *p<0.05, ***p<0.001). DAMGO is unable to promote MOR ubiquitination in the βarr1/2-KO MEFs (two-way ANOVA WT DAMGO vs KO DAMGO; p<0.01 for time, p<0.001 for drug, p<0.001 for the interaction; Bonferroni post-test ##p<0.01, ###p<0.001).

97

A. WT arr1-KO arr2-KO arr1/2-KO

IP:

HA-MOR

Vehicle DAMGO morphine Vehicle DAMGO morphine

Vehicle DAMGO morphine

morphine Vehicle DAMGO IB: UB

IB: MOR

B. * 130 *** Vehicle DAMGO 120 Morphine

110 #

100 ### 90

80 % Ubiquitination Over Vehicle % Ubiquitination WT arr1-KO  arr2-KO  arr1/2-KO

Figure 3.8 Agonist-induced MOR ubiquitination in WT and βarrestin-KO MEFs. The HA-MOR was immunoprecipitated from cell lysates of WT, βarr1-KO, βarr2-KO, and βarr1/2-KO MEFs stably expressing the HA-MOR that were treated with vehicle, DAMGO (1µM), or morphine (10µM) for 60 minutes. The extent of MOR ubiquitination was determined by western blotting. A. Representative immunoblots are shown. B. Densitometric analysis of at least three experiments done in triplicate per cell line (n=9- 23 per condition) shows mean  S.E.M. of ubiquitinated MOR normalized to total MOR. In WT and βarr2-KO MEFs, only DAMGO induces MOR ubiquitination (one-way ANOVA p<0.0001 for WT and p<0.01 for βarr2-KO; Bonferroni multiple comparisons test ***p<0.001, *p<0.05 vs vehicle of same genotype, ###p<0.001, #p<0.05 vs DAMGO of same genotype). For the βarr1-KO and βarr1/2-KO MEFs, agonist-induced MOR ubiquitination is not observed (one-way ANOVA p>0.05). 98

CHAPTER 4

CONCLUSIONS

The data presented in this dissertation demonstrate that different agonists acting at the MOR can promote different βarrestin recruitment profiles, which in turn dictate receptor trafficking and ubiquitination, demonstrating biased agonism at the MOR. We demonstrate both qualitatively and quantitatively that DAMGO promotes robust recruitment of both βarrestin1 and 2 to the MOR, while morphine preferentially recruits βarrestin2, consistent with previous reports. In addition, we characterize several novel MOR agonists that are fully efficacious at promoting G protein coupling and ERK1/2 activation, yet are unable to promote

βarrestin interactions. Further, we provide both qualitative and quantitative data that suggest that the ability of an agonist to promote MOR internalization is contingent on its ability to promote interactions between the MOR and either

βarrestin, while ubiquitination of the MOR specifically requires βarrestin1.

βArrestins are classically described as proteins which desensitize GPCRs.

Therefore, in their absence, MOR coupling to G proteins should be enhanced.

Indeed, βarr2-KO mice show enhanced G protein coupling to the MOR compared

99 to WT mice in several regions [133]. Following chronic morphine- treatment, extensive MOR desensitization is observed in brain regions associated with pain perception, such as the brainstem and periaqueductal grey

(PAG), as assessed by the reduced ability of MOR agonists to stimulate GTP coupling compared to vehicle treated animals [131,136-138,163]. In contrast, chronic treatment with morphine does not promote MOR desensitization of G protein coupling in brains from βarr2-KO mice, further suggesting βarrestin2 plays a critical role in regulating the morphine-bound MOR.

The enhanced ability of the MOR to signal in both naïve and chronic morphine-treated βarr2-KO mice has functional consequences as βarr2-KO mice display dramatically enhanced morphine-induced antinociceptive responses, and the development of tolerance to the antinociceptive effects of morphine are attenuated [131,133]. Collectively, these data suggest that morphine promotes

βarrestin2-mediated desensitization of the MOR, which may contribute to the development of tolerance to the antinociceptive effects of morphine. When

βarrestin2-mediated MOR regulation is disrupted, as in the βarr2-KO mice, the

MOR maintains the ability to couple to G proteins, and therefore morphine antinociception is enhanced and tolerance is attenuated.

On the other hand, βarrestins have been show to promote signaling for a number of GPCRs, by scaffolding components of signaling cascades, such as kinases, to the receptors [72-74,129] [165]. The physiological relevance of

βarrestin-mediated signaling has been suggested in vivo for a number of GPCRs

100

[164], as evidenced by a decrease in behavioral responses in the βarr2-KO mice.

Raehal et al. [139,144] showed that morphine-induced constipation, respiratory suppression, and the display of antagonist-precipitated withdrawal signs were reduced in the βarr2-KO mice, perhaps due to the absence of βarrestin2- mediatated signaling. Indeed, these differential roles for βarrestins may be due to changes in the cellular environment in different tissues.

This bifurcation of βarrestin-mediated MOR regulation and signaling suggests that a functionally selective ligand that activates the MOR but does not promote βarrestin recruitment may have therapeutic advantages. By circumventing βarrestin-mediated desensitization of the MOR, one may achieve enhanced G protein coupling in some neurons, resulting in enhanced analgesic responses. On the other hand, by avoiding βarrestin-mediated MOR signaling in other tissues, one may achieve a reduced occurrence of unwanted side effects, such as respiratory suppression and constipation, due to decreased signaling.

We have described several agonists, herkinorin and three herkinorin derivatives, with such properties, which demonstrate that activation of the MOR can be accomplished without promoting βarrestin recruitment. While these observations are encouraging, one must remember that these studies were done in cultured cells over-expressing the MOR, and that the MOR is subject to complex regulation that differs between tissues. The examples cited above emphasize the cell-type specificity of MOR regulation and signaling by βarrestins, which suggest that βarr2 may negatively regulate the MOR in some tissues (CNS),

101 while promoting MOR signaling in other tissues (ENS). Therefore, in vivo testing of herkinorin and its derivatives will be essential in determining how it may affect

MOR function in endogenous tissues.

The differences observed in vivo may be a consequence of unique intracellular complements of regulatory and signaling proteins expressed in residence with the MOR in particular tissues. Studies in cultured cells as well as neurons have demonstrated that MOR regulation can be dependent on GRK expression levels. Over-expression of GRK2 dramatically enhances morphine- induced βarrestin-mediated MOR regulation in cell culture studies. Further,

Haberstock-Debic et al. [113-114] showed that morphine-induced MOR trafficking can differ between neuronal population and even between different cellular regions of the same neuron, and the authors suggest that the trafficking differences may be due to differential expression levels of GRK2. It is likely that tissues that express greater levels of GRKs, may promote morphine-induced

MOR trafficking via a βarrestin-dependent mechanism.

To this end, we assessed the contribution of the specific complement of intercellular proteins to MOR regulation and signaling by the classical MOR agonists, DAMGO and morphine. We demonstrate the agonist-selective

βarrestin recruitment to the MOR has functional consequences with regards to

MOR internalization and ubiquitination. Either βarrestin1 or βarrestin2 is sufficient to promote agonist-induced MOR internalization. DAMGO, which recruits both βarrestins, is able to utilize either βarrestin to internalize the MOR,

102 while morphine only promotes βarrestin2 recruitment and requires this βarrestin for MOR internalization. Finally, herkinorin does not recruit ether βarrestin and is unable to promote MOR internalization under any condition tested. For ubiquitination, only βarrestin1 is able to mediate MOR ubiquitination, and hence

DAMGO, but not morphine, is able to promote MOR ubiquitination. It is likely that herkinorin does not promote MOR ubiquitination since it does not recruit either

βarrestin, but we have not directly tested this hypothesis.

These results show that MOR regulation is the collective result of both the particular agonist and intracellular complement of proteins available to interact with the MOR. The agonist-specificity of βarrestin interactions with the MOR provides further support to the explanations of the behavioral differences observed with the βarr2-KO mice. Agonists that show robust recruitment of both

βarrestins, such as fentanyl and methadone, have normal MOR-mediated physiological responses in the absence of βarrestin2, presumably due to

βarrestin1 compensation in these animals [123,131,133,140,144]. If indeed

βarrestin1 compensation accounts for the unaltered responses to fentanyl and methadone in the βarr2-KO mice, we would expect to observe responses similar to morphine’s effects in the βarr2-KO mice when fentanyl or methadone is administered to βarr1/2-KO mice. Unfortunately, the deletion of both βarrestins results in embryonic lethality [64]. Studies using conditional knockouts or lentiviral knockdown of βarrestins will be useful for these types of experiments.

103

In contrast to fentanyl and methadone, however, our in vitro data suggest that morphine is unable to promote βarrestin1-mediated regulation of the MOR, even under conditions that may be biased toward βarrestin recruitment (GRK2 over-expression). Therefore, in the absence of βarrestin2, the morphine bound receptor is devoid of any βarrestin regulation, such that acute morphine-induced antinociception is enhanced, morphine tolerance is greatly diminished, and morphine-induced constipation is attenuated [34,123,131,133,139,144].

Moreover, βarrestin1-KO mice do not show altered morphine-induced antinociception, consistent with the lack of morphine-induced βarrestin1 recruitment to the MOR [34].

Further, while chronic treatment with morphine, etorphine, or fentanyl promotes antinociceptive tolerance, differences in the abilities of these treatments to promote MOR downregulation were observed [162]. While chronic etorphine or fentanyl can promote downregulation of the MOR, chronic morphine does not lead to MOR downregulation, which may be due to differential MOR ubiquitination. Since βarrestin1 is required for agonist-induced MOR ubiquitination, perhaps the lack of morphine-promoted MOR ubiquitination prevents robust MOR downregulation. Both fentanyl and etorphine promote

βarrestin1 recruitment to the MOR, and we postulate that both compounds would promote MOR ubiquitination, though these experiments have not yet been performed. These data suggest that MOR downregulation is not required for antinociceptive tolerance to opiates.

104

Instead, MOR desensitization may be a critical factor in tolerance to opiates. In the case of etorphine or fentanyl, perhaps two mechanisms of MOR desensitization, and hence tolerance, exist: first, βarrestin1 and βarrestin2 mediated desensitization to G protein coupling and second, βarrestin1-mediated ubiquitination and downregulation, which may manifest as MOR desensitization in that less receptors would be available to signal. Therefore, disruption of either

βarrestin individually should not affect antinociceptive tolerance to either fentanyl or etorphine. In the βarr2-KO mice, the βarrestin1-mediated mechanism of MOR downregulation and tolerance may predominate. Indeed, fentanyl was shown to produce antinociceptive tolerance in these animals [144]. In the βarr1-KO mice,

βarrestin2 mediated MOR desensitization of G protein signaling would still promote antinociceptive tolerance, though these experiments have not been performed.

In contrast, the morphine-bound MOR is only subject to βarrestin2- mediated mechanisms of MOR desensitization. This is consistent with the dramatically attenuated MOR desensitization and antinociceptive tolerance in morphine treated βarr2-KO mice [131]. Further, the lack of differences in morphine-induced antinociceptive responses between WT and βarr1-KO mice supports this hypothesis, as morphine does not promote βarrestin1 recruitment to the MOR. It remains to be determined how chronic morphine would affect the

MOR in the βarr1-KO, however, it is attractive to speculate that the MOR would utilize βarrestin2 and become desensitized.

105

In conclusion, the results presented in this dissertation show that different ligands at the MOR can promote signaling by certain pathways, while avoiding effects mediated by other pathways, demonstrating functional selectivity at the

MOR. Certainly, work by our lab and others have demonstrated that the environment in which the MOR is expressed is equally important to MOR function and regulation. This information may be useful to future efforts in drug discovery as a means to design ligands that preferentially engage certain signaling pathways. Ligands that target MORs in specific tissues and activate desired pathways while avoiding others may be the future of pain management.

106

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