Differential Regulation of Serotonin 2A Receptor Responsiveness by Agonist

Differential regulation of serotonin 2A receptor responsiveness by agonist-

directed interactions with arrestin2

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Cullen Laura Schmid, B.S.

Neuroscience Graduate Studies Program

The Ohio State University

2011

Dissertation Committee:

Laura M. Bohn, Co-advisor

Georgia A. Bishop, Co-advisor

Candice C. Askwith

Wolfgang Sadee Copyright by

Cullen Laura Schmid

2011

Abstract

The G protein-coupled, serotonin 2A (5-HT2A) receptor is a major drug target for the treatment of a number of mental health disorders, including schizophrenia, anxiety and depression. In addition to modulating several of the physiological effects of the neurotransmitter serotonin, activation of the 5-HT2A receptor mediates the psychotomimetic effects of serotonergic hallucinogenic drugs, such as lysergic acid diethylamide (LSD), 2,5-dimethoxy-4-iodoamphetamine (DOI) and 5-methoxy-N,N- dimethyltryptamine (5-MeO-DMT). Though hallucinogens are agonists at the 5-HT2A receptor, not all 5-HT2A receptor agonists induce hallucinations in humans, including the endogenous ligand serotonin. Therefore, the activation of the 5-HT2A receptor can result in different biological responses depending upon the chemical nature of the ligand, a concept that has been referred to as “functional selectivity.” One way in which ligands can induce differential signaling at GPCRs is through interactions with arrestins, which can act to dampen or facilitate receptor signaling cascades or mediate the internalization of receptors into intracellular vesicles. The overarching hypothesis of this dissertation is that the interaction between the regulatory protein, arrestin2, and the 5-HT2A receptor is a critical point in the divergence of agonist-directed 5-HT2A receptor responsiveness.

Using mice lacking arrestin2, we evaluate 5-HT2A receptor trafficking and signaling in vivo for serotonin, the hallucinogenic agonists DOI and 5-MeO-DMT and the endogenous, hallucinogenic metabolite of serotonin, N-methylserotonin. We find that

arrestin2 mediates 5-HT2A receptor trafficking in primary neuronal cultures and can facilitate 5-HT2A receptor-mediated signaling cascades in the mouse frontal cortex, although its role is entirely dependent upon the agonist acting at the receptor. Serotonin requires arrestins to internalize the 5-HT2A receptor and to scaffold the signaling kinases Akt and Src to the receptor. The formation of this receptor scaffold results in an increase in Akt activity, which is disrupted in the absence of arrestin2, while the reintroduction of arrestin2 into primary cortical neurons rescues serotonin-induced phosphorylation of Akt. Moreover, the disruption of these cellular events, either by the absence of arrestin2 or by inhibiting the kinases, results in the inability of serotonin to induce the head twitch response in mice, which is a behavioral model of 5-HT2A receptor activation in the mouse frontal cortex. In contrast, DOI maintains its ability to internalize the 5-HT2A receptor in the absence of arrestin2 and DOI, 5-MeO-DMT and N- methylserotonin do not activate arrestin2-mediated signaling cascades in mouse embryonic fibrobalsts, primary cortical neurons or the mouse frontal cortex. The activation of the head twitch response by these hallucinogenic agonists is not disrupted in arrestin2-knockout mice or in the presence of inhibitors to Akt. Collectively, these studies advance our understanding of the mechanism through which 5-HT2A receptor activation by different agonists can lead to distinct regulatory and signaling pathways in vivo. These studies suggest that agonist-directed 5-HT2A receptor regulation bifurcates based on interactions with arrestin2. Moreover, the elucidation of these signaling pathways could have far reaching implications for the treatment of those neuropsychiatric disorders which have been associated with the disregulation of the 5-

HT2A receptor.

iii

Dedication

This dissertation is dedicated to my family.

The twitch of the head is telling, shows variance in signaling. „tonin activates, arrestin migrates, not the N-methyltryptamines. -Steve Schmid

Acknowledgments

I am much in debt to a number of individuals who having guided and supported me throughout my graduate school training. First and foremost, I must thank my thesis advisor, Dr. Laura M. Bohn. I am extremely grateful to have had her as a mentor throughout this process. She has guided every aspect of this dissertation and I am thankful for the incredible amount of time, support, encouragement, insight and guidance that she has given me over the years. I am much indebted to Dr. Bohn for all of the opportunities she has provided me with, including allowing me to present at national meetings, to pursue external funding for my project and to publish my findings in scientific journals. I am enormously grateful for her investment in me and for the incredible scientific training with which she has provided me.

I would also like to thank the members of my thesis committee, Dr. Georgia Bishop, Dr.

Candice Askwith and Dr. Wolfgang Sadee, for their time, guidance and patience throughout my graduate training. Special thanks to Dr. Bishop for serving as my co- advisor following Dr. Bohn‟s departure from Ohio State. I am also grateful to Dr. Askwith for allowing me to complete a ten week rotation in her laboratory, during which time I learned how to culture primary cortical neurons under her guidance.

I am also incredibly grateful to the past and present members of Dr. Bohn‟s laboratory. I would like to thank Kirsten Raehal and Chad Groer for their scientific insights into my project and technical expertise. I would like to thank Lori Hudson for her assistance in maintaining the mouse colony and managing the laboratory at Ohio State. I would also like to thank Kim Lovell, John Streicher and Robert Moyer for their intellectual contributions to this work. Most of all I would like to thank all of these individuals for their constant support, encouragement and friendship. They kept me going when I wanted to quit, celebrated with me when I succeeded and made coming to work every day enjoyable.

Additionally, I would also like to thank the faculty and staff at both research institutions where this dissertation work was completed. Namely, Keri Bantz, Cheryl Ring, Gina

Alderson, the faculty members of the Neuroscience Graduate Studies Program and the vivarium staff at The Ohio State University. I would also like to thank Mary Krosky and the vivarium staff at The Scripps Research Institute.

Finally, I would like to acknowledge my friends and family for their constant love and support throughout my doctoral training. None of this would have been possible without them.

Vita

June 2001 ...... Valedictorian Wooster High School Wooster, Ohio

Summer of 2004 ...... Research Intern Department of Medicine Pennsylvania State University College of Medicine Hershey, Pennsylvania

May 2005 ...... B.S. Biochemistry, Summa Cum Laude Messiah College Grantham, Pennsylvania

September 2005 to present ...... Graduate Research Associate Neuroscience Graduate Studies Program The Ohio State University College of Medicine Columbus, Ohio

2005 to 2006 ...... University Fellow The Ohio State University Columbus, Ohio

March 2009 to present ...... External Graduate Student Departments of Molecular Therapeutics and Neuroscience The Scripps Research Institute Jupiter, Florida

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Publications

Schmid CL, Bohn LM (2010) Serotonin, but not N-methyltryptamines, activates the serotonin 2A receptor via a arrestin2/Src/Akt signaling complex in vivo. Journal of Neuroscience 30:13513-13524.

Bohn LM, Schmid CL (2010) Serotonin receptor signaling and regulation via arrestins. Critical Reviews in Biochemistry and Molecular Biology 45:555-566.

Fei G, Raehal K, Liu S, Qu MH, Sun X, Wang GD, Wang XY, Xia Y, Schmid CL, Bohn LM, Wood JD (2010) Lubiprostone reverses the inhibitory action of morphine on intestinal secretion in guinea pig and mouse. Journal of Pharmacology and Experimental Therapeutics 334:333-340.

Schmid CL, Bohn LM (2009) Physiological and pharmacological implications of arrestin regulation. Pharmacology and Therapeutics 121:285-293.

Raehal KM, Schmid CL, Medvedev IO, Gainetdinov RR, Premont RT, Bohn LM (2009) Morphine-induced physiological and behavioral responses in mice lacking G protein-coupled receptor kinase 6. Drug and Alcohol Dependence 104:187-196.

Schmid CL, Raehal KM, Bohn LM (2008) Agonist-directed signaling of the serotonin 2A receptor depends on arrestin2 interactions in vivo. Proceedings of the National Academy of Sciences of the United States of America 105:1079-1084.

Kenney SP, Lochmann TL, Schmid CL, Parent LJ (2008) Intermolecular interactions between retroviral Gag proteins in the nucleus. Journal of Virology 82:683-691.

Fields of Study

Major Field: Neuroscience Graduate Studies Program

viii

Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vii

List of Tables ...... xii

List of Figures ...... xiii

List of Abbreviations ...... xv

Chapter 1 ...... 1

1.2 Biosynthesis and metabolism of serotonin ...... 3

1.3 5-HT2A receptor activity in vivo ...... 5

Neuroanatomical localization of the 5-HT2A receptor ...... 5

The head twitch response: a behavioral model of 5-HT2A receptor activation ...... 6

1.4 5-HT2A receptor signaling ...... 7

Functional selectivity at the 5-HT2A receptor ...... 9

1.5 Classical regulation of GPCRs by arrestins ...... 12

Desensitization of G protein coupling ...... 13

Internalization of GPCRs into clathrin-coated pits...... 14

Facilitation of downstream signaling by arrestins ...... 15

Arrestin bias ...... 16

1.6 Regulation of the 5-HT2A receptor ...... 17

Desensitization of the 5-HT2A receptor ...... 18

Internalization of the 5-HT2A receptor ...... 20

5-HT2A receptor regulation by additional scaffolding proteins ...... 23

1.7 Central hypothesis and outline for chapters ...... 24

1.8 Chapter 1 Figures ...... 28

Chapter 2 ...... 31

2.1 Introduction ...... 31

2.2 Materials and Methods ...... 34

2.3 Results ...... 43

Agonist-directed 5-HT2A receptor regulation in vitro ...... 43

Agonist-directed 5-HT2A receptor regulation in vivo ...... 46

2.4 Discussion ...... 50

2.5 Chapter 2 Tables ...... 56

2.6 Chapter 2 Figures ...... 57

Chapter 3 ...... 66

3.1 Introduction ...... 66

3.2 Materials and Methods ...... 69

3.3 Results ...... 75

High doses of serotonin induce head twitches in arr2-KO mice ...... 75

Serotonin metabolism modulates the head twitch response ...... 77

N-methyltryptamines induce head twitches independent of arrestin2 ...... 79

Facilitation of serotonin-mediated 5-HT2A receptor signaling by arrestin2 in vivo .. 80

Blockade of the Akt signaling complex inhibits serotonin-mediated head twitches .. 85

3.4 Discussion ...... 88

3.5 Chapter 3 Tables ...... 94

3.6 Chapter 3 Figures ...... 95

Chapter 4 ...... 109

Reference List ...... 118

List of Tables

Table 2.1 5-HT2A receptor expression levels in MEFs and mouse frontal cortex ...... 56

Table 3.1 Mouse numbers for behavioral studies ...... 94

xii

List of Figures

Figure 1.1 Biosynthesis and metabolism of serotonin ...... 28

Figure 1.2 Canonical model of GPCR regulation ...... 29

Figure 1.3 Ligand directed signaling at GPCRs ...... 30

Figure 2.1 Serotonin and DOI both recruit arrestin2 to the HA-5-HT2AR ...... 57

Figure 2.2 Serotonin, but not DOI, induces internalization in a arrestin-dependent

manner ...... 58

Figure 2.3 Serotonin utilizes arrestins to activate ERK1/2 in MEFs while DOI does not

...... 59

Figure 2.4 The 5-HT2A receptor is more prominently localized to the plasma membrane

of cortical neurons in the absence of arrestin2 ...... 61

Figure 2.5 5-HTP, but not DOI, activates ERK1/2 in the mouse frontal cortex in a

arrestin2-dependent manner ...... 62

Figure 2.6 5-HTP and serotonin induce fewer head twitches in arr2-KO mice ...... 63

Figure 2.7 DOI induces an equivalent number of head twitches in WT and arr2-KO

mice ...... 65

Figure 3.1 High levels of serotonin and 5-HTP can induce a head twitch response in the

arr2-KO mice...... 95

Figure 3.2 Inhibition of MAO-A enhances the 5-HTP-induced head twitch response in

both WT and arr2-KO mice ...... 96

xiii

Figure 3.3 Pretreatment with an INMT inhibitor significantly attenuates 5-HTP-induced

head twitches in arr2-KO mice ...... 97

Figure 3.4 N-methyltryptamines induce more head twitches in arr2-KO mice than their

WT littermates ...... 98

Figure 3.5 5-HTP, but not 5-MeO-DMT, stimulates the formation of a arrestin2/Src/Akt

complex with the 5-HT2A receptor in mouse frontal cortex ...... 99

Figure 3.6 5-HTP, but not 5-MeO-DMT, induces Akt phosphorylation in the mouse

frontal cortex in a arrestin2-dependent manner ...... 100

Figure 3.7 Serotonin induces Akt phosphorylation in primary cortical neurons in a

arrestin2-dependent manner ...... 101

Figure 3.8 The N-methyltryptamines do not stimulate Akt phosphorylation in primary

cortical neurons ...... 103

Figure 3.9 Inhibitors to PI3-K and Src block serotonin-induced Akt phosphorylation in

WT cortical cultures ...... 104

Figure 3.10 Inhibitors to PI3-K, Src or Akt attenuate 5-HTP-induced, but not 5-MeO-

DMT-induced, head twitches in normal mice ...... 105

Figure 3.11 Inhibitors to PI3-K, Src or Akt have no effect on 5-HTP-induced head

twitches in arr2-KO mice ...... 106

Figure 3.12 Concurrent inhibition of Akt and INMT abrogates 5-HTP-induced head

twitches in WT mice ...... 107

Figure 3.13 Serotonin and N-methyltryptamines induce differential signaling at the 5-

HT2A receptor in the mouse frontal cortex ...... 108

xiv

List of Abbreviations

5-HIAA 5-hydroxyindoleacetic acid (5-HIAA)

5-HT Serotonin, 5-hydroxytryptamine

5-HTX receptor Serotonin X receptor

5-HT2AR-YFP 5-HT2A receptor, tagged on the C-terminus with YFP

5-HTP 5-hydroxytryptophan

5-MeO-DMT 5-methoxy-N,N-dimethyltryptamine

AA Arachadonic acid

AADC Aromatic amino acid decarboxylase

Akt Protein kinase B

ANOVA Analysis of variance

AT1A Angiotensin II type 1A receptor

ARF ADP-ribosylation factor

AP2 Adaptor protein 2

arr1 arrestin1

arr1319-418 Dominant negative arrestin1

arr1R169E Constitutively active arrestin1

arr1/2-KO arrestin1 and arrestin2 double-knockout

arr2 arrestin2

arr2284-409 Dominant negative arrestin2 xv

arr2-GFP Arrestin2, tagged on the C-terminus with GFP

arr2-YFP Arrestin2, tagged on the C-terminus with YFP

CaMKII Ca2+/calmodulin-dependent protein kinase II

CHO Chinese hamster ovary cells

CNS Central nervous system

COS-7 African green monkey kidney cell line

DAG Diacyl glycerol

DMEM Dulbecco's modified eagle medium

DMT N,N-dimethyltryptamine

DOI 2,5-dimethoxy-4-iodoamphetamine

DOB 2,5-dimethoxy-4-bromoamphetamine

DynaminK44A Dominant negative dynamin

ELISA Enzyme-linked immunosorbent assay

FBS Fetal bovine serum

GFP Green fluorescent protein

GPCR G protein-coupled receptor

GRK GPCR kinase

GRK2K22R GRK2 dominant negative

HA Haemagluttenin

HA-5-HT2AR 5-HT2A receptor, tagged on the N-terminus with HA

HEK-293 Human embryonic kidney cells

HT Heterozygous i.c.v. Intracerebroventricular

INMT Indoleamine N-methyltransferase

xvi i.p. intraperitoneal

IP3 Inositol-1,4,5-triphosphate

KO knockout

LSD Lysergic acid diethylamide m-CPP m-chloro-phenylpiperazine

MAO Monoamine oxidase

MAO-I Monoamine oxidase inhibitor

MAP kinase Mitogen activated protein kinase

MAP Microtubule-associated protein

MEF Mouse embryonic fibroblast

MEM Minimal essential medium

MSCV Murine stem cell virus

MTZ N,N‟ Bis-(3-methyl-2-thiazolidinylidene)-succinamide

Myc-arr2 Arrestin2, tagged on the N-terminus with myc

NIH-3T3 NIH mouse embryonic fibroblasts

N-Me-5-HT N-methylserotonin

PBS Phosphate buffered saline

PC12 Rat adrenal medulla pheochromocytoma cells

PCA P-chlorophenylalanine

PET Positron emission tomography

PI Phosphatidylinositol

PI3-K Phosphoinositide-3 kinase

PKC Protein kinase C

PLA2 Phospholipase A2

xvii

PLC Phospholipase C-

PLD Phospholipase D

PCP Phencyclidine

PP2A Protein phosphatase 2A

PSD-95 Post-synaptic density protein-95

RSK p90 kDa ribosomal S6 family of serine/threonine kinases

SAH S-adenosyl-L-homocysteine siRNA Small interfering RNA

SSRI Selective serotonin reuptake inhibitor

TPH Tryptophan hydroxylase

WT Wild-type

YFP Yellow fluorescent protein

xviii

Chapter 1

Introduction

1.1 The 5-HT2A receptor as a clinical target

The biogenic indoleamine neurotransmitter serotonin (5-hydroxytryptamine or 5-HT) is involved in numerous physiological and behavioral systems in the periphery and within the central nervous system. Serotonin modulates diverse physiologies including cardiovascular function, platelet aggregation, respiration, gastrointestinal function, thermoregulation, circadian rhythms, appetite, aggression, pain processing, mood, learning, memory and cognition. Serotonin mediates these effects through the activation of seven major serotonin receptor (5-HT receptor) families, which are comprised of at least 15 genetically unique receptors. With the exception of the 5-HT3 receptor, which is a ligand-gated ion channel, all of the serotonin receptors are G protein-coupled receptors (GPCR). The serotonin receptors have been the focus of extensive research efforts and are prominent targets for pharmacotherapies for the treatment of conditions such as eating disorders, migraines, gastrointestinal diseases and a wide range of psychiatric disorders, including antidepressants, anxiolytics and antipsychotics (1, 2).

One member of the 5-HT2 receptor family (which is comprised of 5-HT2A, 5-HT2B and 5-

HT2C receptors), the 5-HT2A receptor, is of particular interest because disregulation of the

5-HT2A receptor has been implicated in several mental health disorders, including depression, schizophrenia, mania and anxiety (3). Most notably, the 5-HT2A receptor has become a prominent target for the treatment of psychotic disorders. The atypical antipsychotics, such as clozapine, risperidone, olanzapine and ziprasidone, mediate their actions in part through antagonism of the 5-HT2A receptor (4, 5). Furthermore, blockade of the 5-HT2A receptor with the highly selective 5-HT2A receptor antagonist,

M100907, completely prevents phencyclidine (PCP)-induced hyperlocomotion in rats, an animal model of a psychotomimetic state present in schizophrenia (6). Preclinical trials with M100907 have also demonstrated that the selective antagonism of the 5-HT2A receptor is effective for the treatment of some of the symptoms of schizophrenia in hospitalized patients (7). In another double blind study, treatment with the highly selective 5-HT2A receptor inverse agonist pimavanserin decreased the experience of hallucinations in patients experiencing Parkinson‟s disease-associated psychosis (8).

In addition to being a major drug target for the treatment of mood disorders, 5-HT2A receptors mediate the psychoactive effects of serotonergic hallucinogenic drugs, such as lysergic acid diethylamide (LSD), N,N-dimethyltryptamine (DMT), 2,5-dimethoxy-4- iodoamphetamine (DOI) and psilocybin. All serotonergic hallucinogens are agonists at the 5-HT2A receptor, though most have nearly equal potency at both 5-HT2A receptors and 5-HT2C receptors (9). Several lines of evidence exist to strongly suggest that hallucinogens mediate their psychotomimetic effects through the activation of 5-HT2A receptors, specifically. Apart from humans, there are no animal models of hallucinations.

In rodents, however, the behavioral and neuropharmacogical effects of hallucinogenic drugs can be compared through the two-lever drug discrimination test, wherein there is a

2 strong correlation between a hallucinogen‟s potency in humans and its potency in the test in rats (9). Antagonism of the 5-HT2A receptor with ketanserin or M100907 blocks the discriminative cue of hallucinogens in rats (10, 11), while treatment with the selective 5-HT2C receptor antagonist, SB 200646A, has no effect on the discriminative stimulus effects of DOI (12). The most convincing evidence, however, that the psychotomimetic effects of hallucinogens are due to the activation of 5-HT2A receptors was presented in a study by Vollenweider et al. (13), who showed that the 5-HT2A receptor antagonists ketanserin and ritanserin abrogate the hallucinogenic effects of psilocybin in humans.

Though all serotonergic hallucinogens are agonists at the 5-HT2A receptor, not all agonists at the 5-HT2A receptor are hallucinogenic. For instance, serotonin, ergotamine and lisuride have structural similarities to hallucinogenic drugs, are agonists at the 5-

HT2A receptor, but do not induce hallucinations in humans (14, 15). This suggests that there are distinct differences in the 5-HT2A receptor-mediated regulation and signaling that occur for hallucinogenic and non-hallucinogenic 5-HT2A receptor agonists, yet these differences remain unclear. Moreover, these findings indicate that the elucidation of the molecular and cellular mechanisms which underlie such divergence in 5-HT2A receptor function in vivo may reveal novel strategies for the development of drugs for the treatment of 5-HT2A receptor-related disorders.

1.2 Biosynthesis and metabolism of serotonin

As serotonin cannot cross the blood-brain barrier, brain serotonin levels are determined by the balance between the synthesis and metabolism of the neurotransmitter (Figure

1.1). Serotonin is synthesized in the periphery and the brain in a two-step process from the amino acid tryptophan, which is essential to the human diet. Tryptophan is hydroxylated to form 5-hydroxytryptophan (5-HTP) by two isoforms of the enzyme tryptophan hydroxylase (TPH): TPH-1 is expressed in the periphery and TPH-2 is preferentially expressed in the central nervous system (CNS) (16, 17). Once synthesized, 5-HTP is decarboxylated by aromatic amino acid decarboxylase (AADC) to yield serotonin. The primary route for serotonin metabolism is through oxidation by monoamine oxidase A (MAO-A) into 5-hydroxyindoleacetic acid (5-HIAA), an inert metabolite which can then be transported out of the brain and excreted. An alternate route of metabolism involves the N-methylation of serotonin by indoleamine N- methyltransferases (INMT) to generate N-methyltryptamines, including N- methylserotonin and N,N-dimethylserotonin (bufotenine) (18-22). The N- methyltryptamines are also substrates for MAO-A.

Given the multiple steps in the synthesis and metabolism of serotonin, there are several ways to pharmacologically modulate serotonin levels in vivo. Both tryptophan and 5-HTP can be transported across the blood-brain barrier, therefore systemic injection of either compound increases brain serotonin levels (23, 24). Moreover, inhibition of the enzymes in the either the biosynthesis or metabolic pathways can affect brain serotonin concentrations. Treatment with drugs like p-chlorophenylalanine, an irreversible tryptophan hydroxylase inhibitor, or the decarboxylase inhibitor methyldopa, deplete serotonin levels in the brain (25). In contrast, inhibitors of MAO-A (MAO-I), such as clorgyline, or inhibitors of INMT, such as N,N-Bis-(3-methyl-2-thiazolidinylidene)- succinamide (MTZ) or S-adenosyl-L-homocysteine (SAH), enhance serotonin levels in

4 the brain by blocking the metabolic pathways (26-30). The modulation of serotonin levels in the CNS also affects the concentration of its metabolites in the brain, which has physiological relevance because, in contrast to 5-HIAA which is psychoactively inert, the

N-methyltryptamines have psychoactive properties and are agonists at the 5-HT2A receptor (31-34).

1.3 5-HT2A receptor activity in vivo

Neuroanatomical localization of the 5-HT2A receptor

Autoradiography and immunohistochemical studies have shown that the 5-HT2A receptor is expressed in many regions within the CNS, with highest expression in the claustrum and all areas and laminae of the cortex, and lower expression levels in the olfactory system, septum, hippocampus, basal ganglia, amygdala, diencephalon, cerebellum, brainstem and spinal cord (35, 36). A PET study in humans demonstrates that the frontal and temporal cortices have the highest levels of binding of the high affinity 5-HT2A receptor ligand N1-[11C]-methyl-2-bromo-LSD (37). At a cellular level, electron and confocal microscopy of immuno-labeled 5-HT2A receptors show that the 5-HT2A receptor is primarily expressed post-synaptically on the spines and dendrites of pyramidal neurons within the middle layers of the prefrontal cortex, with lesser expression on the dendrites of local circuit gamma aminobutyric acid (GABA)ergic interneurons within this region (38-41).

Hallucinogens appear to induce their effects through the activation of 5-HT2A receptors expressed in specific brain regions. By using Fos activity as a model of neuronal activation, the Sanders-Bush laboratory demonstrated that LSD activates 5-HT2A

5 receptors expressed specifically in the rat prefrontal and anterior cingulate cortices (42).

Similarly, the hallucinogen psilocybin increases glucose metabolism in the frontal cortex, anterior cingulate cortex and temporomedial cortex of healthy human volunteers (43).

These studies suggest that activation of 5-HT2A receptors expressed in the frontal cortex and anterior cingulate cortex primarily underlie the experience of hallucinations.

The head twitch response: a behavioral model of 5-HT2A receptor activation

5-HT2A receptor activation in rodents is manifested as a rapid, discrete shaking of the head, termed the head twitch response. The head twitch response was first described by

Corne et al. (23) as a method for assessing the central actions of serotonin in vivo.

Systemic injection of 5-HTP induces the head twitch response in rodents (23, 44, 45).

This response is presumably due to the resulting increase in brain serotonin levels rather than the direct actions of 5-HTP per se, as it can be significantly attenuated by pretreatment with decarboxylase inhibitors, which prevent serotonin synthesis, or potentiated by pretreatment with MAO-A inhibitors, which block the main degradation pathway (23). Moreover, the time-course of the response closely correlates to the rise and fall of serotonin levels in the brain (23). It was also determined that this response is due to the central action of serotonin receptors, since the systemic injection of serotonin, which is not brain penetrant, does not induce the head twitch response (23, 46), yet head twitches are induced by the direct injection of serotonin into the intracerebroventricular (i.c.v.) space (45, 47, 48).

Extensive pharmacological studies have demonstrated that the 5-HT2A receptor is the target for the head twitch response in rodents. Initial observations that agonists with

6 strong affinity for the 5-HT2A receptor, such as LSD, DMT, DOI, psilocin, mescaline and quipazine, all induce the head twitch response in rodents (46, 49-51), implicated the 5-

HT2A receptor in the activation of this behavioral response. Antagonist studies with

M100907, which has 200-fold selectivity for 5-HT2A receptors over 5-HT2C receptors, demonstrate that the selective blockade of the 5-HT2A receptor is sufficient to prevent the agonist-induced head twitch responses (44, 52-55). Finally, 5-HT2A receptor knockout

(KO) mice do not display head twitches following treatment with a range of hallucinogenic drugs, including LSD, DMT or DOI, or following treatment with 5-HTP (51,

56, 57), J.A. Gingrich and B.L Roth communication to L.M. Bohn).

Several experiments have also demonstrated that the head twitch response is due to the activation of 5-HT2A receptors expressed specifically in the rodent frontal cortex. For example, the direct bilateral administration of the 5-HT2A receptor agonists m-chloro- phenylpiperazine (m-CPP) and DOI into the rat medial prefrontal cortex activates the head twitch response (54). Moreover, the Gingrich laboratory selectively restored 5-HT2A receptor expression to cortical glutamatergic (primarily pyramidal) neurons of 5-HT2A receptor-KO mice by crossing them with a second line of mice expressing cre- recombinase under the control of the Emx1 promoter. The resulting cortical 5-HT2A receptor expression was sufficient to rescue the LSD- and DOI-mediated head twitch responses in mice (51). Collectively, these studies implicate the head twitch response as an in vivo model of selective 5-HT2A receptor activation in the mouse frontal cortex.

1.4 5-HT2A receptor signaling

Like other GPCRs, the 5-HT2A receptor is a seven-transmembrane spanning receptor which transduces extracellular signals by coupling to heterotrimeric G proteins and effectors (Figure 1.2A). The most established 5-HT2A receptor mediated signaling pathway involves the receptor coupling to Gq, resulting in the activation of phospholipase C- (PLC). PLC then hydrolyzes phosphatidylinositol (PI) membrane lipids, generating the second messengers diacyl glycerol (DAG) and inositol-1,4,5-

2+ triphosphate (IP3), the latter causing the release of Ca from intracellular stores. These in turn also activate protein kinase C (PKC). 5-HT2A receptor-mediated activation of PLC has been shown in vivo, as serotonin stimulates PI hydrolysis in the rat cerebral cortex and thoracic aorta, an effect that can be blocked by the 5-HT2A receptor antagonist ketanserin (58, 59).

Activation of 5-HT2A receptors also stimulates other phospholipases, including phospholipase A2 (PLA2) and phospholipase D (PLD) (60, 61). The activation of PLA2 hydrolyzes arachidonic acid (AA) containing phospholipids, producing free AA and lysophospholipids. Serotonin stimulates the release of AA in a manner that is independent of the PLC-mediated signaling cascade in hippocampal neurons (62), although the mechanism by which the receptor mediates PLA2 activation is poorly understood. A study from the Nichols laboratory has demonstrated that in NIH-3T3 mouse embryonic fibroblasts, serotonin activates PLA2 through a complex 5-HT2A receptor signaling cascade, involving both Gi/o/G-mediated and G12/13/Rho-mediated pathways (63). Administration of DOI to rats also leads to an increase in the

3 incorporation of [ H]-AA in the frontal cortex, demonstrating that 5-HT2A receptors can activate PLA2 signaling in vivo (64). 5-HT2A receptor coupling to PLD catalyzes the

8 hydrolysis of phosphatidylcholine, resulting in the generation of phosphatidic acid and choline. In COS-7 African green monkey kidney cells, the mechanism for serotonin- induced activation of PLD is independent of Gq and involves 5-HT2A receptor interactions with the small G protein, ADP-ribosylation factor (ARF), although the pathway is again not well understood (65).

In vitro, 5-HT2A receptor activation also initiates the activation of mitogen activated protein kinase (MAP kinase) cascades, including ERK1/2 and p38 (44, 63). Quinn et al.

(66), showed that in rat adrenal medulla pheochromocytoma (PC12) cells, 5-HT2A receptor-mediated activation of ERK1/2 by serotonin is Ca2+- and calmodulin-dependent, suggesting the involvement of the Gq/PLC pathways. However, they also found that pretreatment with PP1, an inhibitor to the tyrosine kinase Src, reduces ERK1/2 activation in these cells. Moreover, inhibitors to Src and phosphoinositide-3 kinase (PI3-K) also reduce serotonin-induced ERK1/2 phosphorylation in rat aortic myocytes and reduce serotonin-induced contraction of the rat aorta ex vivo, which has been shown to be a 5-

HT2A receptor-mediated event (67, 68). These studies suggest the 5-HT2A receptor uses multiple pathways to stimulate ERK1/2 activation in different cell types.

Functional selectivity at the 5-HT2A receptor

Traditionally, the conventional understanding of receptor pharmacology has been that an agonist fully activates all signal transduction pathways to which a GPCR is coupled, while partial agonists induce sub-maximal activation of these same pathways.

Antagonists do not shift the any of the responses away from basal levels, yet block further signaling, and inverse agonists reduce the basal activities of these pathways.

However, these concepts of receptor pharmacology are too simple to conceptualize the full range of pharmacological profiles that are experimental observed. For example, agonists at a particular GPCR can display full efficacy in certain signaling assays, while only partially activating or having no activity at others (69-71). Current receptor pharmacology is based on the understanding that receptors can exist in multiple, ligand- specific conformational states which allow GPCRs to preferentially and differentially engage a subset of the multiple signaling pathways to which they are coupled. This concept that ligands differentially activate downstream signaling pathways has been referred to as “biased agonism” or “functional selectivity” (Figure 1.3) (72-75). The cellular environment can also influence the signaling that occurs downstream of GPCR activation by determining the complement of intracellular proteins available to couple to a receptor. In this way, the same ligand can induce differential signaling cascades at a particular receptor expressed in different cell types (76).

Functional selectivity has been demonstrated at the 5-HT2A receptor, as the G protein and phospholipase-mediated signaling pathways can be differentially activated by different agonists. For example, Berg et al. (77) used Chinese hamster ovary (CHO) cells expressing the 5-HT2A receptor to compare the abilities of a panel of 5-HT2A receptor agonists to induce AA release and IP3 accumulation. They show that agonists like tryptamine and bufotenine have efficacies equal to or greater than serotonin for stimulating AA release, but lower efficacies for stimulating IP3 accumulation. In contrast,

DOI and LSD have similar efficacies for the AA pathway (both less than serotonin), but differ in their efficacies at the IP3 pathway. A similar study in NIH-3T3 cells also

10 demonstrates that some agonists are more efficacious at the PLA2 pathway than the

PLC pathway, and vice versa (78).

The fact that some, but not all agonists at the 5-HT2A receptor are hallucinogenic provides in vivo relevance to the idea that agonists can differentially direct 5-HT2A receptor signaling. A number of studies have attempted to correlate the activation of specific signaling pathways with the action of hallucinogens. Nearly all of the hallucinogens examined by Berg et al. (77) and Kurrasch-Orbaugh et al. (78) have greater efficacy at stimulating AA release than IP3 accumulation, leading to the hypothesis that AA release is more relevant to the actions of hallucinogens. However, hallucinogenic agonists do not differ from non-hallucinogenic agonists in their ability to activate either pathway (9, 77, 78), suggesting that the mere activation of these pathways is not sufficient for the induction of hallucinations. The Gingrich and Sealfon laboratories have compared changes in gene transcription that occur in the mouse frontal cortex following treatment with the hallucinogens DOI and LSD to those that occur following treatment with the non-hallucinogenic compound lisuride. They found that DOI and LSD induce changes in transcription that are similar to each other, but the changes in transcription induced by hallucinogenic drugs drastically differ from those induced by lisuride (56). For instance, they show that while both LSD and lisuride activate c-fos expression in the mouse frontal cortex and in primary cortical neurons, only LSD induces expression of the transcription factor egr-2 and neither transcription factor is induced in 5-HT2A receptor-KO mice (51). Therefore, hallucinogenic and non- hallucinogenic agonists have differential effects on gene transcription in vivo, yet the

11 signaling pathways underlying these differences and the physiological implication for the activation of these pathways has yet to be identified.

1.5 Classical regulation of GPCRs by arrestins

5-HT2A receptor responsiveness is determined by the both the agonist activated signaling cascades and regulatory mechanisms that interact with the receptor to control the extent and duration of the response. The canonical model of GPCR regulation posits that the agonist bound receptor is regulated through its interactions with the serine/threonine GPCR kinases (GRKs) and the intracellular regulatory proteins,

arrestins (Figure 1.2). Upon agonist activation, GPCRs are phosphorylated by GRKs, which initiate the desensitization of G protein-mediated signaling by promoting the recruitment of arrestins. Once recruited, arrestins can further inhibit G protein- coupling, initiate the internalization of receptors and scaffold additional signaling molecules to GPCRs, thus facilitating G protein-independent signaling cascades (79).

GRKs can be divided into three main families: the visual GRKs (GRK1 and 7), the cytosolic GRKs (GRK2 and 3) and the palmitoylated, membrane associated GRKs

(GRK4, 5 and 6). GRK2, 3, 5 and 6 are widely expressed throughout the CNS, while

GRK4 expression is limited to the purkinje cells of the cerebellum (80). GRKs can interact with both the cytoplasmic regions of GPCRs and the  and  subunits of the heterotrimeric G proteins (81). In this way, GRKs can simultaneously phosphorylate receptors and in some cases can sequester already released G and G subunits, preventing further activation of their effectors.

The phosphorylation of the agonist-bound GPCR by GRKs causes arrestins to translocate to the receptor. Arrestin recruitment to, and interaction with, GPCRs has been visualized by confocal microscopy with fluorescently tagged arrestins (82) and quantified by co-immunoprecipitation, bioluminescence/fluorescence resonance energy transfer, gene reporter and enzyme fragment complementation assays (83-87). There are four arrestin isoforms: the visual arrestins (arrestin1 and arrestin4) and the arrestins

(arrestin1/arrestin2 and arrestin2/arrestin3). Though the two arrestins have very similar amino acid sequences and are, for the most part, ubiquitously expressed, some

GPCRs are preferentially regulated by one isoform over the other (88, 89). The 2- adrenergic receptor,  opioid receptor, endothelin type A receptor, D1 dopamine receptor, and 1B-adrenergic receptor bind arrestin2 with a higher affinity than

arrestin1, while the angiotensin II type 1A (AT1A) receptor, neurotensin receptor 1, V2 vasopressin receptor, thyrotropin-releasing hormone receptor and substance P receptor bind both arrestins with equal affinity (88).

Desensitization of G protein coupling

arrestin interactions with GPCRs serve to desensitize further signaling by sterically blocking further interactions between receptors and their cognate G proteins (Figure

1.2B) (90). In vitro, the role of arrestins in the desensitization of many GPCRs has been shown by over-expressing arrestins, thus enhancing receptor desensitization, or by disrupting GPCR interactions with arrestins, thereby diminishing the desensitization of the signaling pathways (91-93). For example, the over-expression of either arrestin1 or

arrestin2 increases the desensitization of 2-adrenergic receptor coupling to Gs (94).

In contrast, agonist-induced 2-adrenergic receptor signaling is enhanced in human embryonic kidney (HEK-293) cells in which both arrestins have been silenced by small interfering RNAs (siRNA) or in mouse embryonic fibroblasts (MEF) generated from genetically modified mice lacking both arrestins (arr1/2-KO) (89, 95).

This negative regulatory role for arrestins has also been demonstrated in vivo through the use of mice lacking arrestin2 (arr2-KO). Chronic morphine administration desensitizes the  opioid receptor expressed in brainstem from wild-type (WT) mice, while the  opioid receptor retains its ability to couple to G proteins in brainstems isolated from arr2-KO mice (96). Furthermore, arr2-KO mice also display enhanced antinociceptive responses to acute morphine and develop significantly less tolerance to the hot-plate test following chronic treatment with the opioid, compared to their WT littermates (96-98), implicating the desensitization of the opioid receptor by arrestin2 as an integral step in the development of antinociceptive tolerance to morphine.

Enhanced signaling profiles or behavioral responses in the absence of arrestins, either in vitro or in vivo, suggest that arrestins are serving as negative regulators of these receptor-mediated signaling cascades.

Internalization of GPCRs into clathrin-coated pits

Arrestin interactions with GPCRs also facilitate the internalization of receptors into intracellular vesicles (Figure 1.2C). Arrestins promote GPCR internalization by acting as adaptors which link agonist-bound receptors to elements of clathrin-coated pits. Both

arrestin1 and arrestin2 have been shown to directly interact with clathrin and the 2-

14 adaptin subunit of the adaptor protein 2 (AP2) complex (99-102). The arrestin-mediated recruitment of these proteins then targets receptors for endocytosis. Interfering with

GPCR/arrestin interactions has been shown to impair agonist-mediated trafficking. In

HEK-293 cells, a 5-HT2C receptor mutant that has decreased interactions with arrestin2

(5-HT2C receptorP159A) is restricted to the plasma membrane rather than being constitutively internalized in the absence of agonist, like the WT receptor (103).

Furthermore, the 2-adrenergic receptor and protease-activated receptor-2 fail to internalize in response to their respective agonists in arr1/2-KO MEFs (89, 104). The internalization of a GPCR then aids in the determination of receptor fate, as it can direct receptors to endosomes for recycling or to lysosomes for degradation (105), thus implicating arrestins in receptor resensitization and down-regulation.

Facilitation of downstream signaling by arrestins

Although arrestins are traditionally thought to negatively regulate GPCRs, they can also mediate GPCR signaling by scaffolding elements of signal transduction cascades to receptors (Figure 1.2D). arrestins have been shown to act as adaptors between

GPCRs and components of many signaling pathways, including the ERK1/2, p38 and c-

JNK MAP kinases, the Src family of tyrosine kinases, PI3-K and protein kinase B (Akt) in vitro (106-110). In contrast to arrestins‟ role in dampening G protein coupling, the facilitation of signal transduction by arrestins has been demonstrated for a number of

GPCRs in vitro and is manifested as a decreased response in the absence of arrestins

(111-115). Moreover, while both arrestins and G proteins can mediate signaling through the same downstream effectors, the two pathways have different time-courses

15 of activation. For example, the AT1A receptor activates ERK1/2 via both pathways: a rapid G protein-mediated pathway and a slower and more persistent arrestin2- dependent pathway (116). In vivo, arr2-KO mice display decreased behavioral responses to psychostimulants, a response that has been associated with arrestin- mediated signaling through D1 and D2 dopamine receptors (117-119). Therefore, decreased in vivo responsiveness to a drug in the absence of arrestins implicates the proteins in facilitating the GPCR-mediated signaling events underlying the behavioral response.

Arrestin bias

Ligands can also selectively direct interactions between GPCRs and arrestins. For instance, the opioids etorphine, morphine and herkinorin are all agonists at the  opioid receptor, yet while etorphine robustly recruits both arrestin1 and arrestin2 to receptors expressed in HEK-293 cells, morphine only recruits arrestin2 under conditions in which

GRKs are over-expressed and herkinorin does not recruit either arrestin to the receptor, even when GRK2 is over-expressed (84, 120). Interestingly, morphine-induced antinociceptive responses are enhanced in arr2-KO mice, while etorphine-induced responses are unchanged in the absence of arrestin2 (121). In light of the in vitro recruitment studies, the differences between the ligands are presumably due to the etorphine-bound, but not the morphine-bound,  opioid receptor being able to be regulated by arrestin1. Collectively, these studies in the arr2-KO mice demonstrate that the ligand-directed interactions between arrestins and GPCRs can have major implications for receptor responsiveness in vivo.

1.6 Regulation of the 5-HT2A receptor

Like other GPCRs, the 5-HT2A receptor is subject to regulation by arrestins, yet the regulation of the receptor and the roles that arrestins play in these processes have only been partially determined. Dual label fluorescence confocal microscopy has demonstrated that the 5-HT2A receptor and both arrestin1 and arrestin2 are co- expressed in many, but not all pyramidal neurons in the rat frontal cortex (122).

Furthermore, in this same study, Gelber et al. (122) demonstrated that purified

arrestin1 and arrestin2 interact with fusion proteins encoding the third intracellular loop of the 5-HT2A receptor in vitro. Given that the 5-HT2A receptor is co-expressed with and can interact with arrestins, these data support a role for arrestins in the regulation of the receptor.

Arrestins have also been shown to be recruited to the 5-HT2A receptor following agonist-treatment in vitro. Serotonin does induce interactions between the 5-HT2A receptor and arrestin2 in HEK-293 cells, as determined by co-immunoprecipitation of the 5-HT2A receptor and myc-tagged arrestin2 (myc-arr2) (123). Furthermore, confocal microscopy with fluorescently tagged arrestins shows that serotonin induces marginal translocation of arrestin1 to the plasma membrane of HEK-293 cells expressing the 5-

HT2A receptor, while quipazine induces robust translocation of arrestin2 (124). This could indicate that the 5-HT2A receptor has a higher affinity for arrestin2 than

arrestin1, although the assay is neither ratiometric nor quantitative and may simply reflect properties of the transfected arrestin constructs. Moreover, the differences in the

17 degree of recruitment could also be agonist-dependent and the converse studies were not completed. Regardless, the fact that arrestins are recruited to the receptor in the presence of agonist further implicates their involvement in the regulation of the 5-HT2A receptor.

Desensitization of the 5-HT2A receptor

Cell culture studies have demonstrated that exposure to agonist results in the desensitization of 5-HT2A receptor-mediated PI hydrolysis (125, 126). The Roth laboratory systematically mutated all of the serine and threonine residues in the cytoplasmic domains of the 5-HT2A receptor and assessed the agonist-mediated desensitization of the IP3 pathway. They show that the mutation of two serine residues, serine 421 in the C-terminal tail and serine 188 in the second intracellular loop, results in significant inhibition of quipazine-induced desensitization (127). These findings suggest that the phosphorylation of the 5-HT2A receptor can impact agonist-induced desensitization; however, the studies detailed below demonstrate that the mechanism by which the 5-HT2A receptor is desensitized varies for different cell types. In rat C6 glioma cells, which endogenously express the 5-HT2A receptor, quipazine and serotonin- induced desensitization of PI-hydrolysis is attenuated by a dominant negative arrestin1

(arr1319-418), that encodes only the C-terminal tail of the protein and not the GPCR binding domain (128-130). The expression of a dominant negative GRK2 (GRK2K22R), which lacks kinase activity (131), also blocks serotonin-mediated desensitization in C6 glioma cells (130). In contrast, the arr1319-418 dominant negative has no effect on quipazine-induced desensitization in HEK-293 cells transiently transfected with the 5-

HT2A receptor (129). Serotonin-mediated desensitization of PI hydrolysis is also

18 unaffected by GRK2K22R in CHO cells stably expressing the 5-HT2A receptor (132).

Therefore, while the 5-HT2A receptor can be classically desensitized via interactions with

GRKs and arrestins, there also appear to be alternative pathways for the desensitization of the receptor.

GPCRs can also be desensitized through interactions with second messenger- dependent kinases, such as PKC. The 5-HT2A receptor contains five putative PKC phosphorylation sites in its intracellular domains (133), suggesting that PKC may be involved in the desensitization of this receptor. Berg et al. (132) demonstrated that the

PKC inhibitors staurosporine and bisindolylmaleimide significantly inhibit serotonin- induced desensitization of PI hydrolysis in CHO cells stably transfected with the 5-HT2A receptor. However, PKC-mediated desensitization is also cell-type specific, as inhibition of PKC fails to alter serotonin-mediated desensitization of the IP3 pathway in Chinese hamster lung fibroblasts stably expressing the 5-HT2A receptor (133).

Another serine/threonine kinase, p90 kDa ribosomal S6 family of serine/threonine kinases (RSK), has also been implicated in the agonist-mediated desensitization of the

5-HT2A receptor. Studies from the Roth laboratory have shown that RSK2 co- immunoprecipitates with the 5-HT2A receptor in HEK-293 cells, C6 glioma cells and rat cortical homogenates and is co-expressed with the 5-HT2A receptor in neurons in the rat frontal cortex (134, 135). Moreover, purified and activated RSK2 directly phosphorylates serine 314 of the third intracellular loop of the 5-HT2A receptor in vitro (135). The activation of intracellular Ca2+ release, PI hydrolysis and ERK1/2 phosphorylation are all potentiated following treatment with a panel of 5-HT2A receptor agonists in RSK2-KO

MEFs or in WT MEFs expressing a RSK2-insensitive 5-HT2A receptor mutant (5-HT2A receptorS314A) (134-136). The enhanced responses observed in the absence of 5-HT2A receptor interactions with RSK2 suggest that RSK2 may act to dampen 5-HT2A receptor signaling in vitro. Taken together, these data demonstrate that there are multiple pathways by which the 5-HT2A receptor can be desensitized and that both the cell-type and the agonist may determine which pathway is utilized.

Internalization of the 5-HT2A receptor

Agonist stimulation leads to the internalization of the 5-HT2A receptor in vitro. Treatment of NIH-3T3 cells with quipazine induces trafficking of the 5-HT2A receptor from the cell surface to intracellular vesicles that co-express clathrin (137, 138). Moreover, pretreatment with concanavalin A or phenylarsine oxide, two chemical inhibitors of endocytosis, inhibits the quipazine-induced internalization of the 5-HT2A receptor in HEK-

293 cells (129). Finally, a dominant negative to dynamin (dynaminK44A) also completely attenuates serotonin-mediated 5-HT2A receptor internalization in HEK-293 cells (124).

Collectively, these studies suggest that the receptor can be internalized through the clathrin-mediated pathway.

Although arrestins facilitate the clathrin-mediated endocytosis of GPCRs, their involvement in the trafficking of the 5-HT2A receptor is not fully understood. For instance, expression of arr1319-418 or a similar dominant negative for arrestin2 (arr2284-409) (139), has no effect on quipazine-induced internalization of the 5-HT2A receptor as assessed by confocal microscopy (124). The arr1319-418 mutant also does not affect serotonin- mediated internalization of the receptor, as quantified by a cell-surface biotinylation

20 assay (124), suggesting that arrestins are not involved in the internalization process in

HEK-293 cells. However, another study from the Roth laboratory has demonstrated that the transfection of a constitutively active arrestin1 mutant (arr1R169E), which binds to

GPCRs regardless of the phosphorylation state of the receptor (140-142), results in the constitutive internalization of the 5-HT2A receptor in HEK-293 cells, as determined by confocal microscopy. The 5-HT2A receptor also co-immunoprecipitates with the

arr1R169E mutant in the absence of agonist (143), suggesting that the internalization of the 5-HT2A receptor is sensitive to the expression of arrestins in vitro. Although the dominant negative arrestin mutants are thought to compete in receptor/clathrin-coated pit assembly, they may not fully inhibit the function of the endogenous arrestins, which could explain these differential findings.

The 5-HT2A receptor has also been shown to be internalized in vivo. Several immunohistochemical studies have demonstrated that the 5-HT2A receptor is found to be internalized in cortical neurons from untreated rats (36, 40), suggesting that the receptor is constitutively internalized. Moreover, arrestins have been shown to co-localize with the 5-HT2A receptor within intracellular vesicles of rat cortical neurons (122). These studies further indicate that arrestins may be involved in the internalization of the 5-

HT2A receptor in vivo and, in light of the conflicting results of the in vitro studies described above, suggest that the mechanisms employed may be cell-type specific.

Like many GPCRs, the 5-HT2A receptor can be down-regulated following exposure to agonists, both in vitro and in vivo. For instance, daily administration of LSD or DOI for 7 days significantly decreases [3H]-ketanserin binding in the rat cortex (144, 145). 21

However, in vitro studies have shown that down-regulation can differ depending upon cell-type or agonist, as, exposure to serotonin for 8 hours decreases [125I]-LSD binding in

P11 rat pituitary tumor cells (125), while exposure to DOI for up to 24 hours had no effect on [3H]-ketanserin binding in NIH-3T3 cells (126). Interestingly, chronic treatment with 5-HT2A receptor antagonists, such as clozapine, can also down-regulate the receptor in the rat frontal cortex (146, 147). Though receptor endocytosis can play a role in the down-regulation of some GPCRs by directing them towards degradation pathways

(148), the mechanism of 5-HT2A receptor down-regulation by both agonists and antagonists and the involvement of receptor trafficking is not defined.

Internalization of GPCRs can also lead to the de-phosphorylation and recycling of receptors for trafficking back to the plasma membrane, a process termed resensitization.

The 5-HT2A receptor can also be resensitized following agonist treatment in vitro (132).

The arr1319-418 and dynaminK44A dominant negatives significantly inhibit the resensitization of 5-HT2A receptor-mediated PI hydrolysis after quipazine-induced desensitization in C6 glioma cells (129). In contrast, both dominant negatives actually potentiate the resensitization of the 5-HT2A receptor in HEK-293 cells (129, 143). These studies demonstrate that requirement of receptor internalization and the involvement of

arrestins in the resensitization process again appears to be cell-type specific.

Furthermore, the potentiation of 5-HT2A receptor resensitization in HEK-293 cells under conditions in which internalization is blocked, suggests the existence of a mechanism in which the 5-HT2A receptor can be resensitized on the cell-surface, a phenomenon that has also been shown to occur previously for the 2-adrenergic receptor (149).

5-HT2A receptor regulation by additional scaffolding proteins

Interactions with other intracellular proteins can affect the regulation of the 5-HT2A receptor by mediating receptor trafficking and localization, scaffolding components of receptor complexes and modifying receptor signaling (150). To date, the 5-HT2A receptor has been shown to interact with microtubule-associated protein (MAP) 1A (40), postsynaptic density protein-95 (PSD-95) (151), calmodulin (152), caveolin-1 (153) and

ARF1 (154), in addition to arrestins, GRKs, PKC and RSK2. Two of these proteins,

PSD-95 and caveolin-1, have been shown to affect 5-HT2A receptor trafficking and signaling events.

5-HT2A receptor interactions with PSD-95 impact receptor responsiveness. The 5-HT2A receptor contains a PDZ-binding domain within its C-terminus which directly interacts with the PDZ-domain of PSD-95. The two proteins also co-localize on the cell surface of

HEK-293 cells and primary cortical neurons, as determined by confocal microscopy and co-immunoprecipitation studies (151, 155). The interaction between the two proteins appears to localize the receptor to the plasma membrane, as over-expression of PSD-95 attenuates serotonin-mediated 5-HT2A receptor internalization in HEK-293 cells, an effect that can be blocked by mutation of the receptor PDZ-binding domain (151). Further, inhibition of 5-HT2A receptor/PSD-95 interactions through mutation of the receptor PDZ- binding domain or the use of PSD-95-KO mice abrogates normal dendritic targeting of the receptor and sequesters receptors in the soma of primary cortical neurons (155,

156). Interactions with PSD-95 also impact 5-HT2A receptor signal transduction, as the over-expression of PSD-95 augments serotonin-induced IP accumulation in HEK-293 cells, while inhibiting the interaction by mutation of the receptor PDZ-binding domain

23 reduces serotonin-mediated PI hydrolysis (151). Moreover, DOI-induced head twitch responses and ERK1/2 phosphorylation are attenuated in the cortex of PSD-95-KO mice

(156). Collectively, these studies suggest that PSD-95 is involved in properly targeting the receptor to the apical dendrites of cortical neurons, which appears to be integral for the activation of some 5-HT2A receptor-mediated signaling cascades.

Caveolin-1 is also a multifunctional scaffolding protein involved in the targeting and internalization of GPCRs (157, 158). The 5-HT2A receptor co-immunoprecipitates with caveolin-1 in HEK-293 cells, C6 glioma cells and synaptic membranes prepared from rat frontal cortex (153). Caveolin-1 also co-localizes with the 5-HT2A receptor at the plasma membrane of HEK-293 cells (153). Caveolins can facilitate clathrin-independent, but dynamin-dependent endocytosis of receptors (158), and caveolin-1 may promote 5-HT2A receptor internalization, as serotonin induces the co-localization of the two proteins in intracellular vesicles of HEK-293 cells (153). In addition to internalization, caveolins have been shown to target GPCRs to lipid microdomains of the plasma membrane, and both the 5-HT2A receptor and PLC can localize to these caveolin-containing regions (159,

160), suggesting that caveolin-1 may facilitate downstream signaling events. Likewise, the over-expression of caveolin-1 increases 5-HT2A receptor interactions with Gq and siRNA knockdown of caveolin-1 attenuates serotonin-mediated increases in intracellular

2+ Ca (153). Therefore, caveolin-1 interactions with the 5-HT2A receptor may also serve to scaffold the receptor with Gq and/or PLC in lipid rafts, thereby facilitating agonist- mediated signaling.

1.7 Central hypothesis and outline for chapters

The studies detailed in this chapter demonstrate that while the 5-HT2A receptor is capable of being regulated through interactions with GRKs and arrestins, interactions with other proteins can facilitate receptor desensitization and internalization through non-

arrestin-mediated mechanisms. Moreover, the pathways utilized for each of these events may be determined by both the complement of intracellular proteins expressed in residence with the 5-HT2A receptor and the agonist acting at the receptor. The dissertation work presented herein addresses the hypothesis that the interaction between arrestin2 and the 5-HT2A receptor is a critical point in the divergence of agonist-directed 5-HT2A receptor responsiveness.

In Chapter 2, we evaluate the contribution of arrestins to the regulation of 5-HT2A receptor responsiveness for serotonin and the hallucinogenic 5-HT2A receptor agonist,

DOI. We utilize arr1/2-KO MEFs and arr2-KO mice to assess agonist-mediated 5-

HT2A receptor internalization and ERK1/2 kinase activation for the two agonists. The studies presented in this chapter indicate that serotonin requires arrestins to internalize the 5-HT2A receptor and utilizes arrestin2 to promote ERK1/2 signaling, while DOI can internalize the receptor through arrestin-independent mechanisms and does not activate the arrestin-mediated ERK1/2 signaling cascade. Moreover, 5-HT2A receptor interactions with arrestins have in vivo consequences, as 5-HT2A receptor trafficking is abrogated in arr2-KO cortical neurons and the serotonin-mediated head twitch response is significantly attenuated in arr2-KO mice. In contrast, the DOI-mediated head twitch response is unaffected by the absence of arrestins. The majority of the

25 studies presented in this chapter have been published in The Proceedings of the

National Academy of Sciences (44).

In Chapter 3, we evaluate the differences in ligand directed, 5-HT2A receptor-mediated signaling for serotonin and its endogenous, psychoactive N-methyltryptamine metabolites in vivo. We utilize arr2-KO mice to assess the involvement of arrestins in kinase activation and receptor signaling complex formation following treatment with these agonists. The findings presented in this chapter demonstrate that serotonin utilizes

arrestin2 to scaffold particular signaling partners to the 5-HT2A receptor, leading to the phosphorylation of Akt in both primary cortical neurons and in the mouse frontal cortex.

We then demonstrate the functional significance of this signaling cascade in vivo through the use of kinase inhibitors, wherein we demonstrate that inhibition of any component of this complex attenuates the serotonin-mediated head twitch response. In contrast, the N- methyltryptamines do not activate this arrestin-mediated signaling cascade and inhibition of Akt has no impact on the N-methyltryptamine-induced head twitch response.

The studies presented in this chapter have been published in The Journal of

Neuroscience (45).

The studies presented in this dissertation seek to elucidate differences between 5-HT2A receptor regulation and signaling that occur in the mouse frontal cortex for serotonin and agonists that have known hallucinogenic properties in humans. As the cellular environment in which the receptor is expressed has been shown to play a major role in determining receptor signaling and regulatory mechanisms, determining 5-HT2A receptor responsiveness in physiologically relevant tissues may potentially highlight new, or

26 previously unappreciated targets for the treatment of diseases associated with the disregulation of the 5-HT2A receptor.

1.8 Chapter 1 Figures

PERIPHERY CENTRAL NERVOUS SYSTEM BLOOD BRAIN BARRIER

Tryptophan Tryptophan PCA

(1) TPH-1 (1) TPH-2

5-HTP 5-HTP Methyldopa

(2) AADC (2) AADC MTZ or SAH

5-HT 5-HT INMT N-Me-5-HT (4) Bufotenine (3) MAO-A

5-HIAA MAO-I (clorgyline)

Figure 1.1 Biosynthesis and metabolism of serotonin Serotonin (5-HT) is synthesized from the amino acid tryptophan: (1) tryptophan is converted into 5-HTP by tryptophan hydroxylase (TPH1 or TPH2) and (2) 5-HTP is converted into serotonin by aromatic amino acid decarboxylase (AADC). Both tryptophan and 5-HTP can be transported across the blood brain barrier. Serotonin can then either be (3) oxidized by monoamine oxidase A (MAO-A) into 5-hydroxyindoleacetic acid (5-HIAA), which is excreted, or be (4) methylated by indoleamine N-methyltransferases (INMT) into N-methyltryptamines (such as N-methylserotonin, N-Me-5-HTI; or bufotenine). Serotonin and the N-methyltryptamines are endogenous agonists at the 5-HT2A receptor. Inhibitors to each of the enzymatic steps are indicated in italics (p- chlorophenylalanine, PCA; N,N‟ Bis-(3-methyl-2-thiazolidinylidene)-succinamide, MTZ; S- adenosyl-L-homocysteine, SAH; MAO inhibitors, MAO-I).

Figure 1.2 Canonical model of GPCR regulation A. Agonist binding to a GPCR catalyzes the dissociation of the G subunit from the G heterodimer, initiating downstream signaling events. B. Rapidly following agonist binding to a GPCR, the receptor is phosphorylated by GRKs, which facilitates arrestin binding. Arrestins can then desensitize GPCRs by preventing further coupling to G proteins. C. Arrestins also facilitate GPCR internalization by acting as adaptor proteins between the receptor and clathrin and AP2, proteins involved in endocytosis. D. Arrestins also initiate additional signaling by scaffolding components of non-G protein-mediated cascades to GPCRs.

Figure 1.3 Ligand directed signaling at GPCRs Different agonists (ligand A and ligand B) acting at the same GPCR selectively recruit a subset of the signaling proteins (depicted by different colored shapes) expressed in close proximity to the receptor complex, which leads to the activation of different downstream signaling cascades (effector 1 and effector 3). Some proteins are recruited in a conserved manner to activate the same signaling cascades (effector 2), though the degree of activation of these pathways may differ between agonists (depicted by arrows of different thickness).

Chapter 2

Agonist-directed regulation and signaling of the 5-HT2A receptor is dictated by

interactions with arrestin2

2.1 Introduction

Serotonergic hallucinogenic drugs produce their psychotomimetic effects through the activation of 5-HT2A receptors expressed in the frontal cortex (9, 13). However, not all agonists at the 5-HT2A receptor are hallucinogenic. For instance, neither serotonin nor lisuride have psychotic effects in humans, yet both are fully efficacious at inducing 5-

HT2A receptor coupling to G proteins (14, 15). These findings suggest that the 5-HT2A receptor-mediated signaling pathways activated by hallucinogenic drugs differ from those mediating the physiological actions of serotonin, providing in vivo evidence for functional selectivity at the 5-HT2A receptor. Ligand directed signaling at the 5-HT2A receptor has also been demonstrated in vitro. Comparison of the ability of different agonists to induce either PI hydrolysis or AA release in NIH-3T3 cells reveals that LSD, tryptamine and quipazine preferentially activate the PLA2 pathway over the PLC pathway, while serotonin, lisuride, 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and

2,5-dimethoxy-4-bromoamphetamine (DOB) are more potent for the PLC pathway than the PLA2 pathway (78). These in vivo and in vitro findings demonstrate how agonists at

31 the 5-HT2A receptor can direct differential signaling cascades downstream of receptor activation.

One way in which agonists can direct GPCR responsiveness is through dictating receptor interactions with arrestins. arrestins can either negatively regulate GPCR signaling by preventing further receptor coupling to G proteins or positively mediate receptor signaling by facilitating interactions between GPCRs and additional signaling partners. For example, activation of the AT1A receptor stimulates ERK1/2 phosphorylation via two routes in COS-7 cells: a Gq/PLC-mediated pathway and a

arrestin-dependent/G protein-independent pathway. Consequently, the over-expression of either arrestin1 or arrestin2 in COS-7 cells simultaneously decreases coupling to

Gq and decreases PLC-mediated ERK1/2 activation, while enhancing AT1A receptor signaling through the arrestin-dependent pathway (161). These findings show how the phosphorylation of a downstream signaling kinase such as ERK1/2, can reflect the simultaneous activation of multiple upstream signaling cascades and the dual roles that

arrestins can play in the modulation of their activity.

Agonists have also been described which preferentially induce receptor regulation through arrestin-dependent and arrestin-independent mechanisms. Herkinorin is a full agonist at the opioid receptor in terms of activating G protein coupling and inhibiting cAMP accumulation, yet it does not recruit arrestins and does not internalize the

opioid receptor in vitro (84, 162, 163). In contrast to herkinorin, the AT1A receptor agonist Sar1, Ile4, Ile8-AngII selectively stimulates arrestin2-mediated signaling pathways without inducing any detectable coupling to G proteins (70). These studies 32 provide examples of how ligand directed interactions between GPCRs and arrestins can affect receptor sensitivity in vitro.

Differences in interactions between arrestins and the 5-HT2A receptor may account for some of the variance in 5-HT2A receptor responsiveness observed for different ligands.

While arrestins have been implicated in the desensitization and internalization of 5-

HT2A receptors expressed in some cell types in vitro (129, 130, 143), their roles in the regulation of 5-HT2A receptor signaling remain largely unclear. However, both arrestins and the 5-HT2A receptor are highly expressed in the rodent frontal cortex, and the receptor has been shown to be co-expressed with either arrestin1 or arrestin2 in many cortical pyramidal neurons (38, 40, 41, 94, 122, 164). Moreover, Gelber et al. (122) demonstrated that the 5-HT2A receptor and arrestins can be co-localized in intracellular vesicles within rat cortical pyramidal neurons. In light of these studies, we hypothesized that arrestins are involved in the regulation of the 5-HT2A receptor in vivo.

To address this hypothesis, we assessed differences in 5-HT2A receptor trafficking and signaling in the absence of arrestins. We designed studies to determine whether the

arrestin-mediated regulation of the 5-HT2A receptor differs between serotonin and the hallucinogenic agonist DOI. DOI was chosen because it is selective for the 5-HT2A/2C receptors, unlike many other hallucinogenic drugs which have affinity for numerous

GPCRs (9, 165). In this chapter, we show that serotonin-mediated receptor internalization and ERK1/2 activation are disrupted in arr1/2-KO MEFs and in the mouse frontal cortex isolated from arr2-KO mice. In contrast, DOI-mediated receptor trafficking and ERK1/2 activation are unchanged in the absence of arrestins. 33

Finally, to compare how arrestin regulation impacts 5-HT2A receptor responsiveness in vivo, we assessed serotonin- and DOI-induced head twitch responses in arr2-KO mice.

The head twitch response is used as a behavioral indicator of 5-HT2A receptor activation in the rodent frontal cortex, as 5-HT2A receptor antagonists inhibit the head twitch response in normal mice, and the selective restoration of 5-HT2A receptor expression to cortical pyramidal neurons rescues the head twitch response in 5-HT2A receptor-KO mice (51, 54, 55). We find that the genetic deletion of arrestin2 impacts the serotonin- induced, but not the DOI-induced head twitch response. Collectively, these data indicate that arrestins can facilitate 5-HT2A receptor internalization and signaling and suggest that 5-HT2A receptor interactions with arrestin2 may be a critical point in the divergence of the signaling cascades activated by serotonin and DOI. The results reported in this chapter have largely been published in The Proceedings of the National Academy of

Sciences (44).

2.2 Materials and Methods

Drugs. 5-HTP, serotonin and DOI were purchased from Sigma-Aldrich. M100907 [R(+)-

-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol] was provided by Dr. Kenner Rice (National Institute on Drug Abuse/National Institute on Alcohol Abuse and Alcoholism/National Institutes of Health). The PLC inhibitor U73122 was purchased from Calbiochem. For animal experiments, 5-HTP and DOI were prepared in 0.9% saline, serotonin was prepared in 18 Ω purified, distilled, sterile water and M100907 was prepared in 0.9% saline plus 0.02% Tween 80. For cellular experiments, serotonin and

DOI were prepared in 2 M ascorbate and U73122 was prepared in 0.1% DMSO. 34

Antibodies. Total ERK1/2 (1:1000; p44/42 MAPK 137F5) was purchased from Cell

Signaling Technology, phospho-ERK1/2 (1:1000; Tyr 204 E4) was purchased from

Santa Cruz Biotechnology, the N-terminal polyclonal 5-HT2A receptor antibody (1:500 for immunoblotting, 1:100 for immunocytochemistry) was purchased from Neuromics and the MAP2 monoclonal mouse antibody (1:20,000) was purchased from Abcam. The

Alexa Fluor goat anti-mouse 488 (1:5000), goat anti-rabbit 568 (1:5000) and anti-HA-594 conjugate (1:100) antibodies were purchased from Molecular Probes/Invitrogen.

Plasmid DNA. The mouse 5-HT2A receptor (GenBank accession # BC108973) was tagged on the N-terminus with haemagluttenin (HA-5-HT2AR) or on the C-terminus with yellow fluorescent protein (5-HT2AR-YFP). Mouse arrestin2 (GenBank accession #

BC016642) was tagged on the C-terminus with green fluorescent protein (arr2-GFP) or yellow fluorescent protein (arr2-YFP).

Mice. Subjects used in the experiments include male WT, heterozygous (arr2-HT) and

arr2-KO mice between 3 and 6 months of age and were derived from heterozygous breeding (97). Drugs were administered intraperitoneally (i.p.) at a volume of 10 l/g body weight or centrally (i.c.v.), 2 mm caudal and 2 mm lateral from bregma and at a depth of 3 mm in a volume of 5 l (166). Mice were used only once and all experiments were performed with the approval of the Institutional Animal Care and Use Committee of

The Ohio State University.

Behavioral Experiments. For head twitch response studies, mice were injected with 5-

HTP (100 mg/kg, i.p., n = 13 WT, 10 arr2-HT and 8 arr2-KO), DOI (1 mg/kg, i.p., n = 9

WT and 5 arr2-KO) and serotonin (10 g, i.c.v., n = 4 WT and 5 arr2-KO). The 5-HT2A receptor antagonist M100907 (0.05 mg/kg, i.p.) was administered 10 minutes prior to 5-

HTP (100 mg/kg, i.p., n = 5 for each genotype) or DOI (1 mg/kg, i.p., n = 5 for each genotype). Immediately after injection, each mouse was placed individually into a 13.9 x

12.7 x 15.2 cm3 Plexiglas box and the number of head twitches was counted in 5

(serotonin or 5-HTP) or 10 (DOI) minute increments for 30 or 60 minutes. Body temperature was assessed by using an electronic thermometer (TH5; Physitemp) connected to a rectal temperature probe (RET-3; Physitemp) before and immediately after the 30 minute drug treatment for the 5-HTP studies (97). 5-HTP treated animals were also scored for severity of diarrhea concurrently with head twitches as followed: 0, normal or no fecal boli; 1, visibly wet fecal boli (moderate); 2, liquid fecal boli lacking form (severe) (167).

Cell lines. HEK-293 cells were purchased from ATCC and were grown in minimal essential medium (MEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco/Invitrogen). MEFs were generated from WT and

arrestin1/2 double-knockout mouse embryos (89) and were grown in Dulbecco's modified eagle medium (DMEM; Gibco/Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. MEF WT and arr1/2-KO cells were stably and efficiently transfected with the HA-5-HT2AR using murine stem cell virus (MSCV) expression vectors (Clontech/Takara Bio). To avoid post-transfection cellular adaptations, cells were not maintained over three passages after viral transfection.

3 [ H]-Ketanserin saturation binding assay. To determine 5-HT2A receptor expression

3 levels in the HA-5-HT2AR MEF WT and arr1/2-KO stable cell lines, [ H]-ketanserin saturation binding assays were performed (n = 5, performed in triplicate, from two separate MSCV transfections for each genotype). Membranes were prepared using glass-on-glass dounce homogenization, pelleted by centrifugation at 20,000 x g for 30 minutes at 4 C and resuspended in 50 mM Tris, pH 7.4, followed by glass-on-glass homogenization to form a suspension (97). Protein levels were measured and standardized as described above. Binding assays were performed on membranes (20

g) with 2 nM [3H]-ketanserin (PerkinElmer); nonspecific binding was determined in the presence of 1 M M100907. Plates were incubated at 25 C for 45 minutes before filtering through GF/B filters (Brandel), which had been preincubated in 0.2% polyethyleneimine solution (Sigma-Aldrich). Filters were washed and counted using a liquid scintillation counter.

To determine 5-HT2A receptor expression levels in mouse brain, frontal cortex was isolated from the brains of untreated WT and arr2-KO mice (n = 5 for each genotype) and tissue was homogenized with a polytronic tissue grinder. Membranes were prepared and [3H]-ketanserin binding was performed as described above.

Arrestin2 translocation assays. To determine agonist-induced arrestin2 recruitment to the 5-HT2A receptor, HEK-293 cells were transfected by electroporation using the Gene

Pulser II system (Bio-Rad) (121). Cells were resuspended in serum-free MEM containing

5 mM BES to a final concentration of 2 x 106 cells in 0.5 ml per 0.4-cm cuvette

37 containing 5 g HA-5-HT2AR and 2 g arr2-GFP. A single pulse of 260 V, 1000 F and

∞ Ω was used to transfect cells. Cells were plated in fresh media containing 10% FBS onto collagen-coated 35-mm glass-bottomed culture dishes. Two hours prior to imaging, media was replaced with MEM lacking phenol red and serum. Fifteen minutes prior to imaging, surface HA-5-HT2AR was stained by incubation with the anti-HA-594 antibody

(1:100). Cells were treated with 1 M agonist and confocal microscopy was performed on an Olympus Fluoview 300 confocal microscope with green-helium neon and argon lasers (Olympus). Imaging was performed on at least five different transfections, with multiple cells imaged per dish.

Live cell 5-HT2A receptor-internalization assay. To determine agonist-induced 5-HT2A receptor internalization, MEF WT and arr1/2-KO cells were transfected with 2 to 2.5 g

6 5-HT2AR-YFP cDNA by electroporation. Approximately 4 x 10 cells in 0.5 ml per cuvette were subjected to two square wave pulses of 260V, each with a time constant of approximately 20 ms, and a 1 second pulse interval. Cells were imaged in a manner similar to that described for the HEK-293 cells. Imaging was performed on at least four different transfections, with multiple cells imaged per dish.

MAP kinase assays. To assess agonist-induced ERK1/2 phosphorylation in vitro, HA-5-

HT2AR MEF WT and arr1/2-KO cells were serum-starved for 2 hours followed by a 10 minute treatment with vehicle (n = 16 WT and 12 arr1/2-KO), 1 M 5-HT (n = 16 WT and 12 arr1/2-KO) or 1 M DOI (n = 15 WT and 12 arr1/2-KO). For some experiments, cells were pretreated with vehicle (n = 9-10 WT and 6 arr1/2-KO for each drug treatment) or U73122 (1 M; n = 9 WT and 6 arr1/2-KO for each drug treatment) during 38 the last 30 minutes of serum starvation. Following agonist treatment, cells were lysed in solubilization lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1%

SDS, 1% NP-40, 0.25% deoxycholate, 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM

NaF, with Complete Mini, EDTA-free protease inhibitor cocktail tablets (Roche

Diagnostics)) (84). Lysates were clarified by centrifugation at 20,000 x g for 30 minutes at 4 C. Protein levels were determined using the detergent-compatible DC protein assay

(Bio-Rad). Equivalent levels of protein were resolved on 10% Bis-Tris gels and proteins were transferred to polyvinylidene fluoride membranes (Immobilon P; Millipore).

Membranes were immunoblotted for total ERK1/2 and phospho-ERK1/2 levels.

Chemiluminescence was detected and quantified using the Kodak 2000R imaging system (Eastman Kodak Company). For each sample, phospho-ERK1/2 levels were normalized to total-ERK1/2 levels and fold stimulation was determined by normalizing to the average vehicle levels for each experiment.

To assess agonist-induced ERK1/2 phosphorylation in the frontal cortex, WT and arr2-

KO mice were treated with vehicle (n = 11 for each genotype), 5-HTP (100 mg/kg, i.p.; n

= 11 WT and 9 arr2-KO) and DOI (1 mg/kg, i.p.; n = 13 WT and 12 arr2-KO) for fifteen minutes. Mice were sacrificed by cervical dislocation and frontal cortices were isolated and frozen in liquid nitrogen. Tissue was homogenized in solubilization lysis buffer with a polytronic tissue grinder and immunoblotting and quantification were performed as described above.

Cell surface biotinylation assay. To quantify receptor internalization, cell surface biotinylation was performed on HA-5-HT2AR MEF WT and arr1/2-KO cells (84). Cells

39 were serum-starved for 2 hours, washed once with cold phosphate buffered saline (PBS) and then incubated in 600 g/ml sulfo-NHS-SS-biotin (Pierce Biotechnology) for 30 minutes at 4 C. To quench excess biotin, cells were washed in Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) prior to treatment with vehicle (n = 10 WT and 6

arr1/2-KO), 1 M 5-HT (n = 9 WT and 4 arr1/2-KO) and 1 M DOI (n = 10 WT and 8

arr1/2-KO) for 1 hour at 37 C. Surface biotin was then removed via a 15 minute incubation in stripping buffer (50 mM glutathione, 75 mM NaCl, 75 mM NaOH and 10%

FBS) at 4 C. Residual glutathione was quenched with two sequential 15 minute incubations with iodoacetamide buffer (50 mM iodoacetamide, 1% BSA, in PBS). Cells were lysed in Triton X-100 extraction buffer (10 mM Tris-HCl, pH 7.4, 120 mM NaCl, 25 mM KCl, 1 mg/ml iodoacetamide, 1 mM PMSF, 0.5% Triton X-100, with Complete Mini,

EDTA-free protease inhibitor cocktail tablets) for 2 hours at 4 C. Lysates were clarified by centrifugation at 20,000 x g at 4 C for 30 minutes. Protein levels were standardized as described above and applied to immobilized avidin-conjugated agarose beads (Pierce

Biotechnology) overnight at 4 C with rotation. Protein was eluted in Laemmli Sample

Buffer (Bio-Rad) with 10% -mercaptoethanol and 50 mM dithiothreitol. Immunoblotting was performed with the 5-HT2A receptor polyclonal rabbit antibody (1:500).

Primary neuronal cultures. Primary cortical neuronal cultures were obtained from postnatal day one mouse pups generated from both homozygous and heterozygous breeding of βarrestin2 mice. Primary neurons were prepared as described (168).

Briefly, frontal cortex was isolated and digested in a 37.5% papain solution in oxygenated Leibovitz‟s L-15 medium (Gibco/Invitrogen), supplemented with 0.025% bovine serum albumin for 15 minutes at 37 C, followed by trituration with glass pipettes 40 of decreasing diameter. Neurons were plated in neurobasal medium (supplemented with 2% B27, 0.5 mM L-glutamine, 0.25x insulin-transferrin sodium-selenite and 50 µg/ml gentamycin reagent (Gibco/Invitrogen)) onto poly-L-lysine coated 35-mm glass-bottomed culture dishes. One day after plating, 10 l cytosine B-D arabinofuranoside (Sigma-

Aldrich) was added per 1 ml neurobasal medium.

Neuronal transfection. Three (arr2-YFP) or four (HA-5-HT2AR) days after culturing, primary neurons were transfected with 500 ng HA-5-HT2AR or 200 ng arr2-YFP cDNA with 0.5% Lipofectamine 2000 (Invitrogen) in Opti-MEM, according to the manufacturer‟s instructions. Media was changed the next day and neurons were fixed and stained 24 hours after transfection.

Immunocytochemistry. WT and arr2-KO neurons were fixed at 4 C in 4% paraformaldehyde for 10 minutes, followed by 10 minutes in ice cold 1:1 methanol:acetone solution (169). Neurons were then permeabilized in 0.2% Triton X-

100 in PBS for 20 minutes at 4 C and blocked with 10% goat serum, 5% BSA and 5%

FBS in PBS for 1 hour at 37 C. For endogenous receptor staining, neurons were fixed four days after culturing and incubated with the rabbit polyclonal 5-HT2A receptor antibody (1:100) and MAP2 antibody (1:20,000) overnight (MAP2) or for 36 hrs (5-HT2A receptor) at 4 C in 5% BSA, 5% goat serum and 0.02% sodium azide. To determine receptor trafficking, neurons transfected with the HA-5-HT2AR were live cell-labeled with the anti-HA-594 conjugate antibody (1:100) for 45 minutes, 24 hours after transfection.

Cells were then fixed and stained for MAP2 as described above. Localization of the 5-

HT2A receptor was determined through the use of a confocal microscope, as described 41 above. All experiments were performed on at least 4 separate neuronal preparations of each genotype, with multiple neurons imaged for each dish of cells.

Neuronal cell-surface enzyme-linked immunosorbent assay (ELISA). To quantitate the levels of endogenous receptor localized to the plasma membrane, neurons from individual pups from heterozygous litters were plated onto poly-L-lysine coated 24-well tissue cultures dishes and the pups were genotyped. The resulting WT (n = 5), arr2-HT

(n = 4) and arr2-KO (n = 6) neurons were fixed in 4% PFA for 20 minutes, four days after plating. The endogenous 5-HT2A receptor was stained as described above, without permeabilization. Neurons were incubated in an anti-rabbit HRP-conjugated secondary antibody (1:1000). K-Blue Aqueous TMB Substrate (Neogen) was added to each well, followed by 1 M HCl. The absorbance at 450 nM was determined with a spectrophotometer.

Statistical analysis. Two-way analysis of variance (ANOVA) was used to examine group differences in head twitch responses. Comparisons between the total number of twitches across genotypes or between groups within a genotype, as well as comparisons of the biochemical data were completed with a Student‟s unpaired, two-tailed t test. All statistics were conducted with a 95% confidence interval and were performed using

GraphPad Prism 5.0 software (GraphPad Software). Two-way ANOVA are presented in the text, while Student‟s t tests are presented in the figure legends.

2.3 Results

Agonist-directed 5-HT2A receptor regulation in vitro

The drug-induced translocation of arrestin2 from the cytosol to the plasma membrane of cells expressing a given GPCR has been used as a marker of agonist-induced

arrestin2/GPCR interactions in vitro (82, 84). To determine whether agonists recruit

arrestin2 to the 5-HT2A receptor, HEK-293 cells expressing N-terminal HA-tagged 5-

HT2A receptors (HA-5-HT2AR) and C-terminal GFP-tagged arrestin2 (arr2-GFP) were treated with serotonin (1 M) and DOI (1 M) and arrestin2 translocation was imaged by confocal microscopy. In the absence of agonist, the HA-5-HT2AR is localized to the plasma membrane and arr2-GFP is localized in the cytosol of HEK-293 cells (Figure

2.1). Within 10 minutes of exposure to serotonin or DOI, translocation of arr2-GFP to 5-

HT2A receptors localized at the plasma membrane is observed, as evidenced by the punctate pattern of GFP expression co-localizing with HA-5-HT2AR staining on the surface treated cells. These findings suggest that arrestin2 may be involved in the regulation of the agonist-activated 5-HT2A receptor.

Arrestins have been shown to be integral to the internalization of GPCRs through clathrin coated pits. To assess whether agonist-mediated internalization of the 5-HT2A receptor is disrupted in the absence of arrestins, WT and arr1/2-KO MEFs were transfected with a C-terminal YFP-tagged 5-HT2A receptor (5-HT2AR-YFP) and receptor trafficking was observed using confocal microscopy (Figure 2.2A). In complete media which contains 10% FBS, the 5-HT2AR-YFP is internalized in WT MEFs and removal of the serum-containing media for 2 hours returns the 5-HT2AR-YFP to the plasma membrane. Treating the serum-starved WT MEFs with serotonin or DOI internalized the 43

5-HT2A receptor within 30 minutes. In contrast, the 5-HT2AR-YFP is localized to the plasma membrane of arr1/2-KO MEFs regardless of serum content, and the addition of serotonin to the serum-free media did not affect the membrane localization of the receptor. In stark contrast, DOI was able to internalize the 5-HT2A receptor in the arr1/2-

KO MEFs.

The observed differences between serotonin- and DOI-directed trafficking of the 5-HT2A receptor were then quantified by a cell-surface biotinylation assay. For these studies, we utilized WT and arr1/2-KO MEFs that had been stably transfected with the HA-5-HT2AR via viral transfection. In both WT and arr1/2-KO MEFs, DOI significantly increases the amount of biotinylated HA-5-HT2AR protected from glutathione stripping, indicating an increase in receptor internalization. Serotonin, on the other hand, induces internalization in only in the WT cells (Figure 2.2B). Collectively, both the cell-surface biotinylation studies and the confocal imaging studies indicate that serotonin-induces internalization of the 5-HT2A receptor in a arrestin-dependent manner, while DOI is still able to internalize the receptor in the absence of arrestins in vitro.

As the interaction between arrestins and GPCRs can serve to desensitize G protein signaling cascades or facilitate G protein-independent signaling cascades, we next assessed the impact that arrestins have on 5-HT2A receptor-mediated signal transduction in the MEF cells. Since the activation of the 5-HT2A receptor can lead to

ERK1/2 phosphorylation in several cell types (63, 66-68), we assessed whether the phosphorylation of ERK1/2 differs in the absence of arrestins. Both WT and arr1/2-KO

MEFs were serum-starved for 2 hours prior to the addition of drug, as that was shown to 44 be necessary to obtain similar surface expression of the 5-HT2A receptor between the cell lines. Serotonin and DOI both induce robust ERK1/2 phosphorylation in serum- starved WT MEFs stably expressing the HA-5-HT2AR, although DOI activates ERK1/2 to a much lesser extent (Figure 2.3A). In the arr1/2-KO MEFs, both serotonin and DOI still activate ERK1/2, but the degree of stimulation by serotonin is dramatically decreased compared to WT MEFs and differences between the two agonists are no longer present (Figure 2.3A). The genotype-dependent differences in ERK1/2 activation

3 are not due to variations in 5-HT2A receptor expression, as radioligand-binding with [ H]- ketanserin indicates that the levels of HA-5-HT2AR expression are similar between the two genotypes (Table 2.1). Importantly, WT and arr1/2-KO MEFs, which were mock transfected with empty MSCV virus, do not endogenously express the HA-5-HT2AR as determined by [3H]-ketanserin binding (Table 2.1) and do not show activation of ERK1/2 following treatment with serotonin or DOI (Figure 2.3B). These findings indicate that though the 5-HT2A receptor can mediate ERK1/2 phosphorylation through both arrestin- dependent and -independent pathways, DOI only activates the arrestin-independent pathway while serotonin utilizes both cascades to activate ERK1/2.

Serotonin-induced ERK1/2 phosphorylation has been shown to involve 5-HT2A receptor coupling to the Gq/PLC signaling pathway (66). Therefore, we tested whether PLC contributes to either of the pathways utilized by serotonin and DOI to induce ERK1/2 phosphorylation. In HA-5-HT2AR WT MEFs, pretreatment with the selective PLC inhibitor, U73122 (63), significantly decreased serotonin-mediated ERK1/2 activation and completely blocked DOI-mediated ERK1/2 activation (Figure 2.3C). In the HA-5-

HT2AR arr1/2-KO MEFs, U73122 completely blocked the phosphorylation of ERK1/2 for

45 both agonists (Figure 2.3D). Together, these findings suggest that serotonin activates

ERK1/2 through both PLC-mediated and arrestin-mediated signaling pathways, while

DOI only activates ERK1/2 through the PLC-mediated pathway.

Agonist-directed 5-HT2A receptor regulation in vivo

The findings presented thus far demonstrate that interactions with arrestins can determine 5-HT2A receptor trafficking and signaling profiles for serotonin but not DOI. A number of in vitro studies have shown that the regulation of the receptor can differ depending upon the cell-type (129, 130, 132), underscoring the importance of studying the receptor in its endogenous environment. Therefore, we utilized primary cortical neurons isolated from WT and arr2-KO mice to assess the involvement of arrestin2 in the regulation of 5-HT2A receptor trafficking in a more physiologically relevant cellular system. The endogenous 5-HT2A receptor is primarily localized in intracellular vesicles in cortical neurons isolated from WT mice (without serum-starvation) (Figure 2.4A, upper panels). In contrast, the receptor is restricted to the plasma membrane of neurons lacking arrestin2 (Figure 2.4A, lower panels). We then quantified the levels of 5-HT2A receptors localized to the plamsa membrane by staining non-permeabilized neurons with an antibody to the N-terminus of the receptor. This cell-surface ELISA shows that WT neurons have significantly decreased surface expression compared to cortical neurons from arr2-KO mice. Interestingly, while not significant, neurons from mice heterozygous for arrestin2 (arr2-HT) show a trend for levels of surface expression between that of

WT and arr2-KO neurons (Figure 2.4C; p < 0.05, one-way ANOVA), suggesting that there is a gene dosage effect for trafficking of the 5-HT2A receptor.

The intracellular localization of the 5-HT2A receptor in neurons from WT mice suggests that it may be consitutively trafficked from the surface to intracellular vesicles. To determine whether the receptor is being constitutively internalized, we transfected the

HA-5-HT2AR into primary cultures from WT and arr2-KO mice and performed live-cell labeling with an anti-HA antibody. In WT neurons, the fluorescently-labeled HA-5-HT2AR is internalized during the 45 minute incubation with the antibody, while the receptor retained more prominent surface-labeling in arr2-KO neurons (Figure 2.4B). We next transfected arr2-KO neurons with arr2-YFP to further assess whether arrestin2 is responsible for the differential pattern of staining of the endogenous 5-HT2A receptor that is observed between genotypes. Arr2-YFP rescues the intracellular localization of the endogenous 5-HT2A receptor (Figure 2.4D). These studies indicate that arrestin2 is involved in the regulation of the endogenous 5-HT2A receptor in mouse cortical neurons.

We then tested whether the activation of ERK1/2 was affected by the absence of

arrestin2 in vivo. We treated WT and arr2-KO mice with either 5-HTP (100 mg/kg) or

DOI (1 mg/kg) for 15 minutes and then isolated frontal cortex. Although not a direct agonist at the 5-HT2A receptor, 5-HTP activates the receptor by increasing serotonin levels in the CNS (Figure 1.1). Immunoblot analysis reveals that both 5-HTP and DOI induce ERK1/2 phosphorylation in cortical lysates from WT mice, while only DOI induces

ERK1/2 activation in arr2-KO mice (Figure 2.5A). Comparison of ERK1/2 phosphorylation levels between vehicle treated WT and arr2-KO mice demonstrates that the basal phosphorylation levels do not differ between the two genotypes (Figure

2.5B). Moreover, the differences in kinase activation are not due to differential cortical 5-

3 HT2A receptor expression levels between WT and arr2-KO mice, as [ H]-ketanserin binding in cortical membranes is also similar for both genotypes (Table 2.1).

Finally, to determine if the loss of arrestin2 impacts 5-HT2A receptor-mediated physiological responsiveness in vivo, we treated mice with 5-HTP and assessed differences in head twitch responses between WT and arr2-KO mice. 5-HTP (100 mg/kg, i.p.) induces a head twitch response in WT mice. In contrast, the head twitch response is greatly attenuated in arr2-KO mice (Figure 2.6A: WT vs. arr2-KO: for genotype, F(1,114) = 41.77, p < 0.0001; for time, F(5,114) = 4.99, p = 0.0004; interaction genotype x time, F(5,114) = 2.68, p = 0.0250). A gene dosage affect was observed, as

arr2-HT mice displayed significantly fewer twitches than their WT littermates and significantly more head twitches than the arr2-KO mice (WT vs. arr2-HT: for genotype,

F(1, 126) = 10.01, p = 0.0020; for time, F(5,126) = 7.86, p < 0.0001; interaction genotype x time, F(5,126) = 1.33, p = 0.2548; arr2-HT vs. arr2-KO: for genotype, F(1,96) = 24.54, p <

0.0001; for time, F(5,96) = 3.15, p = 0.0113; interaction genotype x time, F(5,96) = 1.08, p =

0.3749). To demonstrate that the head twitch is due to activation of the 5-HT2A receptor, we treated mice with the selective 5-HT2A receptor antagonist M100907 (0.05 mg/kg, i.p.), prior to 5-HTP administration. M100907 completely blocks 5-HTP induced head twitches in both genotypes (Figure 2.6B).

Since, 5-HTP indirectly induces the head twitch response by increasing serotonin levels in the brain, we concurrently scored the mice for other serotonin-mediated behavioral responses to determine whether the arr2-KO mice experience the surge in serotonin that follows 5-HTP administration. We show that there are no differences between the 48

WT and arr2-KO mice in the severity of diarrhea (Figure 2.6C: for genotype, F(1,114) =

0.01, p = 0.9153; for time, F(5,114) = 7.26, p < 0.0001; interaction genotype x time, F(5,114)

= 0.44, p = 0.8163) or the degree of hypothermia (Figure 2.6D) that develops following

5-HTP administration. Importantly, these behaviors are due to activation of other serotonin receptors and not the 5-HT2A receptor (170, 171), so the lack of differences between genotypes does not impact our interpretation of the head twitch data.

To further confirm that the difference in the head twitch response between genotypes was not due to unanticipated effects of arrestin2 on the conversion of 5-HTP into serotonin, we directly injected serotonin (10 g, i.c.v.) into the brain. Central administration of serotonin induces a head twitch response in WT mice, but again, this response is significantly reduced in the absence of arrestin2 (Figure 2.6E: for genotype, F(1,42) = 19.57, p < 0.0001; for time, F(5,42) = 15.69, p < 0.0001; interaction genotype x time, F(5,42) = 8.71, p < 0.0001; Figure 2.6F). Collectively, these data suggest that the 5-HTP and serotonin-mediated head twitch responses are dependent upon

arrestin2.

These biochemical studies indicate that serotonin utilizes arrestin2 to internalize the 5-

HT2A receptor and facilitate signaling, but the agonist DOI does not. We next assessed whether these agonist-dependent differences were conserved for the head twitch response. DOI (1 mg/kg, i.p.) induces a similar number of head twitches in both WT and

arr2-KO mice (Figure 2.7A: for genotype, F(1,72) = 0.06, p = 0.8031; for time, F(5,72) =

5.69, p = 0.0002; interaction genotype x time, F(5,72) = 0.06, p = 0.9976). Pretreatment with M100907 blocks the DOI-induced head twitch response for both genotypes (Figure 49

2.7B). These studies indicate that while serotonin utilizes arrestin2 for the activation of the 5-HT2A receptor-mediated head twitch response, DOI induces the head twitch response independent of arrestin2.

2.4 Discussion

In this chapter, we demonstrate that although both serotonin and DOI recruit arrestin2 to the 5-HT2A receptor in HEK-293 cells (Figure 2.1), they differ in their dependence upon arrestins for receptor trafficking and downstream signaling events. Serotonin is unable to internalize the 5-HT2A receptor in arr1/2-KO MEFs, while DOI maintains its ability to internalize the receptor in the absence of arrestins (Figure 2.2). Furthermore, we demonstrate that the 5-HT2A receptor can activate ERK1/2 in MEFs via two independent pathways, a arrestin-mediated pathway and a PLC-mediated pathway.

Serotonin-induced ERK1/2 phosphorylation is a result of the activation of both signaling pathways, while DOI stimulates ERK1/2 solely through the PLC-mediated pathway

(Figure 2.3).

The arrestin-dependent internalization of the 5-HT2A receptor following serotonin treatment suggests that the serotonin bound receptor is internalized through the classic clathrin-dependent pathway which mediates the endocytosis of most GPCRs (172).

Although DOI does induce translocation of arrestin2 to the 5-HT2A receptor, it does not require arrestins for the internalization of the receptor. This suggests that the 5-HT2A receptor can be internalized through an alternate pathway. One arrestin-independent mechanism of internalization involves interactions with the membrane protein caveolin

(158) and several GPCRs can be internalized through caveolin-dependent mechanisms 50

(173-176). The 5-HT2A receptor has been shown to co-immunoprecipitate with caveolin-1 and co-localize with caveolin at both the plasma membrane and within intracellular vesicles of HEK-293 cells (153). Therefore, we propose that the 5-HT2A receptor may be able to be endocytosed via both arrestin-dependent and caveolin-mediated mechanisms. The cholecystokinin receptor is a GPCR that also can be internalized through multiple endocytic pathways. While the majority of cholecystokinin receptors are normally internalized via arrestin-mediated/clathrin-coated pits, nearly all of the receptors are still internalized in CHO cells through a caveolin-dependent mechanism when the clathrin-mediated pathway is inhibited (177). Similar to the cholecystokinin receptor, the arrestin-mediated pathway may be the preferred mechanism of 5-HT2A receptor internalization. However, some agonists, such as DOI, may preferentially utilize the caveolin pathway, or may engage this alternate endocytic route only under conditions in which the main pathway is inhibited.

For the two agonists studied in this chapter, there appears to be a correlation between their dependence upon arrestins for receptor trafficking and their utilization of arrestins in the phosphorylation of ERK1/2. In other words, serotonin mediates receptor internalization and ERK1/2 phosphorylation through arrestin-mediated pathways, while

DOI does not require arrestins for the internalization of the 5-HT2A receptor or the activation of ERK1/2. This suggests that the arrestin-mediated internalization of the 5-

HT2A receptor could be necessary for the activation of ERK1/2 through the arrestin- dependent pathway. Internalization has been shown to be a prerequisite for the

arrestin-mediated activation of downstream signaling for other GPCRs, wherein the blockade of endocytosis inhibits arrestin-mediated signaling (106, 178, 179). Moreover, 51 in neuronal cultures, the 5-HT2A receptor agonist -methylserotonin induces ERK1/2 activation through a arrestin-dependent mechanism, as siRNA mediated knockdown of either arrestin blocked the phosphorylation of ERK1/2 (180). Interestingly, the authors demonstrate that treatment with a cell-permeable dynamin inhibitory peptide blocks the

-methylserotonin-mediated activation of ERK1/2 (180), thus implicating 5-HT2A receptor endocytosis in the arrestin-dependent ERK1/2 activation in neurons. Although additional studies are necessary to demonstrate causation, these data support the hypothesis that the serotonin-induced arrestin-mediated endocytosis of the 5-HT2A receptor and activation of ERK1/2 observed in the MEFs may be interrelated events.

Both serotonin and DOI activation of the 5-HT2A receptor leads to the phosphorylation of

ERK1/2 in MEFS, although the agonists utilize different pathways to activate the kinase.

Studies with other GPCRs have demonstrated that the activation of ERK1/2 by arrestin- dependent and arrestin-independent pathways can have differential functional outcomes. The AT1A receptor, for example, can stimulate ERK1/2 phosphorylation through both G protein/PKC-mediated and arrestin-mediated/G protein independent pathways (95, 116, 181). The selective inhibition of each pathway by the PKC inhibitor,

Ro-21-8435, or siRNA against arrestin2 reveals that the PKC-mediated ERK1/2 activation has a rapid onset and transient duration, while the arrestin2-mediated phosphorylation of ERK1/2 has a slower onset, but persists for much longer (95, 116,

181). Additional studies with the AT1A receptor and the vasopressin V2 receptor have also demonstrated that arrestin-signaling to ERK1/2 can serve to impact the subcellular localization and physiological consequences of the ERK1/2 activation (114, 161, 182).

These studies show that the arrestin-bound ERK1/2 is retained in the cytosol of COS-7 cells, while the non-arrestin-bound phosphorylated ERK1/2 translocates to the nucleus where it activates transcription factors and stimulates DNA transcription (161, 182).

These findings indicate that ERK1/2, which is phosphorylated through interactions with

arrestins following serotonin treatment, may impact downstream signaling pathways and cellular responses differently than ERK1/2 activated by DOI.

In addition to the findings of the in vitro studies, the data presented in this chapter demonstrate that the ligand-directed interactions between the 5-HT2A receptor and

arrestins also have functional consequences in vivo. We show that the trafficking of the

5-HT2A receptor into intracellular vesicles is attenuated in primary neuronal cultures from

arr2-KO mice (Figure 2.4). Moreover, serotonin-directed signaling pathways in the frontal cortex are disrupted in the absence of arrestin2, as evidenced by the abrogated

ERK1/2 phosphorylation in the arr2-KO mice following treatment with 5-HTP (Figure

2.5). The disregulation of the serotonin-activated 5-HT2A receptor in the absence of

arrestin2 culminates in a significantly attenuated head twitch response following treatment with either 5-HTP or serotonin (Figure 2.6). In contrast to serotonin, the DOI- induced head twitch response is unaffected in the arr2-KO mice (Figure 2.5). This is consistent with the in vivo and in vitro studies demonstrating that the DOI-mediated regulation and signaling at the 5-HT2A receptor does not depend upon interactions with

arrestins. While the mechanisms by which DOI activates the head twitch response remains to be determined, they presumably are composed of G preotein- dependent/arrestin-independent signaling pathways. This is supported by a study

53 demonstrating that Gq-KO mice exhibit decreased DOI-induced head twitches compared to their WT littermates (183).

The decreased head twitch response observed in the arr2-KO mice following treatment with either 5-HTP or serotonin suggests that arrestins may be facilitating the signaling pathways involved in the expression of this behavior. Other GPCR-mediated behaviors have been attributed to arrestin2-dependent signaling in vivo, wherein arr2-KO mice display decreased responses compared to their WT littermates following agonist challenge (184). Through a series of complex biochemical studies, the reduced amphetamine-induced hyperlocomotor activity observed in arr2-KO mice has been correlated to the formation of a arrestin2/protein phosphatase 2A (PP2A)/Akt signaling complex with the D2 dopamine receptor (117). While the decreased serotonin-induced head twitch response in the arr2-KO mice could be related to the disrupted 5-HT2A receptor trafficking and the inability of serotonin to activate ERK1/2 in the mouse frontal cortex, at this time these studies are just correlative and additional studies are necessary to assess causation.

Throughout this chapter, we demonstrate a novel functional difference in how the 5-HT2A receptor responds to a non-hallucinogenic agonist (serotonin) and to a hallucinogenic agonist (DOI). In vitro studies, which have compared the activation of the PLC and the

PLA2 pathways by a panel of hallucinogenic and non-hallucinogenic agonists, have not identified any correlations between the activation of either of these signaling pathways to the psychedelic nature of the drugs (77, 78). The Gingrich laboratory has demonstrated differential signaling at the 5-HT2A receptor for a hallucinogenic and a non-hallucinogenic 54 agonist in vivo. They show that LSD activates the transcription factor egr-2 in the mouse frontal cortex and in primary cortical neurons, yet its non-hallucinogenic analogue, lisuride, does not (51). Interestingly, unlike serotonin, lisuride does not activate the head twitch response in mice (51), which further complicates the functional selectivity of 5-

HT2A receptor-mediated signaling as these studies suggest that lisuride and serotonin also activate distinct signaling pathways downstream of receptor activation.

In summary, the findings presented in this chapter demonstrate that the 5-HT2A receptor is regulated by, and signals through interactions with arrestins, yet the contribution of

arrestins to 5-HT2A receptor responsiveness is determined by the agonist acting at the receptor. These studies further support the concept of functional selectivity, wherein different ligands at the 5-HT2A receptor stabilize distinct receptor conformations, thus engaging interactions with a select complement of the proteins in residence with the receptor, such as G proteins or arrestins. In this way, different agonists can differentially activate a subset of the signaling pathways downstream of a GPCR. Finally, the identification of arrestin2 as a point of divergence in the 5-HT2A receptor mediated signal transduction that occurs for different agonists may direct the development of pharmaceuticals for the treatment of neuropsychiatric disorders.

2.5 Chapter 2 Tables

Membranes fmol/mg protein (± S.E.M.)

HA-5-HT2AR MEFs WT 238.3 ± 33.91 arr1/2-KO 254.1 ± 60.83 Mock transfected MEFs WT 22.19 ± 9.15 arr1/2-KO 19.21 ± 13.49 Mouse frontal cortex WT 209.5 ± 21.98 arr2-KO 202.3 ± 32.41

Table 2.1 5-HT2A receptor expression levels in MEFs and mouse frontal cortex 3 5-HT2A receptor expression levels do not differ in the absence of arrestins. [ H]-Ketanserin (2 nM) binding was performed on membranes prepared from untreated HA-5-HT2AR expressing MEFs or mock transfected MEFs or from frontal cortex isolated from naïve WT and arr2-KO mice. Non-specific binding was determined by competition with the selective 5-HT2A receptor antagonist M100907 (1 M). Receptor number did not differ between genotypes in either model system: p>0.05. Adapted from Schmid et al., (44).

2.6 Chapter 2 Figures

arr2-GFP HA-5-HT2AR Merged

free

Serum



1 1

free + free

Serum

DOI

 1 1

Figure 2.1 Serotonin and DOI both recruit arrestin2 to the HA-5-HT2AR

HEK-293 cells transfected with HA-5-HT2AR (red) and arr2-GFP (green) were serum-starved for

2 hours prior to drug treatment and live cell imaging. The HA-5-HT2AR was stained with an anti- HA 594-AlexaFluor antibody. Serotonin (5-HT) and DOI both induce translocation of arr2-GFP to

5-HT2A receptors localized at the plasma membrane. Representative images of cells taken 10 minutes after drug treatment are shown.

A. Serum-free +

Complete Media Serum-free 1 M 5-HT 1 M DOI

WT MEF WT

KO MEF KO

arr1/2 

B. 3.5 Vehicle 5-HT *** *** 3.0 DOI 2.5

WT -75 2.0 ** R Internalization R

2A 1.5

arr1/2- KO Control Over Fold

-75 1.0 HA-5-HT WT arr1/2-KO

Figure 2.2 Serotonin, but not DOI, induces internalization in a arrestin-dependent manner

A. In complete media, the 5-HT2AR-YFP (green) is internalized in WT MEFs (upper panels) but is restricted to the cellular membrane in arr1/2-KO MEFs (lower panels). Serum-starving the cells for 2 hours returned the 5-HT2AR-YFP to the plasma membrane of WT MEFs. Serotonin treatment (5-HT: 1 M, 30 minutes) internalizes the 5-HT2AR-YFP only in WT MEFs, while DOI (1 M, 30 minutes) internalizes the receptor in both cell types. Representative images are shown. B.

Quantification of internalized HA-5-HT2AR by cell surface biotinylation reveals a similar pattern of internalization, with DOI internalizing the 5-HT2AR in both cell types and serotonin internalizing the receptor only in WT cells. Cells were serum-starved for 2 hours prior to a 1 hour treatment with 1 M serotonin or DOI. Representative 5-HT2AR immunoblots (left) include “100%” (without glutathione stripping) and “strip” (without vehicle or drug treatment) controls. Vehicle vs. drug treated within each genotype: **p < 0.01, ***p < 0.001. Mean ± S.E.M. are shown. Adapted from Schmid et al. (44).

A. B. Mock

KO P-ERK

- WT

T-ERK

arr1/2  P-ERK 25 Vehicle ^^^ 5-HT WT DOI T-ERK 20 ***

KO P-ERK

15 -

T-ERK

10 arr1/2 

5 ***

*** ** P-ERK1/2 P-ERK1/2 / T-ERK1/2

Fold Over Vehicle Control Over Vehicle Fold 0 WT arr1/2-KO

C. WT D. arr1/2-KO

P-ERK P-ERK

T-ERK T-ERK

### ### 5 25 *** ## *** 4 *** 20

3 15 ***

10 2 ###

5 *** 1

P-ERK1/2 P-ERK1/2 / T-ERK1/2

P-ERK1/2 P-ERK1/2 / T-ERK1/2 Fold Over Vehicle Control Over Vehicle Fold 0 Control Over Vehicle Fold 0

5-HT DOI 5-HT DOI Vehicle Vehicle U7 + 5-HT U7 + DOI U7 + 5-HT U7 + DOI

Figure 2.3 Serotonin utilizes arrestins to activate ERK1/2 in MEFs while DOI does not

Figure 2.3 continued

Stably transfected HA-5-HT2AR MEFs were serum-starved for 2 hours prior to a 10 minute treatment with vehicle (2 M ascorbate), 1 M serotonin (5-HT) or 1 M DOI. A. Serotonin activates ERK1/2 phosphorylation (P-ERK) to a much greater extent than DOI in WT but not arr1/2-KO MEFs (T-ERK: total-ERK1/2). Vehicle vs. drug stimulation within each genotype: **p < 0.01, ***p < 0.001. Serotonin vs. DOI treatment within each genotype: ^^^p<0.001. B. Serum, but neither serotonin nor DOI, activates ERK1/2 in mock transfected MEFs. C-D. MEFs were pretreated with vehicle (0.1% DMSO) or the PLC inhibitor U73122 (U7, 1 M) for 30 minutes, prior to a 10 minute treatment with agonist. C. U73122 significantly attenuates serotonin- mediated ERK1/2 phosphorylation and completely blocks DOI-mediated ERK1/2 phosphorylation in MEF WTs. D. U73122 completely blocks both serotonin- and DOI-mediated ERK1/2 phosphorylation in arr1/2-KO MEFs. Vehicle vs. drug stimulation within each genotype: ***p < 0.001. Vehicle pretreatment vs. U7 pretreatment within each agonist treatment: ##p < 0.01, ###p < 0.001. Representative blots and densitometric analysis are provided. Mean ± S.E.M. are shown. Adapted from Schmid et al. (44).

A. Endogenous 5-HT2A receptor B. HA-5-HT2AR

5-HT2AR MAP2 HA-5-HT2AR MAP2

arr2 

C. D. arr2-KO+arr2-YFP 0.4 * arr2-YFP 5-HT2AR Merged

0.3 HT2AR

0.2 -

R R Surface 2A

0.1

5-HT Immunoreactivity

0.0 5 Endogenous

arr2-HTarr2-KO  

Figure 2.4 The 5-HT2A receptor is more prominently localized to the plasma membrane of cortical neurons in the absence of arrestin2

A-B. 5-HT2A receptor (red) localization in untreated primary cortical neurons as determined by endogenous 5-HT2A receptor staining (5-HT2AR; A) or live cell antibody staining of transfected

HA-5-HT2AR (B) and co-staining with the MAP2 neuronal marker (green). A. The endogenous 5-

HT2A receptor is primarily localized in intracellular vesicles in neurons from WT mice (top panels), while it is restricted to the plasma membrane of arr2-KO neurons (bottom panels). B. In WT neurons (top panels), the transfected HA-5-HT2AR is actively internalized from the plasma membrane. In the absence of arrestin2 (bottom panels), the HA-5-HT2AR remains localized to the neuronal membrane. C. Receptor surface expression as determined by N-terminal 5-HT2A receptor immunoreactivity in non-permeabilized neurons reveals that arr2-KO neurons have significantly higher levels of 5-HT2A receptor surface expression than WT neurons. WT vs. arr2-

KO: *p < 0.05. D. The endogenous 5-HT2A receptor (red) is internalized in arr2-KO neurons transfected with arr2-YFP (green) while the 5-HT2A receptor is localized to the cell surface in non-transfected arr2-KO neurons. Representative images and mean ± S.E.M. are shown. Adapted from Schmid et al. (44).

A. B.

WT arr2-KO

KO P-ERK P-ERK

- WT

arr1/2 T-ERK T-ERK 

2.0 0.5 Vehicle 5-HTP ** DOI 0.4 *** 1.5 * 0.3

0.2 1.0

0.1

P-ERK1/2 / T-ERK1/2 P-ERK1/2 P-ERK1/2 / T-ERK1/2

Fold Over Vehicle Control Over Vehicle Fold 0.5 0.0 WT arr2-KO WT arr2-KO

Figure 2.5 5-HTP, but not DOI, activates ERK1/2 in the mouse frontal cortex in a arrestin2-dependent manner Frontal cortex was isolated from WT and arr2-KO mice treated for 15 minutes with vehicle (0.9% saline, i.p.), 5-HTP (100 mg/kg, i.p.) or DOI (1 mg/kg, i.p.). A. 5-HTP leads to significant ERK1/2 phosphorylation (P-ERK) only in the frontal cortex of WT mice, while DOI induces ERK1/2 phosphorylation in both genotypes (T-ERK: total ERK1/2). Vehicle vs. drug treatment within each genotype: *p < 0.05, **p < 0.01, ***p < 0.001. B. Basal ERK1/2 phosphorylation levels do not differ for vehicle treated WT and arr2-KO mice. WT vs. arr2-KO: p = 0.8338. Representative blots and densitometric analysis are provided. Mean ± S.E.M. are shown. Adapted from Schmid et al. (44).

A. 5-HTP B.

12 WT 50 WT arr2-HT arr2-KO arr2-KO 40 9

30 6 20 3 10 ** ##

Head Twitches/ 5 Minutes Twitches/ Head 0 0 5 10 15 20 25 30 Minutes 30 in Twitches Head Vehicle + M100 + Time (minutes) 5-HTP (100 mg/kg, i.p.) C. D.

1.5 0

WT C)  arr2-KO -1 1.0 -2

0.5 -3

-4 Severity of Diarrhea of Severity 0.0

5 10 15 20 25 30 ( Temperature in Change WT arr2-KO Time (minutes) E. Serotonin F. 12 20 WT arr2-KO 9 15

6 10

3 5 ** 0 Head Twitches/ 5 Minutes Twitches/ Head 0 5 10 15 20 25 30 Minutes 30 in Twitches Head WT arr2-KO Time (minutes)

Figure 2.6 5-HTP and serotonin induce fewer head twitches in arr2-KO mice

Figure 2.6 continued Mice were injected with either 5-HTP (A-D) or serotonin (E-F) and head twitches were scored for 30 minutes. A-D. 5-HTP-induced head twitch responses are decreased in arr2-KO mice, while other 5-HTP-mediated responses are unaffected by the absence of arrestin2. A. WT and arr2- HT mice display significantly more 5-HTP induced (100 mg/kg, i.p.) head twitches than arr2-KO mice. B. A 10 minute pretreatment with the 5-HT2A receptor antagonist M100907 (M100: 0.05 mg/kg i.p.) significantly inhibits the head twitches observed in WT mice, when compared to mice pretreated with vehicle (0.02% Tween-80 in 0.9% saline, i.p.). WT vs. arr2-KO within the same pretreatment group: **p < 0.01. Vehicle pretreatment vs. M100 pretreatment within each genotype: ##p < 0.01. C. Severity of diarrhea scored during the observance of the head twitch response is similar between genotypes. WT vs. arr2-KO: p = 0.9576. D. 5-HTP-induces a similar change in body temperature 30 minutes following 5-HTP administration in WT and arr2-KO mice. E-F. WT mice exhibit significantly more serotonin-induced head twitches than arr2-KO mice. Time-course (E) and total number (F) of head twitches observed following administration of serotonin (5-HT: 10 g, i.c.v.). WT vs. arr2-KO: **p < 0.01. Mean ± S.E.M. are shown. Adapted from Schmid et al. (44) and Schmid and Bohn (45).

A. DOI B.

12 50 WT WT arr2-KO 40 9 arr2-KO

30 6 20

3 10 *** ***

Head Twitches/ 10 Minutes 10 Twitches/ Head 0 0 10 20 30 40 50 60 Minutes 60 in Twitches Head Vehicle+ M100+ Time (minutes) DOI (1 mg/kg, i.p.)

Figure 2.7 DOI induces an equivalent number of head twitches in WT and arr2-KO mice Mice were injected with DOI and head twitches were scored for 1 hour. A. WT and arr2-KO mice exhibit similar head twitch response profiles following treatment with DOI (1 mg/kg, i.p.). B. A 10 minute pretreatment with the 5-HT2A receptor antagonist M100907 (M100: 0.05 mg/kg, i.p.) significantly abrogates the number of head twitches observed in both genotypes. Vehicle pretreatment vs. M100 pretreatment with each genotype: ***p < 0.001. Mean ± S.E.M. are shown. Adapted from Schmid et al., (44).

Chapter 3

Serotonin, but not the N-methyltryptamines, activates 5-HT2A receptor signaling

via a arrestin2/Src/Akt complex in vivo

3.1 Introduction

In the previous chapter, we demonstrate that the serotonin-mediated head twitch response, which is due to the activation of 5-HT2A receptors expressed specifically in the mouse frontal cortex (51), is significantly attenuated in the absence of arrestin2 (Figure

2.6). These studies suggest that arrestin2 may act to facilitate 5-HT2A receptor signaling in the mouse frontal cortex. Although we also demonstrate that 5-HTP is unable to stimulate ERK1/2 phosphorylation in the frontal cortex in the absence of arrestin2, we have not causally linked this signaling pathway to the activation of the head twitch response. Therefore, the mechanism by which arrestin2 facilitates the 5-HT2A receptor signaling pathways which underlie the serotonin-mediated head twitch response was left unresolved.

Arrestins can facilitate G protein-independent signaling by functioning as adaptor proteins, to promote the stable association of signaling proteins with GPCRs. This was

66 first demonstrated for the 2-adrenergic receptor, wherein agonist stimulation was shown to recruit Src to the receptor, but only when arrestin1 was also expressed (107). Since then, arrestins have been shown to be integral members of in vitro receptor signaling scaffolds for a number of kinases, including ERK1/2, JNK, p38 and Akt (106, 109, 110).

arrestin2 has also been co-immunoprecipitated out of mouse brain with ERK1/2, Akt,

Src and JNK3 kinases (108, 117, 119, 185), demonstrating the formation of these complexes in vivo. Moreover, by binding multiple components of a signaling cascade simultaneously, arrestins can increase the efficiency of the signaling between successive kinases. When expressed in COS-7 cells, arrestin2 forms a complex with the MAP kinase kinase kinase Ask1, the MAP kinase kinase MKK4 and JNK3, and over- expression of arrestin2 increases Ask1-dependent phosphorylation of JNK3 (108, 186).

As the 5-HT2A receptor has also been shown to activate many of these kinases, including ERK1/2, p38, Src and Akt (63, 66, 187), we hypothesized that serotonin mediates the head twitch response in vivo through the arrestin2-dependent scaffolding of such a kinase to the 5-HT2A receptor. Moreover, we anticipated that the inhibition of this signaling cascade would block the serotonin-mediated head twitch response in WT mice.

In the brain, serotonin can be converted by indoleamine N-methyltransferase (INMT) into

N-methyltryptamines, which include N-methylserotonin, bufotenine and dimethyltryptamine (Figure 1.1) (19, 22, 188). These compounds are also agonists at the

5-HT2A receptor and can induce hallucinations in humans and head twitches in rodents

(32, 34, 57, 189-191). It has previously been difficult to delineate the neuropharmacological effects mediated by serotonin and its N-methyltryptamine 67 metabolites in vivo, since many of these endogenous amines have similar or higher affinities for the 5-HT2A receptor than serotonin (32, 192). However, serotonin and the hallucinogenic compound DOI differ in their dependence upon arrestin2 for the induction of the head twitch response (Figure 2.7). Therefore, we propose that 5-HT2A receptor activation by the hallucinogenic N-methyltryptamines and serotonin may lead to the formation of distinct receptor signaling complexes in the frontal cortex and may differentially depend upon arrestin2 for the induction of the head twitch response.

To address these hypotheses, we assessed the differences in 5-HT2A receptor-mediated head twitch responses and signaling that occur in the mouse frontal cortex and primary cortical neurons between serotonin and its psychoactive metabolites. Moreover, by immunoprecipitating the 5-HT2A receptor from the mouse frontal cortex of WT and arr2-

KO mice, we were able to determine both the agonist-directed differences in receptor interactions with signaling kinases and the dependence upon arrestin2 for these interactions. Our findings demonstrate functional selectivity at the 5-HT2A receptor for serotonin and its psychoactive metabolites in vivo, in that serotonin stimulates the formation of a receptor complex that is contingent upon arrestin2, while the N- methyltryptamines do not. Finally, by using kinase inhibitors, we demonstrate the functional significance of the arrestin2-dependent signaling pathway by assessing the impact that kinase inhibitors have on the serotonin-mediated head twitch response.

Through these studies, we show that the in vivo actions of serotonin and its psychoactive metabolites are functionally distinct. The results reported in this chapter have been published in The Journal of Neuroscience (45)

3.2 Materials and Methods

Drugs. 5-HTP, serotonin, N-methylserotonin oxalate salt, 5-MeO-DMT and clorgyline were purchased from Sigma-Aldrich. MTZ [N,N‟ Bis-(3-methyl-2- thiazolidinylidene)succinamide] was purchased from Life Chemicals. LY294002, AKTi and PP2 were purchased from Cayman Chemical, Calbiochem and Tocris, respectively.

M100907 was generously provided by Dr. Kenner Rice (NIDA/NIAAA/NIH, Bethesda,

MD, USA). For animal experiments, 5-HTP, 5-MeO-DMT and clorgyline were prepared in 0.9% saline. Serotonin, N-methylserotonin and MTZ (for Figure 3.3) were prepared in

18 Ω purified, distilled, sterile water. M100907 was prepared in saline plus 0.02%

Tween-80. The inhibitors LY294002, AKTi, PP2 and MTZ (for Figures 3.4C, 3.4F and

3.12) were prepared in 1% DMSO. For biochemical experiments, serotonin, N-Me-5-HT and 5-MeO-DMT were prepared in 2 M ascorbate, M100907 was prepared in 0.0001%

DMSO and LY294002 and PP2 were prepared in 0.1% DMSO.

Primary Antibodies. Total Akt (1:2000; pan C67E7), phospho-Akt (1:1000; Thr308

C31E5E) and Src (1: 500; L4A1) antibodies were obtained from Cell Signaling

Technology; anti-PSD-95 (1:500; K28/4) was purchased from UC Davis/NINDS/NIMH

NeuroMab Facility; arrestin2 (1:2000; A2CT) antibody was provided by Dr. Robert

Lefkowitz (Duke University, HHMI, Durham, NC;); the polyclonal antibody to the N- terminus of the 5-HT2A receptor (1:500) was from Neuromics; the c-Myc monoclonal antibody (1:500) was from Clontech/Takara Bio Company. Antibody specificity was based on the literature (97, 185, 193, 194). Most-notably, the N-terminal antibody directed at the 5-HT2A receptor does not detect the protein in 5-HT2A receptor-KO mice

(195).

Plasmid DNA. Mouse arrestin2 (GenBank association # BC016642) cDNA was tagged on the N-terminus with myc (myc-arr2).

Mice. Subjects used in the experiments include male WT and arr2-KO mice derived via heterozygous breeding as well as male C57BL/6J mice (Jackson Laboratory) between

2.5 and 9 months of age (44, 97). For systemic injections, drugs were administered i.p. at a volume of 10 l per gram body weight. For injections directly into the CNS, drugs were injected i.c.v. at a volume of 5 l, 2 mm caudal and 2 mm lateral from Bregma at a depth of 3 mm (166). In all cases, mice were used only once. All experiments were performed with the approval of the Institutional Animal Care and Use Committee of The

Ohio State University or The Scripps Research Institute.

Behavioral Experiments. Immediately following injection with agonist, mice were placed in individual Plexiglas boxes (13.9 x 12.7 x 15.2 cm3) and head twitches were counted in

5 minute increments for either 30 or 60 minutes (44). The number of mice used in the experimental design is summarized in Table 3.1. For dose response studies, WT and

arr2-KO mice were treated with either 5-HTP (100, 150, 200 mg/kg, i.p); serotonin (10,

20, 40 g, i.c.v.); N-methylserotonin (10, 20, 40 g, i.c.v.); or 5-MeO-DMT (5, 10, 15 mg/kg, i.p.). To determine the contribution of MAO-A metabolism to 5-HTP-induced head twitches, mice were pretreated (i.p.) with either vehicle (0.9% saline) or clorgyline

(1 mg/kg) 60 minutes prior to treatment with 5-HTP (50, 100 mg/kg, i.p.). The 5-HT2A receptor antagonist, M100907 (0.05 mg/kg, i.p.), was administered 10 minutes prior to 5-

HTP (200 mg/kg, i.p.), 5-HT (40 g, i.c.v.), N-methylserotonin (20 g, i.c.v.) or 5-MeO- 70

DMT (10 mg/kg, i.p.). For inhibition of clorgyline-enhanced head twitches, M100907 was administered 50 minutes following treatment with clorgyline (1 mg/kg, i.p.) and 10 minutes prior to treatment with 5-HTP (100 mg/kg, i.p.). The INMT inhibitor, MTZ (125 ng, i.c.v.), or vehicle was injected 10 minutes prior to treatment with 5-HTP (200 mg/kg, i.p.), N-methylserotonin (20 g, i.c.v.) or 5-MeO-DMT (10 mg/kg, i.p.). Vehicle or the kinase inhibitors LY294002 (125 ng, i.c.v.), PP2 (300 ng, i.c.v.) and AKTi (55 ng, i.c.v.) were injected 10 minutes prior to treatment with 5-HTP (200 mg/kg, i.p.) or 5-MeO-DMT

(10 mg/kg, i.p.). For the studies detailed in Figure 3.12, WT mice were pretreated with either vehicle, AKTi (55 ng, i.c.v.), MTZ (125 ng, i.c.v.) or both ATKi and MTZ for 10 minutes prior to treatment with 5-HTP (200 mg/kg, i.p.). Dosing of the inhibitors was based on the literature: clorgyline (196), MTZ (28, 29), LY294002 (117), PP2 (197), and

AKTi (198).

Akt activation in cortical lysates. Signaling in the mouse frontal cortex was determined following treatment of both WT and arr2-KO mice with either vehicle (0.9% saline, n =

23 per genotype), 5-HTP (100 mg/kg, i.p., n = 16 per genotype) or 5-MeO-DMT (10 mg/kg, i.p., n = 5 WT and 6 arr2-KO). Mice were sacrificed by cervical dislocation and tissue was frozen immediately in liquid nitrogen. Tissue was homogenized through the use of a polytronic tissue grinder, followed by glass-on-glass dounce homogenization and then solubilized overnight at 4°C in immunoprecipitation lysis buffer (20 mM NaF,

10% glycerol, 50 mM Tris, 150 mM NaCl, 0.5% NP-40, 1 mM PMSF, 1 mM EDTA) containing a Mini EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics) (185).

Protein levels were determined by a protein assay kit compatible with detergent-based buffers (Bio-Rad Laboratories) and 25-30 g of protein per lane were resolved on 10%

Bis-Tris gels (Invitrogen). Proteins were transferred to polyvinylidene fluoride membranes (Millipore) and immunoblotted for total-Akt or phospho-Akt.

Chemiluminescence was detected using a Kodak 2000R imaging system (Eastman

Kodak) and fluorescence was detected using an Odyssey Infrared imaging system (LI-

COR Biosciences). Blots were quantified using ImageJ analysis software (NIH). For each sample, phospho-Akt levels were normalized to corresponding total-Akt levels and fold stimulation was determined by normalizing to the average vehicle levels for each experiment.

5-HT2A receptor immunoprecipitation. The 5-HT2A receptor was immunoprecipitated from equivalent aliquots of cortical lysates remaining from the Akt assay by overnight incubation with a polyclonal rabbit antibody against the N-terminus of the 5-HT2A receptor (1:133; Neuromics) at 4°C; complexes were immobilized on Protein A

Sepharose 4 Fast Flow (Amersham Biosciences) beads for 3 hours at room temperature. Beads were washed 3 times in lysis buffer and proteins were eluted by heating in SDS sample buffer containing 5% -mercaptoethanol at 55°C for 20 minutes.

Immunoblot analysis was performed as described above and representative western blots are shown (vehicle: n = 11-24 WT and 19-24 arr2-KO; 5-HTP: n = 4-18 WT and

13-18 arr2-KO; 5-MeO-DMT: n = 5-7 WT and 6-7 arr2-KO). Any alterations to enhance brightness or contrast were applied to the entire gel image. For quantification of the immunoprecipitation studies, the protein levels were first normalized to their corresponding 5-HT2A receptor immunoblots and then drug treatment was normalized to vehicle treated controls.

Primary neuronal cultures. Primary cortical neurons were generated from postnatal day one mouse neonates obtained from homozygous breeding of arr2-KO or WT mice (44,

168). Frontal cortex was removed and digested in Leibovitz‟s L-15 (Gibco/Invitrogen) solution containing 0.025% bovine serum albumin and 0.0375% papain for 15 minutes at

37°C with 95% oxygen/5% carbon dioxide gently agitating the media. Tissue was washed in complete Neurobasal-A media (with 2% B-27 serum supplement, 0.5 mM L- glutamine, 1.675 g/L sodium selenite, 2.5 mg/L insulin, 1.375 mg/L transferrin and 50

g/ml gentamycin; Gibco/Invitrogen). A single cell suspension was generated by triturating the tissue with glass pipettes of decreasing diameter. Neurons were then plated in Neurobasal-A media in 12-well dishes (1 neonate = 2 wells) coated with poly-L- lysine (Sigma-Aldrich).

Activation of Akt in neuronal cultures. Five days after plating, neurons were incubated in serum-free MEM (minimum essential media, Gibco/Invitrogen) for 1 hour prior to a 10 minute drug treatment with 1 M agonist. For each assay, the number of wells of neurons treated over at least 4 separate experiments is provided. For agonist-induced

Akt studies, neurons were treated with “agonist vehicle” (2 M ascorbate in water; n = 56

WT and 35 arr2-KO) or serotonin (n = 35 WT and 23 arr2-KO), N-methylserotonin (n =

19 WT and 12 arr2-KO) or 5-MeO-DMT (n = 13 WT and 12 arr2-KO). For Figure 3.7C,

WT neurons were pretreated with M100907 (10 nM) (187) or vehicle (0.0001% DMSO) during the last 15 minutes of the serum-starvation and prior to agonist stimulation.

Neurons were then treated with either agonist vehicle or serotonin (n = 8 for each of the

4 conditions). For time-course studies, neurons were lysed 0, 1, 3, 5, 10, 20 and 30 minutes following treatment with serotonin (n = 4-8 WT & 4-6 arr2-KO), N- 73 methylserotonin (n = 3-6 per genotype), or 5-MeO-DMT (n = 4-6 per genotype). A concentration curve was completed for serotonin stimulation of Akt, wherein WT neurons were treated with agonist vehicle or 0.001 to 10 M serotonin (n = 8/dose) (Figure 3.7B).

To compare basal phospho-Akt to total-Akt ratios between genotypes, WT and arr2-KO neurons were plated concurrently and lysed following treatment with agonist vehicle (n =

9 per genotype) (Figure 3.7D). For Figure 3.9, inhibitor vehicle (0.1% DMSO) or 1 M

LY294002 or PP2 was added to the serum-free media during the 1 hour incubation.

Neurons were then treated with either agonist vehicle or serotonin (Inhibitor vehicle + agonist vehicle, n = 17; LY294002 + agonist vehicle, n = 9; PP2 + agonist vehicle, n =

10; inhibitor vehicle + serotonin, n = 19; LY294002 + serotonin, n = 10; PP2 + serotonin, n = 11) (199). Following treatment with agonist, primary neurons were washed once in

PBS and lysates were prepared in lysis buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA,

0.1% SDS, 1% NP-40, 0.25% deoxycholate, 1 mM sodium orthovanadate, 1 mM PMSF,

1 mM NaF, protease inhibitors) (44, 84). Lysates were prepared and western blots were performed as described above to assess phospho-Akt and total-Akt levels. For each sample, phospho-Akt levels were normalized to corresponding total-Akt levels and fold stimulation was determined by normalizing to the average vehicle levels for each experiment (or untreated controls for time-course experiments).

Neuronal transfection. Rescue of serotonin-mediated Akt phosphorylation was accomplished by transfecting arr2-KO neurons with 4 g N-terminal myc-arr2 or empty vector (pcDNA3.1) via the calcium phosphate method two days after plating

(Jiang et al., 2004; Clontech/BD Bioscience). After incubation in the DNA/calcium phosphate suspension for 2 hours at 37°C, neurons were washed twice with complete

Neurobasal-A medium. Forty-eight hours following transfection, neurons treated with either agonist vehicle or serotonin and lysed as described above (n = 9 for each condition). Immunoblots were also probed with c-Myc antibody to confirm transfection.

Statistical Analysis. Two-way ANOVA was used to examine group differences in head twitch responses, comparing two main effects (such as genotype and dose); three-way

ANOVA was used to compare three main effects such as pretreatment, genotype and dose. Comparisons between the total number of twitches between drug doses or across genotypes, as well as comparisons of the biochemical data were completed with a

Student‟s unpaired, two-tailed t test. All statistics were conducted with a confidence interval of 95% and were performed using GraphPad Prism 5.0 software (GraphPad

Software) with the exception of the three-way ANOVAs which were performed using

PASW/SSPS 18.0 software (IBM). Three-way ANOVAs were followed by two-way

ANOVA to further examine main effects within groups if visualization prompted the hypothesis that effects may differ within the groups. The use of three-way and one-way

ANOVAs are noted in the text, otherwise, the analyses represent two-way ANOVA.

Unless stated otherwise, Student‟s t tests are presented in the figure legends.

3.3 Results

High doses of serotonin induce head twitches in arr2-KO mice

In the studies presented in the previous chapter, we found that 5-HTP-induced (100 mg/kg, i.p.) head twitch responses are significantly attenuated in mice lacking arrestin2

(Figure 2.6A-B). When the dose of 5-HTP administered was doubled (200 mg/kg, i.p.), however, the response observed in the arr2-KO mice was near that observed for their

WT littermates (Figure 3.1A: for genotype, F(1,156) = 8.15, p = 0.0049; for time, F(11,156) =

7.15, p < 0.0001; interaction genotype x time, F(11,156) = 0.56, p = 0.8550). Moreover, the dose response curve for 5-HTP reveals that as the dose of 5-HTP is increased, the number of twitches observed in both genotypes also increases (Figure 3.1B: for genotype, F(1,42) = 7.99, p = 0.0072; for dose, F(2,42) = 15.30, p < 0.0001; interaction genotype x dose, F(2,42) = 0.76, p = 0.4740). Importantly, pretreatment with the 5-HT2A receptor antagonist M100907 blocked the head twitches induced by the highest dose of

5-HTP (200 mg/kg) in each genotype (Figure 3.1B), demonstrating that the response is due to activation of the 5-HT2A receptor.

The direct injection of serotonin (10 g, i.c.v.) into the brain also induces fewer head twitches in the arr2-KO mice (Figure 2.6E-F). However, similar to the 5-HTP-mediated responses, as the dose of serotonin increases, the number of head twitches observed in the both WT and arr2-KO mice also increases (Figure 3.1D: for genotype, F(1,22) = 0.14, p = 0.7161; for dose, F(2,22) = 21.95, p < 0.0001; interaction genotype x dose, F(2,22) =

9.27, p = 0.0012). In fact, at the highest dose of serotonin administered (40 g, i.c.v.), the arr2-KO mice actually display more head twitches than the WT mice (Figure 3.1C: for genotype, F(1,54) = 16.61, p = 0.0002; for time, F(5,54) = 17.62, p < 0.0001; interaction genotype x time, F(5,54) = 1.67, p = 0.1577). Again, antagonism of the 5-HT2A receptor by

M100907 blocks serotonin-induced head twitches in either genotype (Figure 3.1D). Our initial studies with the lower doses of 5-HTP and serotonin led us to conclude that serotonin requires arrestin2 for the induction of head twitches in mice. The findings presented here call this conclusion into question, as they suggest that the deletion of

arrestin2 might merely shift the relative potency of serotonin at the 5-HT2A receptor. 76

Serotonin metabolism modulates the head twitch response

To begin to explore this apparent discrepancy, we tested the effect that the modulation of serotonin metabolism has on the 5-HTP-induced head twitch response. Clorgyline is an inhibitor of MAO-A and was administered prior to 5-HTP to prevent the conversion of serotonin to 5-HIAA (200). Inhibition of MAO-A increases the number of head twitches observed in both WT and arr2-KO mice treated with a moderate dose of 5-HTP (100 mg/kg, i.p.) (Figure 3.2A: WT: for pretreatment, F(1,156) = 64.82, p < 0.0001; for time,

F(11,156) = 6.61, p < 0.0001; interaction pretreatment x time, F(11,156) = 2.89, p = 0.0017;

arr2-KO: for pretreatment, F(1,132) = 44.07, p < 0.0001; for time, F(11,132) = 2.38, p =

0.0102; interaction pretreatment x time, F(11,132) = 2.18, p = 0.0190). Moreover, the arr2-

KO mice exhibit a response which approaches that observed in the WT mice (vehicle pretreated: for genotype, F(1,108) = 76.38, p < 0.0001; for time, F(11,108) = 10.21, p <

0.0001; interaction genotype x time, F(11,108) = 6.98, p < 0.0001; clorgyline pretreated: for genotype, F(1,180) = 6.48, p = 0.0118; for time, F(11,180) = 10.38, p < 0.0001; interaction genotype x time, F(11,180) = 0.34, p = 0.9753). Analysis of clorgyline‟s effects on the total number of twitches observed for two doses of 5-HTP demonstrates that clorgyline shifts the 5-HTP dose-response curve leftward in both genotypes, such that the arr2-KO mice now display head twitches at doses of 5-HTP that are ineffective when given alone

(Figure 3.2B: three-way ANOVA: for dose, F(1,42) = 6.07, p = 0.0180; for pretreatment,

F(1,42) = 26.00, p < 0.0001; for genotype: F(1,42) = 4.59, p = 0.0380). Again, M100907 blocked the clorgyline-enhanced 5-HTP (100 mg/kg, i.p.) head twitches in both genotypes (Figure 3.2B), demonstrating that the effects are still mediated through activation of the 5-HT2A receptor.

These data further support the conclusion that the absence of arrestin2 merely shifts the potency of serotonin at the 5-HT2A receptor. Alternatively, however, these high doses of 5-HTP and serotonin may elevate the brain concentration of the metabolites of serotonin, which could also impact the head twitch response. The metabolites of serotonin include 5-HIAA, which does not have agonist activity at the 5-HT2A receptor, and the N-methyltryptamines, active neurotransmitters at the receptor (Figure 1.1). High doses of serotonin and 5-HTP or inhibition of the MAO-A metabolic pathway could increase N-methyltryptamine levels in the brain. Therefore, the head twitches observed following these treatment regimens may reflect the actions of both serotonin and the N- methyltryptamines at the 5-HT2A receptor.

To determine the contribution of N-methyltryptamines to the 5-HTP-induced head twitch response, we used the INMT inhibitor MTZ to block the formation of the N- methyltryptamines. MTZ was administered prior to the dose of 5-HTP (200 mg/kg, i.p.) at which we observed the greatest number of head twitches in either genotype (27-29,

201). MTZ significantly decreases the number of head twitches in both the WT and

arr2-KO mice (Figure 3.3A: WT: for pretreatment, F(1,72) = 34.84, p < 0.0001; for time,

F(11,72) = 19.39, p < 0.0001; interaction pretreatment x time, F(11,72) = 1.21, p = 0.2931;

arr2-KO: for pretreatment, F(1,96) = 127.02, p < 0.0001; for time, F(11,96) = 10.77, p <

0.0001; interaction pretreatment x time, F(11,96) = 6.51, p < 0.0001; vehicle pretreated: for genotype, F(1,84) = 22.30, p < 0.0001; for time, F(11,84) = 18.18, p < 0.0001; interaction genotype x time, F(11,84) = 1.59, p = 0.1170; MTZ pretreated: for genotype, F(1,84) =

185.73, p < 0.0001; for time, F(11,84) = 16.53, p < 0.0001; interaction genotype x time,

F(11,84) = 7.47, p < 0.0001). However, inhibition of INMT decreased the head twitches observed in the arr2-KO mice to a greater extent than those observed in the WT mice

(Figure 3.3B). These results indicate the head twitches observed at the highest dose of

5-HTP are in part due to the actions of the N-methyltryptamine metabolites of serotonin.

Moreover, as the MTZ-pretreatment nearly abolished the head twitches observed in the

arr2-KO mice, these findings suggest that the arrestin2-independent head twitches may be solely due to the activation of the 5-HT2A receptor by these metabolites.

N-methyltryptamines induce head twitches independent of arrestin2

To directly evaluate the profile of N-methyltryptamine-induced head twitches in the absence of arrestin2, we injected WT and arr2-KO mice with one of the endogenous metabolites of serotonin, N-methylserotonin. N-methylserotonin (20 g, i.c.v.) induces a greater number of head twitches in the arr2-KO mice than their WT littermates, a response profile which mirrors that observed following the highest dose of serotonin administered (Figure 3.4A: for genotype, F(1,56) = 24.41, p < 0.0001; for time, F(6,56) =

37.89, p < 0.0001; interaction genotype x time, F(6,56) = 4.97, p = 0.0004). Moreover, the

arr2-KO mice display enhanced responses compared to WT mice at all doses of N- methylserotonin tested (Figure 3.4B: for genotype, F(1,20) = 32.99, p < 0.0001; for dose,

F(2,20) = 15.10, p = 0.0001; interaction genotype x dose, F(2,20) = 6.45, p = 0.0069). The 5-

HT2A receptor antagonist M100907 inhibits the N-methylserotonin mediated head twitch response in both genotypes, demonstrating that the head twitches are still due to activation of the 5-HT2A receptor (Figure 3.4B). Importantly, the INMT inhibitor MTZ had no effect on N-methylserotonin-mediated head twitches in WT mice, arguing against a non-specific blockade of the behavior by the enzyme inhibitor (Figure 3.4C: for 79 pretreatment, F(1,49) = 0.83, p = 0.3659; for time, F(6,49) = 29.90, p < 0.0001; interaction pretreatment x time, F(6,49) = 0.28, p = 0.9432).

We also assessed the role of arrestin2 in mediating the head twitch response induced by 5-MeO-DMT, a hydrolysis resistant, psychoactive N-methyltryptamine (190). 5-MeO-

DMT, when tested at multiple doses, induces a similar profile of head twitches as that observed for N-methylserotonin, with arr2-KO mice responding to a greater extent than

WT mice (Figure 3.4D: for genotype, F(1,144) = 32.70, p < 0.0001; for time, F(5,144) = 54.67, p < 0.0001; interaction genotype x time, F(5,144) = 1.84, p = 0.1080; Figure 3.4E: for genotype, F(1,49) = 10.88, p = 0.0018; for dose, F(2,49) = 13.93, p < 0.0001; interaction genotype x dose, F(2,49) = 3.79, p = 0.0294). Moreover, 5-MeO-DMT head twitches in both genotypes are also blocked by pretreatment with M100907 (Figure 3.4E) and MTZ pretreatment has no effect on 5-MeO-DMT induced twitches in C57BL/6J mice (Figure

3.4F: for genotype, F(1,48) = 0.00, p = 0.9657; for time, F(5,48) = 28.66, p < 0.0001; interaction genotype x time, F(5,48) = 1.39, p = 0.2433). Collectively, these studies indicate that the N-methyltryptamines do not require arrestin2 for the induction of the head twitch response. Interestingly, the enhanced responses to N-methyltryptamines that are observed in the arr2-KO mice suggest that arrestin2 may play a negative regulatory role in the signaling cascades involved in this behavioral response for these agonists.

Facilitation of serotonin-mediated 5-HT2A receptor signaling by arrestin2 in vivo

The in vivo findings presented thus far suggest that the head twitches observed in the

arr2-KO mice may be primarily attributed to the actions of the N-methyltryptamine

80 metabolites of serotonin, rather than the direct actions of serotonin at the 5-HT2A receptor. Furthermore, they support our initial conclusion that serotonin-mediated head twitches require arrestin2. Given that arrestins facilitate GPCR signaling by scaffolding signaling proteins to receptors in vivo (117, 185), we assessed the 5-HT2A receptor signaling complexes that are formed in the mouse frontal cortex.

We isolated frontal cortex from WT and arr2-KO following a 10 minute treatment with 5-

HTP (100 mg/kg, i.p.). At this dose of 5-HTP we hypothesize that only serotonin, and not its metabolites, is acting at the 5-HT2A receptor since it induces head twitches only in WT mice (Figure 2.6A and Figure 3.1B). Arrestin2 co-immunoprecipitates with the 5-HT2A receptor following 5-HTP treatment in cortical lysates from WT mice (Figure 3.5A). In addition, 5-HTP causes an increase in 5-HT2A receptor associations with the tyrosine kinase Src and the serine-threonine kinase Akt. The scaffolding protein PSD-95, which has been shown to interact with the 5-HT2A receptor and target the receptor to the plasma membrane of cortical neurons, is also pre-associated with the 5-HT2A receptor in vehicle treated animals and 5-HTP administration results in a decreased interaction between the two proteins. In contrast to the WT mice, 5-HTP treatment did not induce the displacement of PSD-95 from receptors expressed in the frontal cortex of arr2-KO mice, nor did it lead to the recruitment of Akt or Src to the receptor complex in the absence of arrestin2.

We then assessed the 5-HT2A receptor signaling complex that is formed in the frontal cortex of WT and arr2-KO mice following administration of 5-MeO-DMT (10 mg/kg, i.p.).

Contrary to 5-HTP, 5-MeO-DMT administration did not lead to the recruitment of

arrestin2, Src or Akt to the 5-HT2A receptor in either WT or arr2-KO mice, nor did 5-

MeO-DMT induce the disassociation of PSD-95 from the receptor (Figure 3.5B). Since

arrestin2 is not required for N-methyltryptamine-induced head twitches in vivo, these agonist-mediated differences were not wholly unexpected. These data clearly suggest that agonists activate different signal transduction pathways in the mouse frontal cortex, wherein serotonin induces the formation of a 5-HT2A receptor/arrestin2/Src/Akt signaling complex, but the N-methyltryptamines do not.

We hypothesized that the arrestin2-scaffolding of these kinases to the 5-HT2A receptor in the frontal cortex might result in an increase in their activity. Therefore, we assessed the phosphorylation state of Akt in the frontal cortex of WT and arr2-KO mice following treatment with 5-HTP (100 mg/kg, i.p.). Indeed, treatment of WT mice with 5-HTP induces Akt phosphorylation at threonine 308. Conversely, the phosphorylation of Akt is not increased over basal levels in the frontal cortex of arr2-KO mice treated with 5-HTP

(Figure 3.6A). Comparison of vehicle-treated animals from both genotypes reveals no differences in Akt phosphorylation (Figure 3.6B), suggesting that the lack of 5-HTP- induced Akt stimulation in the arr2-KO mice is not attributable to an elevated basal state of activation in the frontal cortex. 5-MeO-DMT (10 mg/kg, i.p.) also does not activate Akt in the mouse frontal cortex, regardless of genotype (Figure 3.6A). From these data, we hypothesized that the serotonin-mediated 5-HT2A receptor signaling complex formed in the mouse frontal cortex is necessary for the activation of Akt.

To further characterize the serotonin-induced Akt phosphorylation that occurs downstream of the 5-HT2A receptor, we assessed agonist-induced kinase activation in 82 primary neuronal cultures prepared from the frontal cortex of WT and arr2-KO neonates. Four days after plating, neurons were serum-starved for 1 hour to reduce basal Akt phosphorylation levels. A 10 minute treatment with serotonin (1 M) stimulates robust Akt phosphorylation in WT cortical neurons but does not modulate Akt phosphorylation levels in neurons from arr2-KO mice (Figure 3.7A). A concentration response curve reveals that serotonin-stimulated Akt phosphorylation is maximal at 1 M

(Figure 3.7B: F(5,38) = 2.83, p = 0.0288, one-way ANOVA). Furthermore, pretreatment with the 5-HT2A receptor antagonist M100907 completely blocks serotonin-mediated Akt phosphorylation in WT neurons, demonstrating that the activation of Akt by serotonin is dependent upon the 5-HT2A receptor (Figure 3.7C).

One possible explanation for serotonin‟s inability to increase the phosphorylation of Akt in the arr2-KO mice is that the arr2-KO neurons may have higher basal levels of Akt activation, which could mask any drug-mediated increases in phosphorylation. To address this concern, we compared Akt phosphorylation levels in neurons from both genotypes which had been plated and treated concurrently. This direct comparison reveals that WT and arr2-KO neurons have similar basal phosphorylation levels

(Figure 3.7D). Another possibility is that in the absence of arrestin2, the kinase is phosphorylated at a different rate than that observed in the WT neurons. Analysis of the time-course of activation, however, reveals that serotonin stimulated Akt phosphorylation peaks at 5 to 10 minutes in WT neurons, but is absent at all time points in the arr2-KO neurons (Figure 3.7E: for genotype, F(1,69) = 55.47, p < 0.0001; for time, F(6,69) = 2.40, p

= 0.0367; interaction genotype x time, F(6,69) = 3.44, p = 0.0050). Therefore, the lack of

Akt activation observed in the arr2-KO neurons is not due to a temporal shift in the 83 drug-induced phosphorylation of the kinase. To further demonstrate the dependence upon arrestin2 for the serotonin-induced phosphorylation of Akt, we transfected arr2-

KO neurons with a myc-tagged arrestin2 (myc-arr2) and assessed Akt phosphorylation two days later. Myc-arr2 rescues serotonin-induced Akt phosphorylation in arr2-KO neurons (Figure 3.7F). Collectively, these studies show that arrestin2 is required the 5-HT2A receptor-mediated phosphorylation of Akt that occurs following the treatment of cortical neurons with serotonin.

In contrast to serotonin, neither of the N-methyltryptamines, N-methylserotonin (1 M) or

5-MeO-DMT (1 M), induce Akt phosphorylation at 10 minutes in cortical neurons from

WT or arr2-KO mice (Figure 3.7A). Moreover, time-course studies show that these compounds do not stimulate Akt phosphorylation at a different rate than serotonin, as an increase in phosphorylation was not observed at any time-point, up to 30 minutes following agonist administration (Figure 3.8A: for genotype, F(1,54) = 3.48, p = 0.0674; for time, F(6,54) = 1.35, p = 0.2532; interaction genotype x time, F(6,54) = 1.20, p = 0.3185;

Figure 3.8B: for genotype, F(1,54) = 0.02, p = 0.8917; for time, F(6,54) = 1.05, p = 0.4047; interaction genotype x time, F(6,54) = 0.11, p = 0.9948). These results further confirm that the N-methyltryptamines do not activate Akt downstream of the 5-HT2A receptor.

The immunoprecipitation data shows that arrestin2 scaffolds Src to the 5-HT2A receptor in addition to Akt. As Src activation has been shown to induce Akt phosphorylation (185,

202, 203), these findings implicated Src in the Akt signaling cascade. To determine if Src activation is upstream of Akt phosphorylation, we pretreated WT neurons with the Src inhibitor (PP2). We found that PP2 blocks serotonin-induced phosphorylation of Akt 84

(Figure 3.9). One mechanism by which Src induces the activation of Akt at threonine

308 is through the activation of PI3-K (199, 204). Therefore, we also pretreated the WT neurons with a chemical inhibitor of PI3-K prior to drug stimulation. Pretreatment with the

PI3-K inhibitor (LY294002) prevents serotonin-induced Akt phosphorylation in WT neurons (Figure 3.9). These inhibitor studies indicate that PI3-K and Src are both involved in the pathway through which the 5-HT2A receptor mediates Akt phosphorylation in neurons.

Blockade of the Akt signaling complex inhibits serotonin-mediated head twitches

There appears to be a direct correlation between the inability of serotonin to induce the head twitch response and its inability to induce the phosphorylation of Akt in the absence of arrestin2. To assess whether these two events are related, C57BL/6J mice were injected with either vehicle or an inhibitor of PI3-K (LY294002) (117), Src (PP2) (197) or

Akt (AKTi) (198) prior to treatment with 5-HTP (200 mg/kg, i.p.). Each of the inhibitors partially blocks the head twitch response observed in C57BL/6J mice (Figure 3.10A: vehicle vs. LY294002: for pretreatment, F(1,156) = 38.52, p < 0.0001; for time, F(11,156) =

5.51, p < 0.0001; interaction pretreatment x time, F(11,156) = 0.88, p = 0.5604; vehicle vs.

PP2: for pretreatment, F(1,156) = 43.02, p < 0.0001; for time, F(11,156) = 6.17, p < 0.0001; interaction pretreatment x time, F(11,156) = 0.86, p = 0.5832; vehicle vs. AKTi: for pretreatment, F(1,156) = 35.94, p < 0.0001; for time, F(11,156) = 6.49, p < 0.0001; interaction pretreatment x time, F(11,156) = 1.09, p = 0.3729). At this high dose of 5-HTP (200 mg/kg, i.p.), we would predict that both serotonin and N-methyltryptamines are acting at the 5-

HT2A receptor to induce head twitches. Moreover, given the findings of the biochemical studies, we would expect that the kinase inhibitors would only block those twitches due

85 to serotonin‟s actions at the receptor. Accordingly, we find that each of the individual kinase inhibitors blocks approximately 50% of the head twitches observed in the

C57BL/6J mice (Figure 3.10B).

Conversely, we predicted that the inhibition of the Akt-signaling cascade should have no effect on N-methyltryptamine-induced head twitches since they do not activate Akt in the mouse frontal cortex. Consistent with this, pretreatment with AKTi does not modulate the head twitch response that follows 5-MeO-DMT (10 mg/kg, i.p.) administration (Figure

3.10C: for pretreatment, F(1,48) = 0.02, p = 0.8759; for time, F(5,48) = 20.62, p < 0.0001; interaction pretreatment x time, F(5,48) = 0.22, p = 0.9536). These data indicate that 5-

MeO-DMT-induced head twitches are independent of the Akt activation and argue against a non-specific blockade of the head twitch response by the kinase inhibitor.

We then pretreated arr2-KO mice with LY294002, PP2 or AKTi prior to 5-HTP administration (200 mg/kg, i.p.). The PI3-K, Src and Akt inhibitors had no effect on the 5-

HTP-induced (200 mg/kg, i.p.) head twitch response in the absence of arrestin2

(Figure 3.11: vehicle vs. LY294002: for pretreatment, F(1,132) = 1.02, p = 0.3136; for time,

F(11,132) = 11.68, p < 0.0001; interaction pretreatment x time, F(11,132) = 0.11, p = 0.9999; vehicle vs. PP2: for pretreatment, F(1,132) = 3.57, p = 0.0609; for time, F(11,132) = 8.31, p <

0.0001; interaction pretreatment x time, F(11,132) = 0.18, p = 0.9983; vehicle vs. AKTi: for pretreatment, F(1,132) = 0.30, p = 0.5830; for time, F(11,132) = 11.81, p < 0.0001; interaction pretreatment x time, F(11,132) = 0.06, p = 1.0000). These results are also consistent with the biochemical studies which demonstrate that 5-HTP does not induce the formation of the Akt signaling complex with the 5-HT2A receptor in the absence of arrestin2 (Figure

3.5). Moreover, our behavioral data suggest that the 5-HTP-induced head twitches observed in arr2-KO mice are due to the actions of the N-methyltryptamine metabolites at the 5-HT2A receptor and not serotonin itself (Figure 3.3). These biochemical and behavioral studies point to a model wherein serotonin induces the head twitch response through a 5-HT2A receptor signaling cascade involving arrestin2, PI3-K, Src and Akt. In contrast, the N-methyltryptamine-induced head twitch response is mediated through an alternate, unidentified, but arrestin2-independent pathway.

The data presented thus far also suggest that the head twitches observed in WT mice following treatment with a high dose of 5-HTP (200 mg/kg, i.p.) are due to the activity of both serotonin and its N-methyltryptamine metabolites at the 5-HT2A receptor. To test this hypothesis, we co-administered the Akt inhibitor (to block the serotonin-mediated twitches) and the INMT inhibitor (to prevent N-methyltryptamine synthesis) to WT mice prior to treatment with this high dose of 5-HTP (200 mg/kg, i.p.). As demonstrated previously, the individual administration of either inhibitor partially attenuates the head twitch response in WT mice (Figure 3.12: vehicle vs. AKTi: for pretreatment, F(1,180) =

96.55, p < 0.0001; for time, F(11,180) = 19.77, p < 0.0001; interaction pretreatment x time,

F(11,180) = 3.72, p < 0.0001; vehicle vs. MTZ: for pretreatment, F(1,168) = 53.73, p < 0.0001; for time, F(11,168) = 18.82, p < 0.0001; interaction pretreatment x time, F(11,180) = 2.28, p =

0.0130). Concurrent administration of the two inhibitors, however, nearly abolishes the 5-

HTP-induced head twitch response (vehicle vs. AKTi + MTZ: for pretreatment, F(1,168) =

172.24, p < 0.0001; for time, F(11,168) = 10.77, p < 0.0001; interaction pretreatment x time,

F(11,168) = 7.13, p < 0.0001). The additive effect of the two inhibitors suggests the

87 involvement of two individual pathways in the induction of the 5-HTP-mediated head twitch response.

3.4 Discussion

In this chapter, we demonstrate functional selectivity at the 5-HT2A receptor whereby serotonin and N-methyltryptamines promote differential signaling in the mouse frontal cortex and in primary cortical neurons (Figure 3.13). We show that serotonin at the 5-

HT2A receptor stimulates the formation of a receptor signaling complex that is composed of arrestin2, Src and Akt and induces Akt phosphorylation through a arrestin2-, PI3-K- and Src-dependent pathway. Moreover, inhibition of any component of this signaling complex prevents the expression of the 5-HTP-induced head twitch response. In contrast, the N-methyltryptamines, N-methylserotonin and 5-MeO-DMT, do not induce

Akt phosphorylation in the mouse frontal cortex or in primary cortical neurons, and while they do mediate the head twitch response through the activation of 5-HT2A receptors, the mechanism appears to be independent of arrestin2 and Akt.

The studies presented in this chapter suggest that arrestin2 facilitates 5-HT2A receptor signaling in the mouse frontal cortex by scaffolding Src and Akt to the receptor. The direct phosphorylation of Akt by Src has been shown to be necessary for the full activation of Akt in some systems (202, 203). In this system, arrestin2 serves to facilitate the phosphorylation of Akt by acting as an adaptor protein and bringing these kinases together in a single complex with the 5-HT2A receptor. Therefore, the scaffolding of these two kinases to arrestin2 may increase the efficiency of the Src mediated phosphorylation of Akt. Interestingly, Beaulieu et al. (117) also demonstrate that

arrestin2 acts to scaffold Akt to the D2 dopamine receptor in the mouse striatum. Yet, for this receptor system, the arrestin2/Akt scaffold also includes the phosphatase

PP2A, and the formation of the complex results in the dephosphorylation of Akt.

Therefore, by scaffolding either Src or PP2A to their respective GPCRs, arrestin2 interactions can ultimately serve to activate or inactivate Akt. These studies exemplify how agonist-induced receptor signaling is finely orchestrated by the proteins expressed in close proximity to a GPCR.

Like arrestin2, PSD-95 is another scaffolding protein that has been shown to regulate

5-HT2A receptor trafficking. Studies from the Roth laboratory have demonstrated that

PSD-95 interactions with the 5-HT2A receptor serve to promote clustering of the receptor to the plasma membrane of HEK-293 cells and are integral for the dendritic targeting of the receptor in cortical pyramidal neurons (151, 155, 156). In Figure 3.5, we demonstrate that following 5-HTP administration, PSD-95 is disengaged from, and arrestin2 is recruited to the 5-HT2A receptor complex in frontal cortex isolated from WT mice.

Moreover, in chapter 2 we demonstrate that the serotonin-mediated recruitment of

arrestin2 induces the internalization of the 5-HT2A receptor (Figure 2.2). In contrast, in the absence of arrestin2, PSD-95 interactions with the 5-HT2A receptor in the frontal cortex remain unchanged regardless of drug treatment (Figure 3.5) and the 5-HT2A receptor is restricted to the plasma membrane of cortical neurons from arr2-KO mice

(Figure 2.4). It is attractive to hypothesize that the interplay between arrestin2 and

PSD-95 may determine the intracellular trafficking of the 5-HT2A receptor, which may ultimately impact the ligand-directed signaling of the receptor in vivo.

While serotonin-mediated head twitches are significantly attenuated in the absence of

arrestin2, an enhanced head twitch response is observed in the arr2-KO mice following treatment with multiple doses of either N-methylserotonin or 5-MeO-DMT

(Figures 3.1 and 3.4). Enhanced physiological responses following a challenge with an agonist have been observed in the arr2-KO mice for other receptor systems in vivo (97,

184, 205-208). In these cases, arrestin2 has been implicated as a negative regulator of

GPCR signaling, thus resulting in enhanced physiological responses in its absence. It is possible that arrestin2 may negatively regulate the N-methyltryptamine-bound 5-HT2A receptor in the mouse frontal cortex, although the direct involvement of arrestin2 in dampening 5-HT2A receptor-mediated G protein-coupling remains to be determined. This hypothesis is confounded by the fact that arrestin2 does not co-immunoprecipitate with the 5-HT2A receptor in brain lysates following 5-MeO-DMT administration (Figure 3.5B).

However, the lack of co-immunoprecipitation does not exclude the possibility that the receptor and arrestins may interact, as the interaction induced by the N- methyltryptamines may be more transient or less stable than the serotonin-mediated interactions, and thereby not preserved in our immunoprecipitation studies.

The head twitch response studies presented in this chapter also suggest that arrestin2 both facilitates and dampens 5-HT2A receptor signaling in the frontal cortex, depending upon the agonist bound to the receptor. These data suggest that the agonist not only dictates whether or not arrestin2 is recruited to a receptor, but also determines the functional consequence of the interaction: serotonin recruits arrestin2 to facilitate signaling, while N-methyltryptamines recruitment of arrestin2 may act to desensitize the

5-HT2A receptor. The chemokine receptor CCR7 agonists, CCL19 and CCL21, provide in 90 vitro precedence for the agonist-directed divergence in arrestin2 function at a single receptor. Both agonists recruit arrestins and stimulate ERK1/2 phosphorylation through a arrestin2-dependent pathway, yet only CCL19 induces arrestin-dependent receptor desensitization and internalization (209-212). Previously, arrestins have been implicated in both facilitating and desensitizing the same GPCR in vivo; however the regulatory role has appeared to be tissue or region specific. While arrestin2 acts to desensitize the opioid receptor expressed in brain regions associated with antinociception (96, 97), data suggest that it may facilitate signaling in neurons in the gastrointestinal tract which are involved in the development of opioid-induced constipation (213). The studies presented in this chapter may be the first in vivo example of arrestins both negatively and positively regulating a particular receptor expressed within the same neuronal population. Furthermore, serotonin and the N- methyltryptamines serve as in vivo examples of ligands which specifically target distinct actions of arrestins to either dampen specific signaling cascades or stimulate the activation of others.

Although the absence of arrestin2 differentially affects the head twitch responses mediated by serotonin and the N-methyltryptamines, all of the head twitches observed in either genotype can be blocked by the 5-HT2A receptor antagonist, M100907 (Figures

3.1 and 3.4). These studies indicate that these agonists induce the head twitch response via the activation of distinct signaling pathways downstream of the 5-HT2A receptor.

However, the activation or inhibition of other serotonin receptor subtypes, such as the 5-

HT1A receptor and 5-HT2C receptor, can modulate the head twitch response in mice

(214-217). It is possible that the N-methyltryptamine-stimulated head twitch response 91 could be confounded by the activation of non-target GPCRs, as these agonists are not selective for the 5-HT2A receptor. On the other hand, serotonin is also not selective for the 5-HT2A receptor and therefore if receptors other than the 5-HT2A receptor were impacting N-methyltryptamine-mediated head twitches, we might expect a similar impact on serotonin-mediated head twitches as well. Furthermore, the immunoprecipitation data demonstrate a stark distinction between the signaling complexes that are formed for the serotonin-bound 5-HT2A receptor versus the 5-MeO-DMT bound receptor (Figure 3.5) and support our conclusion that N-methyltryptamines and serotonin differentially activate signaling cascades at the level of the 5-HT2A receptor in the mouse frontal cortex.

The findings presented in this chapter demonstrate physiological relevance for the functional selectivity observed at the 5-HT2A receptor, as serotonin and its endogenous, hallucinogenic metabolites differentially utilize arrestin2 to signal and induce the head twitch response. Although our data suggest that N-methyltryptamines have limited roles in the activation of the 5-HT2A receptor until serotonin levels are extremely elevated in the CNS, these findings may still have clinical implications as many of the traditional therapies for the treatment of depression involve elevating endogenous serotonin levels in the brain, either by MAO inhibitors (MAO-I) or selective serotonin reuptake inhibitors

(SSRIs). Exposure to a these serotonergic-augmenting drugs can cause a potentially life-threatening condition termed “serotonin syndrome” in humans (218-220).

Interestingly, in some patients, hallucinations have been reported to accompany the syndrome (221, 222), which is brought about by excessive serotonin levels, and presumably N-methyltryptamine levels, in the CNS. Endogenous N-methyltryptamines have also been implicated in schizophrenia (27, 223, 224), wherein elevated levels have

92 been detected in the urine of schizophrenic patients (225-228). However, a definitive role for these endogenous hallucinogens and the psychotic states associated with the disease remains to be determined. In addition, the head twitch responses in the arr2-

KO mice provide an in vivo distinction between serotonin-induced and the N- methyltryptamine-induced activation of 5-HT2A receptor and therefore could be utilized to further differentiate receptor activation by these endogenous agonists.

Overall, the findings presented in this chapter further demonstrate how 5-HT2A receptor responsiveness diverges based on ligand-directed interactions with arrestins.

Moreover, serotonin and its metabolic products stimulate the formation of distinct the 5-

HT2A receptor signaling complexes in the mouse frontal cortex, which impact physiological responses in vivo. Serotonin‟s dependence upon arrestin2 for the activation of the head twitch response also suggests that arrestin2-dependent signaling may be as essential as G protein-mediated signal transduction for physiological responses activated by 5-HT2A receptors expressed in the mouse frontal cortex. The elucidation of these differences could impact future drug development efforts which target the 5-HT2A receptor.

3.5 Chapter 3 Tables

Figure Treatment Serotonergic dose and mouse number (n) (mouse line) 3.1A, B 5-HTP 100 150 200 M100 + 200 WT, arr2-KO n = 13, 8 n = 7, 5 n = 8, 7 n = 6, 6 3.1C, D 5-HT 10 20 40 M100 + 40 WT, arr2-KO n = 4, 5 n = 7, 5 n = 5, 6 n = 4, 4 3.2A, B Veh/Clor + 5-HTP Veh + 50 Clor + 50 Veh + 100 Clor + 100 M100 + Clor + 100 WT, arr2-KO n = 6, 5 n =5, 6 n = 6, 5 n = 9, 8 n = 4, 4 3.3A, B Veh/MTZ + 5-HTP Veh + 200 MTZ + 200 WT, arr2-KO n = 4, 5 n = 4, 5 3.4A, B N-Me-5-HT 10 20 40 M100 + 20 WT, arr2-KO n = 4, 4 n = 5, 5 n = 4, 4 n = 4, 4 3.4C Veh/MTZ + N-Me-5-HT Veh + 20 MTZ + 20 WT n = 5 n = 4 3.4D, E 5-MeO-DMT 5 10 15 M100 + 10 WT, arr2-KO n = 9, 10 n = 10, 16 n = 5, 5 n = 5, 5 3.4F Veh/MTZ + 5-MeO-DMT Veh + 10 MTZ + 10 C57BL/6J n = 6 n = 4 3.10A, B Veh/LY/PP2/AKTi + 5-HTP Veh + 200 LY + 200 PP2 + 200 AKTi + 200 C57BL/6J n = 20 n = 5 n = 5 n = 5 3.10C Veh/AKTi + 5-MeO-DMT 10 AKTi + 10 C57BL/6J n = 6 n = 4 3.11A, B Veh/LY/PP2/AKTi + 5-HTP Veh + 200 LY + 200 PP2 + 200 AKTi + 200 arr2-KO n = 7 n = 6 n = 6 n = 6 3.12A, B Veh/(AKTi±MTZ) + 5-HTP Veh + 200 MTZ + 200 AKTi + 200 (MTZ + AKTi) + 200 WT n = 11 n = 6 n = 5 n = 5

Table 3.1 Mouse numbers for behavioral studies The number of mice used in each of the different head twitch response studies are provided. Abbreviations not used in text: M100907 (M100), serotonin (5-HT), N-methylserotonin (N-Me-5- HT), vehicle (veh), clorgyline (clor) and LY294002 (LY). Serotonergic dosing routes: 5-HTP and 5-MeO-DMT (mg/kg, i.p.); serotonin and N-methylserotonin (g, i.c.v.). For inhibitor and antagonist dosing information, see the Materials and Methods section. Adapted from Schmid and Bohn (45).

3.6 Chapter 3 Figures

A. 5-HTP B.

50 WT 300 WT arr2-KO arr2-KO 40 225 30 150 20

10 75 *

0 ** ## ## Head Twitches/ 5 Minutes Twitches/ Head

Head Twitches in 60 Minutes 60 in Twitches Head 0 10 20 30 40 50 60 100 150 200 M100+200 Time (minutes) 5-HTP (mg/kg, i.p.) C. Serotonin D.

50 WT 125 WT * arr2-KO arr2-KO 40 100

30 75

20 50

10 25 ** ##

0 ** ## Head Twitches/ 5 Minutes Twitches/ Head Head Twitches in 30 Minutes 30 in Twitches Head 0 5 10 15 20 25 30 10 20 40 M100+40 Time (minutes) 5-HT (g, i.c.v.)

Figure 3.1 High levels of serotonin and 5-HTP can induce a head twitch response in the arr2-KO mice Mice were injected with either 5-HTP (A-B) or serotonin (5-HT; C-D) and head twitches were scored for 60 or 30 minutes, respectively. In some cases, the 5-HT2A receptor antagonist M100907 (M100: 0.05 mg/kg, i.p.) was administered 10 minutes prior to agonist treatment. Low doses of 5-HTP or serotonin induce fewer twitches in the arr2-KO mice, however as the dose increases the arr2-KO mice twitch as much as or even more than their WT littermates. A. Time- course of head twitches counted following treatment with 200 mg/kg 5-HTP (i.p.) B. 5-HTP- induced dose response curve and blockade of 5-HTP-induced (200 mg/kg, i.p.) head twitches by M100907 pretreatment. WT vs. arr2-KO within the same dose: *p < 0.05; **p < 0.001. 200 mg/kg 5-HTP vs. M100 + 200 mg/kg 5-HTP within each genotype: ##p < 0.001. C. Time-course of head twitches counted following treatment with 40 g serotonin (i.c.v.). D. Serotonin-induced dose response curve and blockade of serotonin-induced (40 g, i.c.v.) head twitches by M100907 pretreatment. WT vs. arr2-KO within the same dose: *p < 0.05; **p < 0.001. 40 g 5-HT vs. M100 + 40 g 5-HT within each genotype: ##p < 0.001. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

A. Clorgyline + 5-HTP B. WT: Vehicle Clorgyline arr2-KO: Vehicle Clorgyline 50 WT 250 ## arr2-KO 40 200 # 30 150 ### 20 100

10 ### 50 *** 0 ^ ^ Head Twitches/ 5 Minutes Twitches/ Head 0 *** *** 10 20 30 40 50 60 Minutes 60 in Twitches Head Veh Clor Veh Clor Clor + Time (minutes) M100 5-HTP: 50 mg/kg 100 mg/kg Pretreatment

Figure 3.2 Inhibition of MAO-A enhances the 5-HTP-induced head twitch response in both WT and arr2-KO mice An hour pretreatment with the MAO-A inhibitor clorgyline (Clor: 1 mg/kg, i.p.) enhances the number of 5-HTP-induced head twitches observed in both WT and arr2-KO mice, compared to mice pretreated with vehicle (Veh: 0.9% saline, i.p.). A. Time-course of 5-HTP-induced (100 mg/kg, i.p.) head twitches following clorgyline pretreatment. B. Total number of twitches observed for two doses of 5-HTP, with or without clorgyline pretreatment. In some cases, M100907 (M100: 0.05 mg/kg, i.p.) was administered in the last 10 minutes of the clorgyline pretreatment to inhibit the 100 mg/kg dose of 5-HTP. WT vs. arr2-KO within the same pretreatment group: ***p < 0.0001. Vehicle pretreatment vs. clorgyline pretreatment within each genotype: #p < 0.05, ##p < 0.001, ###p < 0.0001. Clorgyline + 100 mg/kg 5-HTP vs. Clorgyline + M100 + 100 mg/kg 5-HTP: ^p < 0.05. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

A. 5-HTP + B.

WT: Vehicle MTZ arr2-KO: Vehicle MTZ 50 300 WT arr2-KO 40 225 30 #

20 150

10 75 ## ***

0 Head Twitches/ 5 Minutes Twitches/ Head Head Twitches in 60 Minutes 60 in Twitches Head 0 10 20 30 40 50 60 Veh MTZ Time (minutes)

Figure 3.3 Pretreatment with an INMT inhibitor significantly attenuates 5-HTP-induced head twitches in arr2-KO mice

Mice were pretreated with MTZ (125 ng, i.c.v.) or vehicle (Veh: 5 l dH2O, i.c.v.) for 10 minutes prior to treatment with 5-HTP (200 mg/kg, i.p.) and head twitches were counted. MTZ significantly decreases head twitches in WT mice, but almost completely eliminates head twitches in arr2-KO mice. Time-course analysis (A) and the total number of twitches (B) observed over the 60 minute period following injection with 5-HTP are shown. WT vs. arr2-KO with each pretreatment group: ***p < 0.001. Vehicle pretreatment vs. MTZ pretreatment within each genotype: #p < 0.05, ##p < 0.01. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

A. N-methylserotonin B. C.

12 WT 6 WT 60 ** Vehicle arr2-KO arr2-KO MTZ 9 45 4

6 30 * 2 3 15 **

0 0 Head Twitches/ 5 Minutes Twitches/ Head Head Twitches/ 5 Minutes Twitches/ Head 0 5 10 15 20 25 30 Minutes 30 in Twitches Head 10 20 40 M100 5 10 15 20 25 30 Time (minutes) +20 Time (minutes) N-Me-5-HT (g, i.c.v.)

D. 5-MeO-DMT E. F.

20 WT 60 WT 8 Vehicle arr2-KO arr2-KO MTZ

15 45 *** 6

10 30 * 4

5 15 2

0 ##### 0 Head Twitches/ 5 Minutes Twitches/ Head 0 5 Minutes Twitches/ Head

Head Twitches in 30 Minutes 30 in Twitches Head M100 5 10 15 20 25 30 5 10 15 5 10 15 20 25 30 +10 Time (minutes) Time (minutes) 5-MeO-DMT (mg/kg, i.p.)

Figure 3.4 N-methyltryptamines induce more head twitches in arr2-KO mice than their WT littermates Mice were injected with either N-methylserotonin (N-Me-5-HT: A-C) or 5-MeO-DMT (E-G) and head twitches were scored for 30 minutes. In some cases, the 5-HT2A receptor antagonist

M100907 (M100: 0.05 mg/kg, i.p.) or the INMT inhibitor MTZ (125 ng, i.c.v. in 5 l dH2O) were administered 10 minutes prior to agonist treatment. Arr2-KO mice treated with N- methylserotonin and 5-MeO-DMT display significantly more head twitches than WT mice. Pretreatment with M100907 blocks head twitches induced by both compounds, while MTZ pretreatment has no effect on the number of head twitches observed. A. Time-course of head twitches counted following treatment with 20 g N-methylserotonin (i.c.v.) B. N-methylserotonin- induced dose response curve and blockade of N-methylserotonin-induced (20 g, i.c.v.) head twitches by M100907 pretreatment. WT vs. arr2-KO within the same dose: *p < 0.05; **p < 0.001. 20 g N-Me-5-HT vs. M100 + 20 g N-Me-5-HT: values for M100907 treatment were 0  0. C. Time-course of N-methylserotonin-induced (20 g, i.c.v.) head twitches in WT mice pretreated with either MTZ or vehicle. D. Time-course of head twitches counted following treatment with 10 mg/kg 5-MeO-DMT (i.p.) E. 5-MeO-DMT-induced dose response curve and blockade of 5-MeO- DMT-induced (10 mg/kg, i.p.) head twitches by M100907 pretreatment. WT vs. arr2-KO within the same dose: *p < 0.05; ***p < 0.0001. 10 mg/kg 5-MeO-DMT vs. M100 + 10 mg/kg 5-MeO- DMT: ##p < 0.01, ###p < 0.0001. F. Time-course of 5-MeO-DMT-induced (10 mg/kg, i.p.) head twitches in C57BL/6J mice pretreated with either MTZ or vehicle. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45). 98

A. 5-HTP B. 5-MeO-DMT

PSD-95 PSD-95

arr2 arr2

Src Src

Akt Akt

5-HT R 2A 5-HT2AR WT arr2-KO WT arr2-KO

2.0 2.0 PSD-95 PSD-95 ** arr2 arr2 ** ** Src Src 1.5 Akt 1.5 Akt

1.0 1.0

P-Akt/ P-Akt/ T-Akt P-Akt/ P-Akt/ T-Akt ***

0.5 0.5 Fold Over Vehicle Control Over Vehicle Fold Fold Over Vehicle Control Over Vehicle Fold 5-HTP - + - + - + - + - + - + - + 5-MeO-DMT - + - + - + - + - + - + - + WT arr2-KO WT arr2-KO

Figure 3.5 5-HTP, but not 5-MeO-DMT, stimulates the formation of a arrestin2/Src/Akt complex with the 5-HT2A receptor in mouse frontal cortex

The endogenous 5-HT2A receptor (5-HT2AR) was immunoprecipitated from frontal cortex isolated from WT and arr2-KO mice treated for 10 minutes with vehicle (0.9% saline, i.p.), 5-HTP (A, 100 mg/kg, i.p.) or 5-MeO-DMT (B, 10 mg/kg, i.p.). A. In WT mice, 5-HTP induces the disassociation between PSD-95 and the 5-HT2A receptor and increases 5-HT2A receptor associations with

arrestin2, Src and Akt. In arr2-KO mice, 5-HTP treatment has no effect on 5-HT2A receptor interactions with PSD-95, Src or Akt. Vehicle vs. 5-HTP within each genotype: **p < 0.01, ***p <

0.001. B. A 10 minute treatment with 5-MeO-DMT has no effect on 5-HT2A receptor interactions with PSD-95, arrestin2, Src or Akt, regardless of genotype. A “no protein” control (NP: antibody + beads) is shown for each immunoblot (IB). Representative blots and densitometric analysis are provided. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

A. B.

WT arr2-KO KO - P-Akt P-Akt

WT T-Akt T-Akt

arr1/2 

Vehicle 1.5 ** 5-HTP 1.5 5-MeO-DMT 1.0

1.0

0.5

P-Akt/ P-Akt/ T-Akt P-Akt /P-Akt T-Akt

0.5 Over WTFold Control

Fold Over Vehicle Control Over Vehicle Fold 0.0 WT arr2-KO WT arr2-KO

Figure 3.6 5-HTP, but not 5-MeO-DMT, induces Akt phosphorylation in the mouse frontal cortex in a arrestin2-dependent manner Frontal cortex was isolated from WT and arr2-KO mice treated with vehicle (0.9% saline, i.p.), 5- HTP (100 mg/kg, i.p.) or 5-MeO-DMT (10 mg/kg, i.p.) for 10 minutes. A. 5-HTP induces Akt phosphorylation (P-Akt) in WT mice, but not arr2-KO mice. 5-MeO-DMT does not activate Akt in either genotype (T-Akt: total Akt). Vehicle vs. drug treatment within each genotype: **p < 0.01. B. Basal Akt phosphorylation levels do not differ between vehicle treated WT and arr2-KO mice. Representative blots and densitometric analysis are provided. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

100

A. B. C.

5-HT Veh M100 V 5 V 5 P-Akt P-Akt P-Akt T-Akt T-Akt T-Akt WT arr2-KO WT WT

2.5 *** Vehicle 2.5 2.5 Vehicle 5-HT 5-HT N-Me-5HT * 2.0 5-MeO-DMT 2.0 2.0 *** *

1.5 1.5 1.5

P-Akt /P-Akt T-Akt P-Akt /P-Akt T-Akt 1.0 /P-Akt T-Akt 1.0 1.0

0.5 0.5 0.5

Fold Over Vehicle Control Over Vehicle Fold

Fold Over Vehicle Control Over Vehicle Fold Fold Over Vehicle Control Over Vehicle Fold 0 1 10 Vehicle M100 WT arr2-KO 0.01 0.1 0.001 Pretreatment [5-HT] (M)

D. E. F. 0 1 3 5 10 20 30 minutes Myc- P-Akt Mock arr2

WT T-Akt V 5 V 5 WT arr2-KO

P-Akt KO

P-Akt - P-Akt T-Akt

T-Akt arr2 T-Akt Myc  arr2-KO

1.5 2.5 *** WT Vehicle * 5-HT *** ** arr2-KO 1.5 2.0 ** 1.0

1.5 1.0

0.5

P-Akt /P-Akt T-Akt P-Akt /P-Akt T-Akt

P-Akt /P-Akt T-Akt 1.0 Fold Over Basal Fold

Fold Over WTFold Control 0.5

0.0 0.5 Control Over Vehicle Fold WT arr2-KO 0 5 10 15 20 25 30 Mock arr2 Time (minutes) Transfection

Figure 3.7 Serotonin induces Akt phosphorylation in primary cortical neurons in a arrestin2-dependent manner

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Figure 3.7 continued Four days after plating, primary cortical neurons were serum-starved for 1 hour prior to a 10 minute treatment with vehicle (V; 2 M ascorbate), 1 M serotonin (5-HT or 5), 1 M N- methylserotonin (N-Me-5-HT) or 1 M 5-MeO-DMT. A. Serotonin induces Akt phosphorylation (P- Akt) in WT, but not arr2-KO neurons. N-methylserotonin and 5-MeO-DMT do not activate Akt in either genotype (T-Akt: total-Akt). Vehicle vs. 5-HT: ***p < 0.001. B. Serotonin-induced Akt phosphorylation is maximal at 1 M, as determined by a concentration response curve in WT neurons. Vehicle vs. 5-HT: *p < 0.05, Bonferroni post-hoc analysis. C. Pretreatment of WT neurons with M100907 (M100; 10 nM) for 15 minutes inhibits Akt phosphorylation induced by serotonin, compared to neurons pretreated with vehicle (Veh; 0.0001% DMSO). Vehicle vs. 5-HT within vehicle pretreatment group: ***p < 0.001. D. Basal Akt phosphorylation levels do not differ between untreated WT and arr2-KO neurons plated concurrently. E. Time-course studies reveal that serotonin induces Akt phosphorylation in primary cortical neurons from WT, but not arr2-KO neonates. WT vs. arr2-KO: **p < 0.01, ***p < 0.001, Bonferroni post-hoc analysis. F. Transfection of myc-tagged arrestin2 (myc-arr2) rescues serotonin-induced Akt phosphorylation in arr2-KO primary cortical cultures. Neurons transfected with empty vector (mock) were used as control. arrestin2 expression was verified by probing immunoblots for myc. Vehicle vs. 5-HT within each transfection group: *p < 0.05. Representative blots and densitometric analysis are provided. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

102

A. N-methylserotonin B. 5-MeO-DMT 0 1 3 5 10 20 30 minutes 0 1 3 5 10 20 30 minutes

P-Akt P-Akt WT

WT T-Akt T-Akt

KO KO -

- P-Akt P-Akt arr2

arr2 T-Akt T-Akt

  2.5 2.5

2.0 2.0

1.5 1.5 P-Akt /P-Akt T-Akt

1.0 /P-Akt T-Akt 1.0

Fold Over Basal Fold Fold Over Basal Fold

0.5 0.5 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (minutes) Time (minutes)

Figure 3.8 The N-methyltryptamines do not stimulate Akt phosphorylation in primary cortical neurons Four days after plating, primary cortical neurons were serum-starved for 1 hour prior to treatment with 1 M N-methylserotonin (N-Me-5-HT) or 1 M 5-MeO-DMT. Time-course studies reveal that neither N-methylserotonin (A) nor 5-MeO-DMT (B) induces Akt phosphorylation in primary cortical neurons from WT or arr2-KO neonates. Representative blots and densitometric analysis are provided. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn,(45).

103

Veh LY PP2 V 5 V 5 V 5 P-Akt T-Akt

Vehicle *** 5-HT 1.5

1.0 P-Akt /P-Akt T-Akt

0.5 Fold Over Vehicle Control Over Vehicle Fold Vehicle LY PP2 Pretreatment

Figure 3.9 Inhibitors to PI3-K and Src block serotonin-induced Akt phosphorylation in WT cortical cultures Four days after plating, primary cortical neurons from WT mice were pretreated with vehicle (Veh: 0.1% DMSO) or with 10 M inhibitors to P13-K (LY294002, LY) or Src (PP2) for 1 hour in serum- free media prior to treatment a 10 minute treatment with vehicle (V: 2 M ascorbate) or 1 M serotonin (5 or 5-HT) (P-Akt: phosphorylated-Akt; T-Akt: total Akt). Both LY294002 and PP2 block serotonin-induced Akt phosphorylation. Vehicle vs. 5-HT within each pretreatment group: ***p < 0.001. Representative blots and densitometric analysis are provided. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

104

A. 5-HTP + B. C. 5-MeO-DMT +

Vehicle LY294002 PP2 AKTi Vehicle 200 30 6 AKTi

150 20 4

100 * * 10 * 2 50

0 Head Twitches/ 5 Minutes Twitches/ Head 0 5 Minutes Twitches/ Head 0 10 20 30 40 50 60 Minutes 60 in Twitches Head Veh LY PP2 AKTi 5 10 15 20 25 30 Time (minutes) + 5-HTP (200 mg/kg, i.p.) Time (minutes)

Figure 3.10 Inhibitors to PI3-K, Src or Akt attenuate 5-HTP-induced, but not 5-MeO-DMT- induced, head twitches in normal mice

C57BL/6J mice were pretreated with vehicle (Veh: 1% DMSO in 5 l dH2O, i.c.v.) or inhibitors to PI3-K (LY294002, LY: 125 ng, i.c.v.), Src (PP2: 300 ng, i.c.v.) or Akt (AKTi: 55 ng, i.c.v.) 10 minutes prior to treatment with 5-HTP (A-B) or 5-MeO-DMT (C). Time-course (A) and the total number (B) of head twitches observed in C57BL/6J mice shows that the kinase inhibitors decrease the number of head twitches observed following treatment with 5-HTP (200 mg/kg, i.p.). Vehicle vs. inhibitor: *p < 0.05. C. Pretreatment with AKTi has no effect on 5-MeO-DMT-induced (10 mg/kg, i.p.) head twitches in C57BL/6J mice, compared to vehicle pretreated mice. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

105

A. 5-HTP + B. Vehicle LY294002 40 PP2 AKTi 250

200 30

150 20 100 10 50

0 Head Twitches/ 5 Minutes Twitches/ Head 0 10 20 30 40 50 60 Minutes 60 in Twitches Head Veh LY PP2 AKTi Time (minutes) + 5-HTP (200 mg/kg, i.p.)

Figure 3.11 Inhibitors to PI3-K, Src or Akt have no effect on 5-HTP-induced head twitches in arr2-KO mice

Arr2-KO mice were pretreated with vehicle (Veh: 1% DMSO in 5 l dH2O, i.c.v.) or inhibitors to PI3-K (LY294002, LY: 125 ng, i.c.v.), Src (PP2: 300 ng, i.c.v.) or Akt (AKTi: 55 ng, i.c.v.) 10 minutes prior to treatment with 5-HTP (200 mg/kg, i.p.). Time-course (A) and the total number (B) of head twitches observed demonstrate that 5-HTP induces a similar head twitch response in arr2-KO mice, regardless of pretreatment. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

106

A. 5-HTP + B. Vehicle MTZ 30 AKTi AKTi + MTZ 200

150 ** 20 *** 100 ## ### 10 50 ###

Head Twitches/ 5 Minutes Twitches/ Head 0 0 10 20 30 40 50 60 Minutes 60 in Twitches Head Veh AKTi MTZ AKTi Time (minutes) + MTZ + 5-HTP (200 mg/kg, i.p.)

Figure 3.12 Concurrent inhibition of Akt and INMT abrogates 5-HTP-induced head twitches in WT mice

WT mice were pretreated with vehicle (Veh: 1% DMSO in 5 l dH2O, i.c.v.) or inhibitors of Akt (AKTi: 55 ng, i.c.v.) or INMT (MTZ: 125 ng, i.c.v.) 10 minutes prior to treatment with 5-HTP (200 mg/kg, i.p.). Pretreatment with each inhibitor individually reduces 5-HTP-induced head twitches. Co-administration of both inhibitors is additive. Time-course (A) and total number (B) of head twitches are shown. Vehicle vs. Inhibitor: ##p < 0.01, ###p < 0.001. AKTi vs. AKTi + MTZ: **p < 0.01; MTZ vs. AKTi + MTZ: ***p < 0.001. Mean ± S.E.M. are shown. Adapted from Schmid and Bohn (45).

107

A. Serotonin B. N-methyltryptamines

5-HT2AR 5-HT2AR K -  PSD-95 PI3 arrestin2 Src ? arrestin2 PSD-95 P Akt  

M100907 M100907 LY294002 PP2 arr2-KO mice AKTi arr2-KO mice AKTi

Head Twitch Response

Figure 3.13 Serotonin and N-methyltryptamines induce differential signaling at the 5-HT2A receptor in the mouse frontal cortex

A. Serotonin at the 5-HT2A receptor induces the disengagement of PSD-95 from the receptor and the recruitment of a signaling complex comprised of arrestin2, Src and Akt in the frontal cortex. This complex acts in conjunction with PI3-K, to phosphorylate Akt and induce the head twitch response. Antagonism of the 5-HT2A receptor (M100907), inhibition of PI3-K (LY294002), Src (PP2) or Akt (AKTi), or the use of genetic deletion of arrestin2 (arr2-KO mice) abrogates the serotonin-mediated head twitch response in mice. B. N-methyltryptamines at the 5-HT2A receptor do not induce either the displacement of PSD-95 or the recruitment of a arrestin2-mediated signaling complex to the receptor. Nor do N-methyltryptamines induce Akt phosphorylation in the frontal cortex, although they still are capable of inducing the head twitch response in mice.

Antagonism of the 5-HT2A receptor (M100907) abrogates the N-methyltryptamine head twitch response. Genetic deletion of arrestin2 (arr2-KO mice) enhances the N-methyltryptamine head twitch response, suggesting that arrestin2 may act to desensitize the G protein-coupling pathways involved in mediating the behavioral response for these agonists. Inhibition of Akt (AKTi) has no effect on the number of head twitches observed following 5-MeO-DMT administration, further demonstrating how serotonin and the N-methyltryptamines utilize different signaling pathways to induce this behavioral response. Adapted from Schmid and Bohn (45).

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Chapter 4

Conclusions

The cellular and animal studies presented in this dissertation demonstrate that the interaction between the 5-HT2A receptor and arrestin2 is a critical point of divergence in agonist-directed 5-HT2A receptor signaling. By utilizing the head twitch response as a model of 5-HT2A receptor activation in vivo, we show that this differential regulation has physiological consequences, which become evident in mice lacking arrestin2. By utilizing a multi-disciplinary approach to elucidate receptor pharmacology, the work presented in this dissertation has advanced the understanding of the role that arrestins play in the agonist-directed regulation and signaling of the 5-HT2A receptor.

We provide evidence which shows that serotonin, the endogenous neurotransmitter at the 5-HT2A receptor, induces 5-HT2A receptor interactions with arrestin2 both in HEK-

293 cells and in the mouse frontal cortex, and that these interactions then facilitate receptor internalization and signaling (44, 45). Both quantitative and qualitative studies show that serotonin-mediated internalization of the 5-HT2A receptor is disrupted in the absence of arrestin2. Similarly, 5-HT2A receptor trafficking to intracellular vesicles is significantly attenuated in primary cortical neurons from arr2-KO mice (44). We also demonstrate that serotonin-induced Akt phosphorylation is dependent upon arrestin2 in 109 both primary neurons and in the mouse frontal cortex, wherein arrestin2 facilitates this signaling by scaffolding components of the complex to the 5-HT2A receptor. Finally, our studies show that the activation of this arrestin2-mediated signaling complex is integral for the serotonin-induced head twitch response (45).

In contrast to serotonin, the hallucinogenic agonists DOI, 5-MeO-DMT and N- methylserotonin are capable of inducing arrestin2-independent signaling via the 5-HT2A receptor. Although DOI clearly induces arrestin2 interactions with the 5-HT2A receptor in

HEK-293 cells, it does not require arrestins to promote receptor internalization in MEFs

(44). DOI and the N-methyltryptamines also do not induce arrestin2-dependent signaling in multiple cellular systems, including the mouse frontal cortex, nor do they require arrestin2 for the induction of the head twitch response (44, 45).

One of the key conclusions that can be drawn from examining the current findings in context with past reports is that the mechanisms underlying 5-HT2A receptor regulation and signaling are cell-type dependent. For instance, 5-HT2A receptor desensitization is

arrestin-independent in HEK-293 cells transfected with the 5-HT2A receptor (129, 132), but arrestin-dependent in C6 glioma cells, where the receptor is endogenously expressed (129, 130). This emphasizes the necessity to study the 5-HT2A receptor in its endogenous environment, wherein we demonstrate that 5-HT2A receptor internalization and serotonin-mediated signaling is disrupted in the absence of arrestin2 in primary cortical neurons and the mouse frontal cortex (44, 45). An attempt to model the serotonin-mediated activation of Akt in vitro only serves to further highlight the need to study the 5-HT2A receptor in physiologically relevant systems, as we have screened 110 numerous immortal cell lines and to date, we have only observed the agonist-differences in 5-HT2A receptor-mediated Akt activation in the frontal cortex and primary cortical cultures (data not shown).

The finding that 5-HT2A receptor regulation differs based on cell-type is further complicated by the fact that 5-HT2A receptors are expressed in multiple neuronal types throughout the central, enteric and peripheral nervous systems, as well as in platelets, smooth muscle and mesangial cells (35, 36, 170, 229, 230). There is evidence to suggest that the same GPCR can be subject to different regulatory mechanisms when expressed in different brain regions or tissues. The  opioid receptor serves as example of this complexity, wherein arrestin2 negatively regulates the morphine-bound receptor expressed in regions associated with the analgesic effects of the drug, such as the brainstem and periaqueductal gray. In contrast, arrestin2 is thought to facilitate morphine-mediated signaling underlying the development of constipation in the enteric nervous system (96-98, 184, 213, 231). While we demonstrate that arrestin2 facilitates serotonin-mediated signaling at 5-HT2A receptors expressed in the mouse frontal cortex, which are involved in the modulation of mood and perception and are the targets of hallucinogenic drugs (43), 5-HT2A receptors could be subject to many regulatory mechanisms in the different cellular environments in which they are expressed in vivo.

In addition to being dictated by the cellular environment, we demonstrate that the regulation of the 5-HT2A receptor is a function of the agonist bound to the receptor.

Comparison between the abilities of serotonin and DOI to induce 5-HT2A receptor trafficking in MEFs reveals that serotonin requires arrestins to internalize the receptor,

111 but DOI is able to internalize the receptor in the absence of both arrestin1 and

arrestin2 (44). Similarly, serotonin leads to ERK1/2 phosphorylation via both a PLC- dependent and a arrestin-mediated pathway in MEFs, while DOI activates ERK1/2 exclusively through a non-arrestin, PLC-mediated pathway (44). The differential involvement of arrestin2 in the mediation of 5-HT2A receptor signaling was also observed in vivo, where we show that serotonin requires arrestin2 to promote 5-HT2A receptor-mediated Akt phosphorylation in the mouse frontal cortex and requires

arrestin2 for the induction of the head twitch response. In contrast, the N- methyltryptamines do not activate this pathway, nor are the head twitches observed following treatment with these agonists dependent upon arrestin2 (45). This demonstration of agonist-directed 5-HT2A receptor signaling indicates that there may be opportunities to develop 5-HT2A receptor ligands which selectively activate distinct signaling cascades. Moreover, as the agonist-directed signaling has physiological implications, these studies suggest that the development of compounds which selectively activate specific 5-HT2A receptor signaling pathways in vivo may allow for the fine-tuning of receptor signaling toward desired pathways and away from those that may induce unwanted side effects, such as hallucinations.

Ligand directed-regulation of the 5-HT2A receptor is further complicated by the finding that, unlike other GPCRs, the 5-HT2A receptor is internalized and down-regulated by antagonists (138, 146, 232-234). The Roth laboratory has shown that 5-HT2A receptor internalization induced by the 5-HT2A receptor antagonist clozapine, is unaffected by the

arr1319-418 dominant negative in HEK-293 cells (124), suggesting that antagonists may utilize the arrestin-independent pathway for internalization. If antagonist-mediated 112 internalization of the 5-HT2A receptor is indeed independent of arrestins, it will be interesting to determine if antagonists induce receptor trafficking through a mechanism shared by agonists such as DOI. Additional studies are needed to fully assess the role that arrestins play in the internalization of the 5-HT2A receptor following antagonist treatment.

Considerable efforts have focused on identifying differences in 5-HT2A receptor-mediated signaling cascades activated by hallucinogenic and non-hallucinogenic compounds. The most well-characterized 5-HT2A receptor signaling cascades are the hydrolysis of PI through the coupling to Gq and its effectors and the PLA2-mediated release of AA (9).

While 5-HT2A receptor agonists differ in their ability to stimulate these pathways in cell- culture, there are no clear differences in the pattern of activation between hallucinogenic and non-hallucinogenic drugs (77, 78). The Gingrich and Sealfon laboratories demonstrated that hallucinogenic and non-hallucinogenic compounds induce differential activation of transcription factors downstream of the 5-HT2A receptor in the mouse frontal cortex and in primary cortical neurons (51, 56); however, the signaling cascades linked to these changes in transcription have not been described.

In the studies presented in this dissertation, we demonstrate that serotonin-mediated signaling at the 5-HT2A receptor differs from that induced by hallucinogenic compounds, based upon their interactions with arrestin2. This functional divergence in signaling is evident in the molecular mechanisms by which serotonin and hallucinogenic compounds induce the head twitch response in mice. We find that serotonin activates the head twitch response through a 5-HT2A receptor signaling cascade involving arrestin2, PI3-K,

113

Src and Akt. In contrast, our data indicate that neither DOI nor the N-methyltryptamines require arrestin2 for the induction of the head twitch response and the inhibition of Akt has no affect on the number of twitches observed following 5-MeO-DMT administration, although the mechanism by which these hallucinogenic agonists induce the head twitch response remains to be determined (44, 45). The DOI-mediated head twitch response has been shown to involve 5-HT2A receptor coupling to Gq, as the Gq-KO mice have significantly attenuated responses compared to their WT littermates (183). The DOI- mediated head twitch response is also attenuated in PSD-95-KO mice (156).

Interestingly, 5-HT2A receptor interactions with PSD-95 have been shown to facilitate

Gq-mediated signal transduction in HEK-293 cells (151), indicating that the two pathways may be interrelated. Moreover, we demonstrate that, unlike serotonin, 5-MeO-

DMT does not promote the displacement of PSD-95 from the 5-HT2A receptor complex in the mouse frontal cortex (45), implicating PSD-95 in the arrestin2-independent head twitch response. Collectively, these findings suggest that 5-HT2A receptor signaling by serotonin and hallucinogenic compounds may diverge based upon interactions with scaffolding proteins in vivo, wherein serotonin scaffolds arrestin2 to the 5-HT2A receptor, thus facilitating the activation of Akt, while hallucinogenic drugs may utilize 5-

HT2A receptor interactions with PSD-95 to promote 5-HT2A receptor coupling to Gq.

Our studies also show that N-methylserotonin and 5-MeO-DMT-induced head twitches are enhanced in arr2-KO mice compared to their WT littermates (45). This enhanced behavioral response implicates arrestin2 in the desensitization of the signaling pathways involved in the induction of the head twitch response for these agonists.

Although this has yet to be observed for the 5-HT2A receptor in vivo, there is certainly 114 precedence for enhanced behavioral responses observed in the arr2-KO mice being due to increased G protein-signaling for other GPCRs (96, 97, 184). Moreover, dominant negatives to arrestins inhibit the desensitization of PI hydrolysis for 5-HT2A receptors endogenously expressed in C6 glioma cells (129, 130), indicating that arrestins can dampen 5-HT2A receptor signaling in some cell types. Future experiments assessing genotypic differences in the activation of G protein-mediated signal transduction pathways downstream of the 5-HT2A receptor in the frontal cortex will be useful in correlating the enhanced response to N-methyltryptamines observed in the arr2-KO mice to neurochemical adaptations in vivo.

The opposing effects that the absence of arrestin2 has on serotonin- and N- methyltryptamine-mediated head twitch responses suggest that arrestins can both facilitate and negatively regulate 5-HT2A receptor signaling within the same neuronal population. These studies indicate that the agonist somehow dictates the functional implications of arrestin2-recruitment, whether it be to assist in the activation of addition signal transduction pathways by scaffolding them to the receptor or to inhibit further coupling to G proteins. GRKs may serve as cofactors which regulate the functional consequences of arrestin interactions with GPCRs, as interactions with GRK2 and 3 have been shown to promote arrestin-mediated desensitization of receptors while interactions with GRK5 and 6 facilitate arrestin-mediated signaling cascades (212, 235,

236). From these studies, we might infer that serotonin and the N-methyltryptamines may recruit specific GRKs to the 5-HT2A receptor, which may impact the selective engagement of arrestin2 to either facilitate or desensitize signal transduction.

Therefore, GRK interactions with the 5-HT2A receptor may represent another target 115 through which 5-HT2A receptor signaling could be modulated in vivo. In addition, the differential involvement of arrestin2 in the induction of the head twitch response suggests that 5-HT2A receptor agonists could be developed to specifically target distinct functions of arrestin2, to either dampen specific signaling cascades or stimulate the activation of others in vivo.

The body of work presented in this dissertation demonstrates that ligand directed signaling at the 5-HT2A receptor bifurcates based on interactions with arrestin2 in vivo.

These studies have broadened our understanding of 5-HT2A receptor signaling by further elucidating the differences in signaling cascades activated by serotonin and serotonergic hallucinogenic drugs. By determining differences in agonist-directed 5-HT2A receptor responsiveness in physiologically relevant systems, we can begin to correlate specific signaling events with either the therapeutic actions or the adverse side effects of drugs.

This could have major implications for drug development, as it suggests that pharmacotherapeutics could be developed to selectively activate or avoid receptor- mediated signaling cascades responsible for the desired responses or unwanted effects of 5-HT2A receptor ligands. In other words, “serotonin mimetics” at the 5-HT2A receptor may preferentially engage the arrestin2-mediated Akt signaling pathway. In contrast, 5-

HT2A receptor antagonists might be designed to selectively inhibit those pathways activated by hallucinogens while leaving the serotonin-activated signaling cascades intact. Therefore, by designing 5-HT2A receptor agonists which selectively target specific signaling events downstream of the 5-HT2A receptor, it may be possible to develop drugs with enhanced therapeutic efficacy and fewer side effects for the treatment of

116 psychological disorders in which disregulation of the 5-HT2A receptor has been implicated, such as depression and schizophrenia.

117

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