Decoding the Signaling of the D2R-2AR Heteromer: Relevance to Schizophrenia

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Theses and Dissertations Graduate School

2018

Decoding the signaling of the D2R-2AR heteromer: relevance to schizophrenia

Miao Huang

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This Dissertation is brought to you for free and open access by the Graduate School at VCU Scholars Compass. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of VCU Scholars Compass. For more information, please contact [email protected]. DECODING THE SIGNALING OF THE D2R-2AR HETEROMER: RELEVANCE TO SCHIZOPHRENIA

Miao Huang

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University

Director: DIOMEDES E. LOGOTHETIS, PH.D. Professor Department of Pharmaceutical Sciences, School of Pharmacy, Bouvé College of Health Sciences, Northeastern University, Boston, MA. 02115

Virginia Commonwealth University Richmond, Virginia December, 2018

ABSTRACT

Decoding the signaling of the D2R-2AR heteromer: relevance to schizophrenia

Miao Huang

Advisor: Dr. Diomedes Logothetis

Schizophrenia is a severe mental disorder affecting ~1% of world population. Two G- protein coupled receptors (GPCRs): Gi-coupled dopamine 2 receptor (D2R), and Gq-coupled serotonin 2A receptor (2AR), are targeted by the typical and atypical antipsychotic drugs to treat schizophrenia. These two receptors have been shown to co-localize in brain regions relevant to schizophrenia, including the ventral tegmental area (VTA), striatum, and prefrontal cortex

(PFC). Studies in our lab characterized the integrated signaling of the D2R-2AR heteromer and found that both the Gi activity of D2R and the Gq activity of 2AR were potentiated in response to dopamine (DA) and serotonin (5-HT), whereas the potency of the typical antipsychotic drug

(APD) haloperidol antagonizing Gi and Gq signaling was also enhanced. Using a peptide mimicking the transmembrane (TM) domain 5 of D2R, we showed disruption of the formation and function of the D2R-2AR heteromer in heterologous systems and ex vivo brain slices. Our functional and mutagenesis data suggested that D2R and 2AR heteromerize though a symmetric

TM5,6-TM5,6 interface, and a network of Pi-Pi stacking interaction among eight conserved aromatic residues of D2R and 2AR may underlie the mechanism for the functional cross-talk between D2R and 2AR. Based on these results, we built a structural model for the D2R-2AR

iii heteromer recapitulating its functional cross-talk characteristics. We are presently pursuing behavioral experiments to investigate the effectiveness of antipsychotic drugs on the function of the D2R-2AR heteromer in animal models of psychosis. Our overall study shows a dual role of the D2R-2AR heteromer in schizophrenia-associated psychosis and sheds light on the development of future therapeutic drugs for schizophrenia and other psychotic diseases.

This dissertation work is gratefully dedicated to my beloved parents, without whose unconditional love, support and encouragement, none of this would be possible.

TABLE OF CONTENTS

Abstract …………………………………………………………………………………………..iii List of Figures ...………………………………………………………………………………….xi List of Tables ...…………………………………………………………………………………xiv List of Abbreviation ……………………………………………………………………………. xv Acknowledgements…………………………………………………………………………….xvii Chapters 1. Introduction 1.1 Schizophrenia and hypotheses 1.1.1 The dopamine hypothesis …………………………………………………………….1 1.1.2 The glutamate hypothesis …………………………………………………………….2 1.1.3 The serotonin hypothesis ……………………………………………………………..4 1.2 Antipsychotic drugs …………………………………………………………………………..6 1.3 The dopamine system 1.3.1 Dopamine in the brain …………………………………………………...…………...8

1.3.2 Distribution of D2R in the brain ……………………………………………………...8 1.3.3 Signaling of D2R …………………………………………………………………….8 1.3.4 Function of the dopamine system ………………………………………………..…..9 1.4. The serotonin system 1.4.1 Serotonin in the brain ……………………………………………………………….11 1.4.2 Distribution of 2AR in the brain ……………………………………………...…….11 1.4.3 Signaling of 2AR …………………………………………………………………...12 1.4.4 Function of the serotonin system …………………………………………………...12

1.5 Evidence for D2R-2AR heteromerization …………………………………………………...14 1.6 Purpose of this study ………………………………………………………………………...15 1.7 References ………………………………………………………………………...…………16

2. The integrated Gi and Gq signaling profile of D2R-2AR 2.1 Background introduction 2.1.1 Monitoring Gi and Gq activity in an oocyte electrophysiology assay and a mammalian cell calcium mobilization assay ………………………………………..39

2.1.2 Co-expression of D2R with 2AR potentiates Gi and Gq signaling in response to 1uM DA and 5-HT ……………………………………………………………….40

2.1.3 Ratio of D2R and 2AR expressed in oocytes and HEK293 cells ……………………40 2.1.4 Aims …………………………………………………………………………………41 2.2 Results and discussion

2.2.1 Co-expression of D2R with 2AR potentiates DA induced Gi signaling …………….41

2.2.2 Co-expression of D2R with 2AR enables 5-HT induced Gi signaling ………………42

2.2.3 Integrated Gi signaling of D2R-2AR co-expression in response to DA and 5-HT…..42

2.2.4 Co-expression of D2R with 2AR potentiates 5-HT-induced Gq signaling ………….43

2.2.5 Co-expression of D2R with 2AR enables DA-induced Gq signaling ……………….43

2.2.6 Integrated Gq signaling of D2R-2AR in response to DA and 5-HT ……………...…44

2.2.7 Co-expression of D2R with 2AR potentiates Gi and Gq signaling ………………….44

2.2.8 D2R synthetic ligands cross-talk to 2AR …………………………………………....44

2.2.9 D2R synthetic ligands cross-talk to modulate 2AR agonist-induced Gq signaling….45 2.3 Summary …………………………………………………………………………………….46 2.4 References …………………...………...…………………………………………………….59

3. Developing tools to disrupt the formation of D2R-2AR in heterologous system and native tissue 3.1 Background introduction 3.1.1 Use of cell-penetrating peptides as tools to disrupt GPCR heteromers ……………..60 3.1.2 The role of the pre-frontal cortex (PFC) in schizophrenia …………………………..61 3.13 The role of the ventral tegmental area (VTA) in schizophrenia ……………………..62

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3.14 Aims ….………………………………………………………………………………63 3.2 Results and discussion

3.2.1 CPPs mimicking TM5 and 6 of D2R and 2AR disrupt the formation of

D2R-2AR heteromer …………………………………………………………………63

3.2.2 D2R TM5 peptide disrupts D2R-2AR heteromeric signaling ……...………………..63

3.2.3 D2R TM5 peptide reverses the enhancement of field Excitatory postsynaptic potential (fEPSP) in PFC neurons …..………………………………………………64

3.2.4 D2R TM5 peptide disrupts DA desensitization in VTA neurons ……………………65

3.2.5 A mini gene method to disrupt the D2R-2AR heteromer ……………………………66 3.3 Summary …………………………………………………………………………………….67 3.4 References …………………………………………………………………………………...77

4. Structure model of D2R-2AR demonstrating its coupling mechanism 4.1 Background introduction 4.1.1 GPCR homodimer interface …………………………………………………………80 4.1.2 Putative GPCR heteromer interface …………………………………………………82

4.1.3 The R-rich motif at IL3 of D2R is necessary for the co-localization of D2R and 2AR …………………………………………………………………………...... 84 4.14 Aims ………………………………………………………………………………….84 4.2 Results and discussion 4.2.1 Pi-Pi stacking interaction among conserved aromatic residues 5.47, 5.48, 6.51 and 6.52 ……………………………………………………………………………..85 4.2.2 The role of residues 6.51 and 6.52 in agonist binding and receptor activation ……..85 4.2.3 The 5.48 residue is localized at the outer surface of the helical bundle …………….86

4.2.4 Hypothesis of the coupling mechanism of D2R-2AR heteromer ……………………87

5.48 4.2.5 D2R(Y199 L) abolishes the Gi signaling functional cross-talk ………..………….87 4.2.6 2AR(F2445.48L) abolishes the Gi signaling functional cross-talk …………………...88

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4.2.7 Computational model of D2R-2AR ………………………………………………….88 4.2.8 The positively charged R-rich motif is necessary to the heteromer formation between dopamine receptor and 2AR ……………………………………………….89

4.2.9 The electrostatic interaction between the R-rich residues of D2R and the EE

residues of 2AR is necessary for the D2R-2AR heteromeric signaling ……………..90 4.3 Summary …………………………………………………………………………………….91 4.4 References ……………………………………………………………..…………………...106

5. The function of D2R-2AR on antipsychotic drugs’ effectiveness 5.1 Background introduction 5.1.1 2AR is necessary to haloperidol’s full antipsychotic effectiveness ……………….110 5.1.2 Combination of antipsychotic drugs (APDs) exerts enhanced antipsychotic effect and reduced side effect ……………………………………………………...110

5.1.3 Typical and atypical APDs potentiate the heteromerization of D2R-2AR ………...111 5.1.4 Aims ………………………………………………………………………………..112 5.2 Results and discussion

5.2.1 The potency of HLP antagonizing DA is enhanced by the D2R-2AR heteromer ….112 5.2.2 The enhanced potency of HLP antagonizing DA depends on the cross-talk

between D2R and 2AR through the 5.48 residue …………………………………..113

5.2.3 The potency of HLP antagonizing Gq signaling is enhanced in the D2R-2AR heteromer …………………………………………………………………………..113 5.2.4 The enhanced potency of HLP antagonizing Gq signaling depends on the

cross-talk between D2R and 2AR through the 5.48 residue ………………………..114

5.2.5 Pimavanserin cross-talks to antagonize D2R ………………………………………114 5.3 Summary …………………………………………………………………………………...116 5.4 References ………………………………………………………………………………….122

6. Discussion and concluding remarks

6.1 Is D2R-2AR officially a heteromer? ……………………………………………………….124 6.2 A common coupling mechanism for aminergic GPCR heteromers? ………………………126

6.3 Unifying model of D2R-2AR’s dual role in psychosis …………………………………….128

6.4 Putative bivalent ligands of the D2R-2AR heteromer ……………………………………...129 6.5 References ……………………………………………………………………...…………..136

A. Materials and methods A1. Molecular biology ……………..…………………………………………………………..147 A2. Drugs and chemicals …………………………...………………………………………….147 A3. Cells and transfection …………………………...………...……………………………….147 A4. Oocytes preparation and injection …………………………………………………………148 A5. Two electrode voltage clamp …………………………………...…………………………148 A6. Statistics …………………...………………………………………………………………148

LIST OF FIGURES

Figure 1.1 Key dopamine pathways in the brain ………………………………………………...16 Figure 1.2 Neuron circuits indicating the interaction between 2AR and glutamate receptors in response to psychoactive and antipsychotic agents ……………………………….17 Figure 1.3 Integrated hypothesis of schizophrenia showing the role of dopamine, serotonin and glutamate ………………………………………………………………...... 18 Figure 1.4 Integrated hypothesis of schizophrenia showing the role of dopamine, serotonin and glutamate ………………………………………………………………………..19

Figure 1.5 Evidence for D2R-2AR heteromerization ……………………….…………………...21 Figure 1.6 2AR is necessary for the inhibition of MK-801-induced locomotor activity by

D2R antagonist haloperidol on channel reporters for G-protein activity ……………22 Figure 2.1 Monitoring Gi and Gq Signaling with Inward-Rectifying Potassium Channels in Xenopus Oocytes and Calcium mobilization assay with Ca2+ indicator GCaMP ….47

Figure 2.2 Co-expression of D2R with 2AR potentiates Gi and Gq …………………………….48

Figure 2.3 ratio of D2R and 2AR expressed in oocytes and HEK293 cells ……………………..49

Figure 2.4 Co-expression of D2R with 2AR potentiates DA-induced Gi signaling …………….50

Figure 2.5 Co-expression of D2R with 2AR enables 5-HT-induced Gi signaling ………………51

Figure 2.6 Integrated Gi signaling of D2R-2AR in response to DA and 5-HT ………………….52

Figure 2.7 Co-expression of D2R with 2AR potentiates 5-HT-induced Gq signaling ………….53

Figure 2.8 Co-expression of D2R with 2AR enables DA-induced Gq signaling ………………..54

Figure 2.9 Integrated Gq signaling of D2R-2AR in response to DA and 5-HT …………………55

Figure 2.10 Co-expression of D2R with 2AR potentiates Gi and Gq signaling ………………...56

Figure 2.11 D2R synthetic ligands cross-talk to 2AR …………………………………………..57

Figure 2.12 D2R ligands cross-talk to modulate 2AR agonist induced Gq signaling …………...58

Figure 3.1 Cell-penetrating peptides mimicking D2R TM5 and A2AR TM5 but not D2R TM7

and A2AR TM7 disrupt A2AR-D2R heteromerization in HEK293 cells ……………..68

Figure 3.2 A2AR and D2R TM4-TAT and TAT-TM5 peptides disrupt A2AR-D2R heteromerization and function in striatal neurons …………………………………...69

Figure 3.3 TAT-MOR1DCT peptide disrupts MOR1D/GRPR heteromerization and function in mice ……………………………………………………………………………….70

Figure 3.4 Cell-penetrating peptides mimicking D2R TM5 and TM6 but not TM4 and TM7

disrupt D2R-2AR heteromerization in HEK293 cells ………………………………..71

Figure 3.5 D2R TM5 peptide disrupts the heteromeric Gi signaling of D2R-2AR in oocytes …..72

Figure 3.6 D2R TM5 peptide disrupts the heteromeric Gq signaling of D2R-2AR in HEK cells 73

Figure 3.7 D2R TM5 peptide disrupts field Excitatory postsynaptic potential (fEPSP) in PFC neurons ………………………………………………………………………………74

Figure 3.8 D2R TM5 peptide disrupts DA desensitization in VTA neurons ……………………75

Figure 3.9 A mini gene method to disrupt the D2R-2AR heteromer …...……………………….76 Figure 4.1 Putative GPCR homodimer interfaces inferred by existing crystal structures ………92

Figure 4.2 Putative heterodimer helical interface of A2AR-D2R and mGlu2-2AR ……………...94 Figure 4.3 The electrostatic interaction between the positively charged cluster of R-rich

residues at the IL3 of D2R and the negatively charged pS or DD residues at the

C terminus of A2AR is necessary to the A2AR-D2R heteromer formation …………..95

Figure 4.4 The R-rich motifs at IL3 of D2R is necessary to the colocalization of D2R and 2AR 96 Figure 4.5 Pi-Pi stacking interaction among conserved aromatic residues 5.47, 5.48, 6.51 and 6.52 of dopamine receptors and serotonin type 2 receptors ………………………...97 Figure 4.6 The role of residues 6.51 and 6.52 in agonist binding and receptor activation ……...98 Figure 4.7 The 5.48 residues locate at the out surface of the helical bundle ……………………99

5.48 Figure 4.8 D2R(Y199 L) abolishes the Gi signaling functional cross-talk ………………..100 Figure 4.9 2AR(F2445.48L) abolishes the Gq signaling functional cross-talk ……………….101

Figure 4.10 The positively charged R-rich motif is necessary to DxR-2AR formation ………102

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Figure 4.11 The electrostatic interaction involving the positively charged cluster of R-rich

motif at the IL3 of D2R and the negatively charged EE residues at the C terminus

of 2AR is necessary to the D2R-2AR heteromer formation ………………………103 Figure 4.12 The TM5-TM6 dimerization interface of Mu-opioid receptor ……………………104

Figure 4.13 Computational model of D2R-2AR recapitulating its functional cross-talk……….105

Figure 5.1 The potency of HLP antagonizing DA is increased on D2R-2AR………………….117 Figure 5.2 The increased potency of HLP antagonizing DA depends on the cross-talk

between D2R and 2AR through the 5.48 residue …………………………………..118

Figure 5.3 The potency of HLP antagonizing 5-HT is increased on D2R-2AR heteromer ……119 Figure 5.4 The increased potency of HLP antagonizing 5-HT depends on the cross-talk

between D2R and 2AR through the 5.48 residue …………………………………..120

Figure 5.5 Pimavanserin cross-talks to antagonize D2R ………………………………………121 Figure 6.1 Criteria for GPCR heteromers ……………………………………………………...132 Figure 6.2 Aromatic residues are highly conserved at positions of 5.47, 5.48, 6.51 and 6.52 in aminergic receptors ……………………………………………………………...133

Figure 6.3 Unifying model of psychosis: D2R-2AR …………………………………………...134

Figure 6.4 Putative D2R-2AR bivalent ligand potentiating D2R-2AR heteromerization and antagonism …………………………………………………………………….135

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LIST OF TABLES

Table 1.1. Physical and function evidence for dopamine receptor heteromer ………………….20

Table 4.1. Putative GPCR heterodimer interface inferred by disruptive peptides, genetic mutation, and computational methods ………………………………………93

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LIST OF ABBREVIATIONS

Abbreviation Full Name APD Anti-Psychotic Drug 2AR Serotonin 2A Receptor BiFC Bimolecular Fluorescence Complementation Ca2+ Calcium cAMP Cyclic Adenosine Monophosphate CNS Central Nervous System cRNA Complementary Ribonucleic Acid DA Dopamine DAG Di-Acyl Glycerol

D2R Dopamine 2 Receptor DOI 2,5-Dimethoxy-4-iodoamphetamine DIR Dopamine Inhibition Reversal EPS Extra-Pyramidal Symptoms fEPSP field excitatory postsynaptic potential 5-HT Serotonin GABA Gamma-Amino Butyric Acid GDP Guanosine Diphosphate GIRK G-Protein Coupled Inwardly Rectifying potassium channels GRK G-Protein Coupled Receptor Kinase GTP Guanosine Triphosphate GPCR G-Protein Coupled Receptor

HEK-293 Human Embryonic Kidney cells HLP Haloperidol

IP3 Inositol trisphosphate LSD lysergic acid diethylamide K+ or K Potassium mGluR Metabotropic Glutamate Receptor mGlu2R Metabotropic Glutamate 2 Receptor NMDAR N-Methyl-D-Aspartate Receptor PCP Phencyclidine PFC Prefrontal cortex PIMA Pimavanserin

PIP2 Phosphatidylinositol 4,5 bisphosphate

PI3K Phosphoinositide 3 Kinase PLA Proximity Ligation Assay PKA Protein Kinase A PKC Protein Kinase C PLC-β Phospholipase C-Beta PTX Pertussis Toxin QP Quinpirole VTA Ventral Tegmental Area

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ACKNOWLEDGEMENTS

First and for most, I would like to thank my advisor, Dr. Logothetis, for his excellent guidance on my research, his trust to have me working on this project independently, and his rigorous attitude toward science that added to my valuable graduate experiences. I could not have progressed so much without his support and mentoring.

I would like to thank my committee members: Dr. Gonzalez-Maeso, whose collaboration with us has tremendous meaning to this work; Dr. Salley, from whose fantastic courses and advices I learned and applied good pharmacology in my work; Dr. McQuiston, who enriched my knowledge in neuron anatomy; Dr. Farrell and Dr. Rutan, whose advice from the aspect of chemistry is invaluable to me.

I would like to thank Dr. Cui for teaching me to use the calcium assay, and collaborating on building the structural models; Dr. Baki for her help in tissue culture work; and Dr. Plant for teaching me to use the patch clamp.

Last but not least, I would like to thank my parents, who ignited my enthusiasm and passion for science, and cheered for my every little step. I am deeply indebted for their constant love, encouragement and faith in me.

xvii

Chapter 1. INTRODUCTION

1.1 Schizophrenia and hypotheses

Schizophrenia is a debilitating mental disorder that affects approximately 1% of the world population. Symptoms are generally divided into three broad categories: positive symptoms

(hallucinations, delusions, disorganized thoughts and behaviors), negative (social withdrawal, emotion flat, lack of interest), and cognitive (impaired working memory, attention deficits).1

Schizophrenia is among the most costly mental disorders with annual costs exceeding 100 billion in the US alone.2 The etiology of schizophrenia remains unknown, although research suggests a combination of physical, genetic, psychological and environmental factors contributing to its causation.3-8 Among all the hypotheses of schizophrenia put forth, three have been studied the most: the dopamine, the glutamate, and the serotonin hypotheses.

1.1.1 The dopamine hypothesis

The dopamine hypothesis is the classic and most established hypothesis of schizophrenia. It evolves from two observations: 1. Sustained exposure to D2R agonists or amphetamines (which increase dopamine release) induce schizophrenia-like positive symptoms. 2. Drugs which block

9 dopamine or D2R function could reduce psychotic symptoms. In the dopamine hypothesis, hyperfunction of the DA mesolimbic pathway causes increased level of DA in the striatum, resulting in the positive symptoms of schizophrenia, whereas hypofunction of the DA

1 mesocortical pathway causes deficient release of DA in the prefrontal cortex (PFC), resulting in the negative and cognitive symptoms of schizophrenia (Figure 1.1).10 The dopamine hypothesis has been supported by evidence from postmortem, neuroimaging and clinical studies.

A large number of early postmortem studies have shown consistent findings of an increase

11-22 in both striatal DA level and striatal D2R density in patients of schizophrenia. A recent study including 176 post mortem samples from patients of schizophrenia showed significantly

23 decreased levels of D2R in PFC. The hypothesis that the level of D2R is altered in schizophrenia is also supported by genome-wide association study (GWAS) that have shown a clear association between the DRD2 gene and schizophrenia.24

The striatal D2R density has been extensively studied by PET and SPECT imaging. A major meta-analysis of 17 imaging studies revealed a small but significant elevation of striatal

25-37 D2R in drug-naïve patients with schizophrenia. An increased striatal binding with D2R/D3R radiotracer [3H] spiperone but not [3H] raclopride has been reported in many studies.38-45 A more recent meta-analysis confirmed these observations, showing a small (d=0.26, p=0.049) elevation

46 of D2R/D3R density in schizophrenia.

Early clinical studies have shown correlations between low DA activity in the PFC, low cerebral blood flow to PFC and poor working memory tasks involving white matter in

47-48 schizophrenia. Imaging studies with PET and SPECT have shown administration of D2R agonists occupied D2R and have beneficial effects on the PFC activation whereas administration of antipsychotic drugs crossed the blood-brain-barrier and blocked striatal D2R/D3R in vivo at clinically effective doses.49-61

1.1.2 The glutamate hypothesis

2 The role of glutamate and the N-methyl- D-aspartate (NMDA) receptor is based on two observations: 1. psychotomimetic drugs such as phencyclidine (PCP), dizocilpine (MK-801), and ketamine induce psychotic and schizophrenic symptoms by blocking neurotransmission at

NMDA glutamate receptors;62 2. agents that stimulate NMDA receptor-mediated neurotransmission have shown encouraging results in preclinical studies. 62 In the glutamate hypothesis, hypofunction of glutamate receptor in the cortex causes schizophrenia-like symptoms and links to downstream dopamine networks.63,64

The glutamate hypothesis is supported by a number of studies. Early postmortem studies showed reduced NMDAR1 subunit density in the superior frontal and temporal cortex.65,66

Proton Magnetic Resonance Spectroscopy (1H-MRS) studies showed reduced cortical glutamate levels and elevated glutamine:glutamate (Gln/Glu) ratio in the anterior cingulate cortex found in patients with chronic schizophrenia.65-72 Consistent morphological alterations were observed in dendrites of glutamatergic neurons in the cerebral cortex of schizophrenic subjects.73 Neurotoxic changes in cortical brain regions were observed in animals given NMDA receptor antagonists, which resembled the reductions in brain volume observed in schizophrenic subjects.74 NMDA receptor antagonists have been shown to reduce cortical GABAergic interneuron function, alter the prefrontal DA neuron firing patterns, and increase DA release to a challenge such as amphetamine.75-80 Activation of mGluR2/3 has been shown to reverse NMDA receptor antagonist-induced cortical neuronal injury and NMDA receptor hypofunction in PFC.81,82

Integrating the glutamate and dopamine hypotheses produces a more thorough theory underlying the mechanism of schizophrenia: a deficit in cortical glutamate or hypofunctional

NMDA receptors on GABA interneurons in the PFC cause overactive downstream glutamate release by glutamate neurons projecting from PFC to VTA, which lead to hyperfunctional DA

3 mesolimbic pathway and hypofunctional DA mesocortical pathway, resulting in schizophrenic symptoms.64

1.1.3 The serotonin hypothesis

The role of serotonin (5-HT) and 5-HT2AR (2AR) in schizophrenia originates from two observations: 1. atypical antipsychotics have highest affinity for 2AR and block the schizophrenia-like psychotic effects of serotonergic hallucinogens such as LSD;83 2. postmortem studies have found up-regulated levels of 2AR in the cortical regions of young untreated schizophrenic subjects.84 In the serotonin hypothesis, hyperfunction of 2AR in the cortex causes schizophrenia-like symptoms and links to downstream dopamine networks.64, 85

The serotonin hypothesis is supported by a series of studies and evidence. Psychedelic drugs such as psilocybin and lysergic acid diethylamide (LSD) induce schizophrenia-like psychosis through excessive 2AR activation.86-89 Psilocybin produces activation in the prefrontal cortex and produces hyperfrontality.90,91 Atypical APDs block the serotonin and glutamate release in PFC and reverse the cellular responses produced by PCP-like drugs in cortical pyramidal neurons.92-94 2AR at the cortical regions mediates the response induced by LSD-like hallucinogens.95,96 Increased levels of 2AR have been found in the temporal cortex of patients with visual hallucinations.97 2AR in the cortex has been found to modulate the activation of

98 mGlu2/3 receptor and glutamate release from cortex to VTA. The potential role of 2AR in mediating the behavioral responses induced by NMDA antagonism is illustrated by PCP models

(Figure 1.2).98 2AR in the medial prefrontal cortex controls the excitability of VTA dopamine neurons.99,100

4 The studies and evidence for the serotonin hypothesis have developed it to a comprehensive theory for schizophrenia in which the role of dopamine, glutamate and serotonin are linked. In this theory, excess serotonin, upregulated 2AR, or psychedelic hallucinogens could all cause hyperactivation of 2AR on glutamatergic neurons in PFC, which leads to glutamate release from PFC to VTA. Excessive glutamate in VTA causes hyperactivity of DA mesolimbic pathway and hypoactivity of DA mesocortical pathway, resulting in schizophrenic symptoms

(Figure 1.3).64,85

5 1.2 Antipsychotic drugs

Consistent with the dopamine, serotonin and glutamate hypotheses of schizophrenia, three types of antipsychotic drugs have been developed to treat schizophrenia and reduce psychosis.

Typical antipsychotic drugs (APDs) (e.g. haloperidol, loxapine) are high-affinity

101 antagonists of D2R. They function though blocking the D2R-Gi signaling pathway and thereby reduce the hyperdopaminergic mesolimbic pathway and improve the positive symptoms of schizophrenia. While effective against positive symptoms, typical APDs demonstrate limited efficacy against negative symptoms and cognitive impairments. Moreover, these drugs are associated with a number of side effects including extrapyramidal symptoms (EPS), hyperprolactinemia, and obesity.102

Atypical APDs (e.g., clozapine, pimavanserin) differ pharmacologically from the typical

103,104 APDs in that they possess higher affinity to 2AR and low affinity to D2R. They antagonize the 2AR-Gq signaling pathway and are effective in treating all three symptoms of schizophrenia, though the efficacy against negative and cognitive symptoms is only modest.104 Atypical APDs have less potential to induce EPS or hyperprolactinemia, but they are also associated with side effects, usually metabolic problems including weight gain and diabetes.105 In a meta-analysis of randomized controlled trials with 2522 participants, no significant differences between typical

APDs and atypical APDs were found for effect on symptoms or discontinuation rates; typical

APDs caused more EPS whereas atypical APDs caused more weight gain.106

6 A third class of APDs (e.g., LY379268, RG1678), involve the agents that stimulate or improve the function of glutamate receptors. Ly3792768, an agonist of mGluR2/3, rescued

NMDA and GABAA activity and reduces psychotic behavior in animal models.107 RG1678, a drug currently in Phase II trials, increased NMDA receptor function via inhibition of type 1 glycine transporters (GlyT1).108 SAR218645, an mGluR2 positive allosteric modulator (PAM), improved memory and attention deficits in models of cognitive symptoms.109

Despite the availability of several typical and atypical APDs, fewer than 35% of people with schizophrenia showed complete remission.110 20%~30% of people diagnosed with schizophrenia do not respond to any antipsychotic therapy. 111 Novel therapeutics with enhanced efficacy and reduced side effects are needed.

7 1.3 The dopamine system

1.3.1 Dopamine in the brain

Dopamine (DA) is synthesized from the amino acid tyrosine; tyrosine transported to DA neurons is converted to the direct precursor L-DOPA and decarboxylated to DA. DA is distributed in the brain through five DA pathways, among which two are most important and associated with schizophrenia and psychosis: the mesolimbic pathway, which drives DA from

VTA to striatum, plays an important role in pleasure and reward seeking behaviors, addiction, emotion, and perception; the mesocortical pathway, which drives DA from VTA to PFC, is critical for attention, learning, memory, cognition and emotional behavior.10,111

1.3.2 Distribution of D2R in the brain

D2R is widely distributed in the brain. Various studies including immunostaning and in situ

112,113 hybridization have found high density of D2R in VTA (in rat and mouse tissue samples), striatum especially nucleus accumbens (in rat and human striatum tissue samples),112, 114-118 and

112, 117 substantia nigra (in rat tissues). mRNA of D2R has also been found highly expressed in the

118 112,114 112 VTA, the striatum, and the substantia nigra. The distribution of D2R in PFC is more sparse but has been found in human tissue samples, especially in layers V and VI.114 mRNA of

119 D2R has been found in rat PFC pyramidal and GABAergic neurons.

1.3.3 Signaling of D2R

D2R couples to and activates the Gi protein, facilitates the exchange of GDP (within the Gα subunit) for GTP, leads to dissociation of the Gα and Gβγ subunits, and induces various

8 downstream signaling pathways. D2R inhibits cyclic AMP (cAMP) levels and various signaling proteins such as PKA and CREB via inhibition of adenylyl cyclase. D2R directly activates G protein-inwardly rectifying potassium channels (GIRK) through the Gβγ subunits and has also been reported to suppress L-type voltage-gated calcium channels and activate muscarinic K+ channel and Kv1 channels through the Gβγ subunits.120-23 The activation of GIRK and

Kv1channels both induce influx of K+, resulting in decreased DA neuronal firing rates or

124 inhibited DA release. Besides the canonical Gi signaling pathway, D2R also couples to β- arrestin2 and activates the downstream PP2A-Akt-GSK3 signaling pathway (Figure 1.4 A).125

1.3.4 Function of the dopamine system

D2R has been found to regulate a broad range of physiological and behavioral processes, including reward-motivation, addiction, emotion, perception, attention, working memory, learning, blood pressure, gastrointestinal motility, vasodilatation, and locomotion.111 Many studies have suggested the function of dopamine receptor heteromers in mental disorders such as schizophrenia, addiction, and ADHD (Table 1.1).126

The mesolimbic system plays an important role in addiction and reward-motivation. DA increase in the striatum induced by various stimulants (e.g. nicotine, alcohol and amphetamine) has been shown to be linked to the subjective experience of euphoria by human brain imaging studies, using PET.127-129 The greatest DA increases following drug exposures have been found

127-129 to cause the most intense euphoria. Moreover, D2R increase in the nucleus accumbens has been found to increase motivation by decreasing inhibitory transmission to the ventral pallidum by in vivo physiological studies.130

9 The mesocortical system is critical for learning and memory. Cognitive processes of learning and memory depend on PFC circuits that are innervated by DA neurons originating in the VTA in the mid brain. PFC activity during tasks associated with working memory has been

131-133 found to correlate with phasic DA signals in VTA in fMRI studies. Blockage of D2R in the lateral PFC has been shown to impair learning of new stimuli (response associations and cognitive flexibility) in monkeys.134 A direct role of DA in PFC-dependent memory was indicated by observations that subjects that were off the DA precursor L-DOPA made more errors in a spatial working memory task compared to when they had received L-DOPA.135,136

Due to the extensive distribution and function of the dopamine and D2R, dopaminergic dysfunction has been implicated in various mental disorders, including schizophrenia, OCD,

ADHD, addiction, mood disorder, autism spectrum, Tourette’s syndrome, and Parkinson’s disease.111

10 1.4 The serotonin system

1.4.1 Serotonin in the brain

Serotonin (5-HT) is synthesized from the amino acid L-tryptophan by neurons of the raphe nuclei in the mid brain. The raphe nuclei contain the majority of 5-HT neurons that innervate the forebrain. 5-HT neurons originating from the raphe nuclei project to prefrontal cortex, cerebral cortex, hippocampus as well as VTA and striatum.137,138

1.4.2 Distribution of 2AR

2AR is widely distributed in the brain. In the rat brain, 2AR is found to be abundantly expressed in the cerebral cortex (especially in layers I, IV, V), and the piriform and entorhinal cortex, the claustrum, endopiriform nucleus, and olfactory bulb/anterior olfactory nucleus, brainstem, as well as the striatum and the basal ganglia (e.g., nuclear accumbens and caudate nucleus).139,140 In the human brain, high density of 2AR is found in laminae III and V of the frontal, parietal, temporal, occipital, anterogenual cortices, and entorhinal area by [3H] ketanserin studies; 141 mRNA of 2AR is found in all neocortical areas (especially in layer V pyramidal neurons and interneurons) by in situ hybridization studies;142 the presence of 2AR in pyramidal neurons and interneurons is confirmed by morphological and double immunofluorescence studies.143-1145 Besides the cortex, 2AR is also expressed in the striatum, substantia nigra, nucleus accumbens, and hippocampus. In both rat and human brain, 2AR is found to be expressed in DA neurons in the VTA by immunochemistry studies.146-148 At the subcellular level, 2AR is found both pre- and post-synaptically but predominantly at postsynaptic dendritic spines and shafts of non-5-HT neurons.149

11 1.4.3 Signaling of 2AR

2AR couples to the Gq protein, activates phospholipase C (PLC) via the α subunit of the

Gq protein, induces the hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2) into inositol trisphosphate (IP3) and di-acyl glycerol (DAG).150 Hydrolysis of PIP2 in general inhibits ion channels, including the G protein-inwardly rectifying potassium channels (GIRK). IP3 stimulates calcium release from endoplasmic reticulum (ER), whereas DAG stimulates protein kinase C

(PKC). In the prefrontal cortex, activation of 2AR suppresses membrane Cav1.2 L-type Ca2+

151 currents via Gαq-PLC-IP3-calcineurin signaling pathway. Generally, 2AR increases intracellular Ca2+ level via Gq-PLC-IP3 signaling pathway, enhances neurotransmission and acts as inhibitory or excitatory neuronal modulators.

Besides the canonical Gq-PLC-IP3 signaling pathway, 2AR activation also induces the

Ras-MEK-ERK pathway, RSK2-tyrosine kinase pathway and β-arrestin2-PI3K-Src-Akt pathway

(Figure 1.4 B).150,153-154

1.4.4 Function of the serotonin system

The serotonergic system regulates a broad range of physiological and behavioral processes, including learning and memory, cognition, aggression, respiration, endocrine regulation, feeding, sleep-wake cycle, pain sensitivity, vascular function, and emesis.155,156

The function of 2AR in learning and memory has been shown in both invertebrate and vertebrate model systems. Increase in 5-HT levels and 2AR activity has been found to improve memory in rodents and in Alzheimer and schizophrenic patients.157,158 In contrast, reduction of 5-

HT and 2AR activity has been found to impair contextual fear memory in mice, object memory in rats, and declarative memory in humans.159-161 A large number of studies have shown the

12 learning and memory effects after 2AR pharmacological manipulations across distinct tasks and different species.162

Polymorphisms and alterations in the human HTR2A gene are associated with altered memory processes and disordered cognitive functions. Carriers of the polymorphism-induced

His452Tyr substitution exhibited impairment in memory recall, poor verbal delayed recall and recognition in humans.163-166 Carriers of the homozygous T102C and A-1438G and T-allele exhibited significantly impaired short-term verbal memory and spatial working memory.167,168

The role of 2AR in mediating hallucination and spatial cognition has been shown in both animal models and humans. Serotonergic psychedelics have been found to cause visual or auditory hallucinations and paranoid delusions, an effect that can be blocked by administration of

2AR antagonists.169-171 The 2AR TT genotype (rs6313) has been found to be associated with better spatial cognitive performance.172 The 2AR agonist DOI has been found to decrease rat performance measured by swim time in both a new maze and a well-learned maze.173

Besides schizophrenia associated psychosis, the level of 2AR in the PFC has been found to correlate with neuropsychiatric disorders such as anxiety, Alzheimer’s, depression and

OCD.174-177

13 1.5 Evidence for D2R-2AR heteromerization

Common distribution of D2R and 2AR occurs in the VTA, striatum and PFC, providing the prerequisite for D2R and 2AR heteromeric complex formation. In the last decade, a few reports have shown evidence for the co-localization and heteromerization of D2R and 2AR in both native and heterologous expression systems.

Confocal microscopy image studies and single cell FRET studies revealed the co-

178 localization of D2R and 2AR in HEK cells co-transfected with YFP-D2R and CFP-2AR.

Immunofluorescence staining studies exhibited the co-localization of native D2R and 2AR in rat medial prefrontal cortex and substantia nigra.178,179 Co-immunoprecipitation (Co-IP) studies suggested interaction between D2R and 2AR, as in HEK293 cells co-transfected with Rluc-D2R and GFP-2AR, a GFP antibody pulled down Rluc-D2R and an Rluc antibody pulled down GFP-

180 2AR. BRET studies suggested formation of heteromeric complex between D2R and 2AR, as a

BRET saturation curve was obtained in HEK293 cells co-transfected with Rluc-D2R and increasing amounts of GFP-2AR.181 Proximity ligation assay (PLA) studies revealed a heteromeric complex of D2R-2AR in rat striatum (nucleus accumbens and caudate putamen)

(Figure 1.5).182,183

In an animal behavioral study, the effect of haloperidol on reversing MK-801 induced locomotion was significantly attenuated in 2AR KO mice, indicating 2AR is necessary for the

180 D2R antagonist haloperidol to exert its full antipsychotic effects (Figure 1.6).

14 1.6 Purpose of this study

The hypothesis and findings that D2R and 2AR may directly interact and form a functional heteromeric complex raise a series of questions including:

1. Would the signaling properties of D2R and 2AR in the heteromeric complex change compared to the homomeric ones?

2. How can one manipulate the formation of D2R-2AR heteromeric complex?

3. How do the D2R and 2AR couple to each other?

4. Does the effectiveness of antipsychotic drugs depend on the D2R-2AR putative heteromer?

The purpose of this study is to answer these questions. We have pursued four major aims for this study:

1. To profile the Gi and Gq signaling properties of the D2R-2AR putative heteromer

2. To develop tools to manipulate the formation and function of the D2R-2AR putative heteromer in heterologous system and native tissue

3. To build a structural model of the D2R-2AR putative heteromer recapitulating its coupling mechanism.

4. To investigate the effectiveness of antipsychotic drugs on the function of the D2R-2AR putative heteromer.

We hope that this study provides insights to the function of the D2R-2AR putative heteromer in schizophrenia and psychosis, and sheds light towards the long-term goal of therapeutic development of more effective antipsychotic drugs.

15 Figure 1.1 Key dopamine pathways in the brain The neuroanatomy of dopamine neuronal pathways in the brain can explain the symptoms of schizophrenia as well as the therapeutic effects and side effects of antipsychotic drugs. (a) Nigrostriatal dopamine pathway (b) Mesolimbic dopamine pathway (c) Mesocortical dopamine pathway (d) Tuberoinfundular dopamine pathway (e) Brainstem projections to the thalamus Adapted from Stahl (2013)

16 Figure 1.2 Neuronal circuits indicating the interaction between 2AR and glutamate receptors in response to psychoactive and antipsychotic agents. a. The serotonin and glutamate release induced in the cortex by PCP-like psychoactive drugs targeting subcortical NMDA receptors might affect the signaling properties of the cortical 5- HT2A–mGlu2 receptor complex b. Concurrently, these results indicate that both LSD-like and PCP-like psychoactive drugs dysregulate the signaling properties of cortical pyramidal neurons and affect cognition and perception processes in the brain cortex. Adapted from Gonzalez-Maeso et al, Trends in Neurosciences (2009)

17 Figure 1.3 Integrated hypothesis of schizophrenia showing the role of dopamine, serotonin and glutamate Excess serotonin, upregulated 2AR, or psychedelic hallucinogens all could cause hyperactivation of 2AR on glutamatergic neurons in PFC, which leads to glutamate release from PFC to VTA. Excessive glutamate in VTA causes hyperactivity of DA mesolimbic pathway and hypoactivity of DA mesocortical pathway, resulting in schizophrenic symptoms. Adapted from Stahl et al, CNS Spectrums (2018)

18 Figure 1.4 D2R and 2AR signaling pathways A. D2R signaling pathways B. 2AR signaling pathways Adapted from Saunders et al, (2017)

19 Table 1.1 Physical and functional evidence for dopamine receptor heteromer Dopamine receptor heteromers are involved in many mental disorders such as schizophrenia, addition, and ADHD. Adapted from Perreault et al, Neuropsychopharmacology (2014)

20 Figure 1.5 Evidence for D2R-2AR heteromerization A. BRET saturating curve indicating the formation of D2R-2AR heteromeric complex in HEK cells co-transfected with D2R and 2AR. Adapted from Borroto-Escuela et al, BBRC (2010) B. PLA results indicating the formation of D2R-2AR heteromeric complex in rat striatum (caudate putamen) samples. Adapted from Borroto-Escuela et al, Methods in Enzymology (2013)

21 Figure 1.6 2AR is necessary for the inhibition of MK-801-induced locomotor activity by the D2R antagonist haloperidol. A. The time course of MK-801-induced locomotion is measured in 5 min blocks. Time of injections of MK-801 is indicated by arrows. Horizontal activity is measured in wild-type (WT) and 5-HT2AR-KO mice. B. Corresponding bar graph of the total MK-801-induced locomotion as a summation of horizontal activity from t=10 min to t=120 min. Mice were administered haloperidol (1 mg/kg) or vehicle followed by MK-801 (0.5 mg/kg; i.p.) (n=6; **p < 0.01; error bars shows SEM). Adapted from Albizu et al, Neuropharmacology (2011)

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38 Chapter 2. THE INTEGRATED GI AND GQ SIGNALING PROFILE OF D2R-2AR

2.1 Background introduction

2.1.1 Monitoring Gi and Gq activity in an oocyte electrophysiology assay and a mammalian cell calcium mobilization assay

Fribourg et al. used the oocyte assay to monitor mGluR2-elicited Gi activity and 2AR-

1 elicited Gq activity. In the present study, we have used the same assay to monitor D2R-elicited

Gi activity and 2AR-elicited Gq activity (Figure 2.1). G protein-gated Inward-Rectifying

Potassium (K) 4* (GIRK4*) channel was used as reporter. D2R activation opened GIRK4*, resulting in current increase, whereas 2AR activation inhibited the opening of GIRK4*, resulting in current inhibition.2 The current change upon receptor activation was measured by Two-

Electrode Voltage Clamp (TEVC) and normalized to the basal current induced by 96 mM K+ solution to represent Gi and Gq activity. When monitoring Gi activity, RGS2 was used to eliminate Gq signaling; when recording Gq activity, PTX was used to inhibit Gi signaling.

To avoid the long recording time for Gq current inhibition that made it a challenge to construct concentration-response relationships, we adapted the calcium mobilization assay as a second method to monitor 2AR-elicited Gq activity. GCaMP, a fusion protein of calmodulin

3 (CaM) and permutated GFP was used as calcium indicator. 2AR activation led to IP3-induced

Ca2+ release from the endoplasmic reticulum (ER). Ca2+ binds to the CaM moiety and causes a structural shift of GFP, resulting in bright fluorescence signals.3 The peak of the fluorescence trace was measured and normalized to the basal line to represent the 2AR-elicited Gq activity.

39 2.1.2 Co-expression of D2R with 2AR potentiates Gi and Gq signaling in response to 1uM DA and 5-HT

Previous work of Younkin et al. explored the effect of co-expression of D2R with 2AR on

D2R-elicited Gi activity and 2AR-elicited Gq activity (Figure 2.2). D2R and 2AR were expressed in oocytes alone and together. The presence of 2AR significantly increased the Gi activity of D2R in response to 1 µM DA, compared to D2R alone (dashed line). Co-addition of

1 µM DA and 1 µM 5-HT further enhanced the Gi activity of D2R. Similar results were obtained for 2AR-elicited Gq activity. Co-expression significantly potentiated the Gq activity of 2AR in response to 1 µM 5-HT compared to the 2AR homomer (dashed line). When both ligands were applied, the Gq activity of 2AR was further augmented. These results revealed a functional cross-talk between D2R and 2AR in response to endogenous ligands.

2.1.3 Ratio of D2R and 2AR expressed in oocytes and HEK293 cells

To explore whether the ratio of D2R to 2AR expressed in oocytes has an impact on the potentiation of Gi and Gq activity, Youkin et al. injected cRNA of D2R and 2AR with different ratios in oocytes and tested the DA-induced Gi activity and 5-HTinduced Gq activity (Figure 2.3

A and B). The results show the greatest Gi and Gq potentiation was obtained when the cRNA of

D2R to 2AR injected was at a ratio of 1:2.

To determine the ratio of D2R and 2AR that gives the greatest potentiation of Gq activity in

HEK293E cells, cDNA of D2R and 2AR was co-transfected with different ratios in HEK293E cells and the Gq activity was measured (Figure 2.3 C and D). The results showed the greatest potentiation of Gq activity in response to 0.1 µM 5-HT or 10 µM DA is when cDNA of D2R and

2AR was co-transfected at a ratio of 1:1.

40 Based on these results, D2R and 2AR were co-injected at a ratio of 1:2 in oocytes, and co- transfected at a ratio of 1:1 in HEK293E cells in all subsequent experiments, to achieve maximal functional cross-talk.

2.1.4 Aims

The work of Youkin et al. showed functional cross-talk between D2R and 2AR in response to 1 µM DA and 5-HT. However, it was not determined whether the positive modulation of Gi and Gq signaling would be observed only in response to 1 µM DA and 5-HT or whether the potentiation was dependent on the concentration of DA and 5-HT. In order to obtain the Gi and

Gq signaling properties of D2R-2AR putative heteromer, we decided to perform full dose- response curves for D2R-2AR co-expression and compare them to those from D2R and 2AR expressed alone. We expected that dose-response relationships would characterize the Gi and Gq activities of the D2R-2AR putative heteromer in response to ligands at a broad range of concentrations, and provide pharmacological information of its signaling properties, such as potency and efficacy and show where the 1 µM concentration fell in the dose-response curves.

2.2 Results and discussion

2.2.1 Co-expression of D2R with 2AR potentiates DA induced Gi signaling

To obtain the Gi signaling profile of D2R homomer and D2R-2AR putative heteromer, seven doses of DA were applied to oocytes expressing either D2R alone or D2R and 2AR together (Figure 2.4). In the full range of the dose-response curves, Gi activity obtained through

D2R-2AR co-expression was markedly enhanced compared to D2R alone. The Emax of the two

41 curves indicated that the presence of 2AR doubled the efficacy of DA. The EC50 values of the two curves were similar, indicating that the presence of 2AR did not exert a major effect on the potency of DA. In both curves, DA saturated at 1 µM.

2.2.2 Co-expression of D2R with 2AR enables 5-HT induced Gi signaling

When repeating Youkin’s experiments, I observed that Gi signaling could be obtained when 1 µM 5-HT was applied to oocytes co-expressing D2R and 2AR. To explore whether 5-HT induced D2R-elicited Gi signaling in the presence of 2AR, six doses of 5-HT were applied to oocytes expressing D2R and 2AR alone or together (Figure 2.5). The results show that in the absence of DA, 5-HT generated Gi signaling in a dose-dependent manner in oocytes co- expressing D2R and 2AR. Because RGS2 was co-expressed to eliminate Gq signaling when monitoring Gi activity, the activation of D2R upon addition of 5-HT could not be due to the

2AR-Gq downstream signaling pathway. Thus, we interpret the 5-HT-induced Gi signaling as 5-

HT stimulating 2AR, then somehow the stimulated 2AR further activating D2R and inducing Gi signaling. Interestingly, here 5-HT saturates at 10 µM, the same as the saturating concentration of 5-HT acting on the 2AR homomer. In control experiments, 5-HT does not generate Gi- signaling on the 2AR homomer because the 2AR homomer does not couple to the Gi protein; only a small amount of Gi activity was observed when micromolar doses of 5-HT were applied to the D2R homomer.

2.2.3 Integrated Gi signaling of D2R-2AR co-expression in response to DA and 5-HT

Comparison of Emax in Figure 2.6 A and B shows that co-expression of D2R and 2AR enhanced the efficacy of DA or 5-HT-induced Gi activity compared with the efficacy of DA on

42 D2R homomer. Inclusion of 0.5 µM 5-HT (close to the EC50 of 5-HT) with DA further potentiated the Gi signaling through D2R-2AR co-expression and enhanced the potency of DA.

These results suggest that 2AR positively modulates the Gi signaling of D2R.

2.2.4 Co-expression of D2R with 2AR potentiates 5-HT-induced Gq signaling

To obtain the Gq signaling profile of 2AR homomer and D2R-2AR putative heteromer, eight doses of 5-HT were applied to HEK293E cells expressing 2AR alone and D2R with 2AR respectively (Figure 2.7). In the full range of the dose-response curves, the presence of D2R significantly increased the 2AR-elicited Gq signaling compared with 2AR homomeric Gq signaling. The Emax values of the two curves indicates the presence of 2AR significantly enhances the efficacy of DA. EC50 of the two curves are comparable, indicating the presence of

2AR does not change the potency of 5-HT. In both curves, 5-HT saturates at 0.1 µM.

2.2.5 Co-expression of D2R with 2AR enables DA-induced Gq signaling

To explore whether DA induces 2AR-elicited Gq signaling in the presence of D2R, eight doses of DA were applied to HEK293E cells expressing D2R and 2AR alone or together (Figure

2.8). The results showed that in the absence of 5-HT, DA generated Gq signaling in a dose- dependent manner in cells co-expressing D2R and 2AR. In control experiments, the DA-induced

Gq-signaling on the D2R homomer was negligible due to the fact D2R homomer does not couple to Gq proteins. A small amount of Gq activity was detected when DA above the micromolar range was applied to the 2AR homomer. This observation is consistent with previous findings of

Woodward et al. and Bhattacharyya et al. that DA at micromolar concentrations acts as a partial efficacy agonist of 2AR in Xenopus oocytes and HEK293 cells.4,5 Notably, the DA induced Gq

43 signaling of 2AR homomer is markedly smaller than that of D2R-2AR co-expression. Thus, we interpret the DA-induced Gq signaling as stimulated D2R by DA activating 2AR and eliciting Gq signaling.

2.2.6 Integrated Gq signaling of D2R-2AR in response to DA and 5-HT

Comparison of Emax values in Figure 2.9 A and B shows that co-expression of D2R and

2AR enhanced the efficacy of 5-HT or DA-induced Gq activity compared with the efficacy of 5-

HT acting on the 2AR homomer. Co-addition of 0.75 µM DA (the EC50 of DA) with 5-HT further potentiated the Gq signaling through D2R-2AR co-expression and enhanced the potency of 5-HT. These results suggest that D2R positively modulates the Gq signaling of 2AR.

2.2.7 Co-expression of D2R with 2AR potentiates Gi and Gq signaling

The dose-response results in Figures 2.4-2.9 reveal both Gi and Gq signaling are potentiated upon D2R-2AR co-expression (Figure 2.10). The presence of 2AR increases the Gi signaling of D2R in response to either DA (cis-signaling) or 5-HT (trans-signaling); co-addition of both ligands further increases Gi signaling (combination of cis and trans-signaling).

Symmetrically, the presence of D2R increases the Gq signaling of 2AR in response to either 5-

HT (cis-signaling) or DA (trans-signaling); when both ligands are applied, the Gq signaling is further augmented (combination of cis and trans-signaling).

2.2.8 D2R synthetic ligands cross-talk to 2AR

Quinpirole (QP), a selective D2R/D3R agonist and psychoactive drug has been found to induce D2R super-sensitivity, and therefore it has been used in animal models of schizophrenia.

44 6-8 Haloperidol (HLP), a typical antipsychotic drug with high affinity to D2R and strikingly low affinity to 2AR has been widely used in the treatment of schizophrenia.9-11 In this study, we applied multiple doses of QP or HLP to HEK cells co-expressing D2R and 2AR, to assess whether D2R synthetic ligands cross-talk to 2AR (Figure 2.11). As the dose-response curve shows, quinpirole generates robust Gq signaling in the absence of 5-HT whereas haloperidol decreases 5-HT induced Gq signaling. Although control experiments testing Gq signaling in response to QP on 2AR homomer has not been performed, it has not been reported in the literature that QP can act as partial or full agonist of 2AR. Results in Figure 5.3 suggest that the presence of D2R significantly increases the potency of HLP antagonizing the 5-HT-induced

2AR-elicited Gq signaling. Thus, we conclude that QP and HLP cross-talk to act on 2AR.

2.2.9 D2R synthetic ligands cross-talk to modulate 2AR agonist-induced Gq signaling

To explore whether QP modulates 2AR agonist-induced Gq signaling, 0.1 µM QP was applied with 5-HT or DOI to cells co-expressing D2R and 2AR (Figure 2.12). The upward- and left-shift of the dose-response curves shows that QP potentiated Gi signaling and enhanced the potency of 5-HT and DOI acting on the D2R-2AR co-expression, indicating that QP cross-talks to positively modulate 2AR agonist-induced Gq signaling.

To assess HLP’s effect on modulating 2AR agonist-induced Gq signaling, 15 µM and 10

µM HLP was applied with 5-HT and DOI respectively to cells co-expressing D2R and 2AR

(Figure 2.12). The right shift of the dose-response curves shows that HLP decreases the potency of 5-HT and DOI acting on D2R-2AR co-expression, indicating that HLP cross-talks to negatively modulate 2AR agonist-induced Gq signaling.

45 2.3 Summary

In this study, we obtained full-dose response curves of DA and 5-HT on oocytes and

HEK cells co-expressing D2R and 2AR. Co-expression of D2R and 2AR potentiates Gi and Gq signaling compared to D2R homomer and 2AR homomer and reveals the signaling properties of

D2R-2AR putative heteromer.

We summarize our results and conclusions as follows:

1. Co-expression of D2R and 2AR potentiates Gi cis-signaling in response to DA

2. Co-expression of D2R and 2AR enables Gi trans-signaling in response to 5-HT

3. Co-expression of D2R and 2AR potentiates Gq cis-signaling in response to 5-HT

4. Co-expression of D2R and 2AR enables Gq trans-signaling in response to DA

5. Co-addition of DA and 5-HT further potentiates Gi and Gq signaling.

6. QP cross-talks to activate 2AR and positively modulate 2AR agonist-induced Gq signaling

7. HLP cross-talks to antagonize 2AR and negatively modulate 2AR agonist-induced Gq signaling.

46 Figure 2.1 Monitoring Gi and Gq Signaling with Inward-Rectifying Potassium Channels in Xenopus Oocytes and Calcium mobilization assay with Calcium indicator GCaMP A. Stimulation of a Gi-coupled receptor, such as D2R, activates Gβγ subunits to stimulate GIRK channel currents. PTX is shown to inhibit Gi signaling. B. Stimulation of a Gq-coupled receptor, such as 2AR, activates PLC to hydrolyze PIP2 via Ga subunit, resulting in inhibition of the IRK3 (or GIRK) channel currents. RGS2 is shown to inhibit Gq signaling. Adapted from Hatcher-Solis et al., Current Pharmaceutical Biotechnology (2014) C. Stimulation of a Gq-coupled receptor, such as 2AR, activates PLC to hydrolyze PIP2 to IP3, resulting in IP3 induced Calcium release from endoplasmic reticulum (ER). Calcium binds to calmodulin (CaM) and activate the GPF in GCaMP, resulting in the generation of fluorescence signals. Adapted from Lindenburg et al., Sensor (2014) D. Representative barium-sensitive traces of GIRK4* currents obtained in oocytes expressing D2R in response to 96 mM potassium solution (basal), 1 µM DA, and 3mM barium solution. Barium inhibits GIRK4* currents and allows for subtraction of GIRK4*-independent currents. E. Representative barium-sensitive traces of GIRK4* currents obtained in oocytes expressing 2AR in response to 96 mM potassium solution (basal), 1 µM serotonin (5-HT), and 3 mM barium solution. F. Representative fluorescence traces of GCaMP obtained in HEK293E cells expressing 2AR in response to 0.1 µM 5-HT. The time point of 5-HT application is indicated by the arrow.

A B C

D E

47 Figure 2.2 Co-expression of D2R with 2AR potentiates Gi and Gq signaling in response to 1 µM DA and 5-HT Bar graph summary of Gi activity (yellow) and Gq activity (red) measured in oocytes. Dashed line represents the Gi and Gq activity of D2R homomer and 2AR homomer. The presence of 2AR increases the Gi activity of D2R in response to 1 µM DA, co-addition of 1 µM DA and 1µM 5-HT further enhances Gi activity. The presence of D2R increases the Gq activity of 2AR in response to 1uM 5-HT, co-addition of 1 µM DA and 1 µM 5-HT further enhances Gq activity. Adapted from Younkin et al.

48 Figure 2.3 The ratio of D2R and 2AR expressed in oocytes and HEK293 cells A. and B. The largest Gi and Gq potentiation is obtained when oocytes co-expressing D2R and 2AR with raio of 1:2. Gi activity (GIRK4* current activation) was measured relative to the basal currents and was normalized relative to the activation of D2R by 1 µM DA (100% or 1) (dashed line). Gq activity (GIRK4* current inhibition) was measured relative to the basal currents and was normalized relative to the activation of 2AR by 1 µM 5-HT (100% or 1) (dashed line). (Mean ± SEM. N=7-8/condition. *p<0.05, **p<0.01) Adapted from Younkin et al. C. and D. Bar graph summary showing the greatest Gq activity in response to 0.1 µM 5-HT or 10 µM DA is obtained when D2R and 2AR is co-expressed with ratio of 1:1 in HEK293 cells. Gq activity was measured relative to the basal florescence and was normalized relative to the activation of 2AR by 0.1 µM 5-HT (100). (Mean± SEM. N=8/condition)

A B

C D

49 Figure 2.4 Co-expression of D2R with 2AR potentiates DA induced Gi signaling A. Representative barium-sensitive traces of GIRK4* currents obtained in response to sequential addition of 1 nM, 10 nM, 32 nM, 100 nM, 320 M, 1 µM and 10 µM DA in oocytes expressing D2R alone (left panel), D2R with 2AR (right panel). (orange: outward current; blue: inward current) B. Dose-response curves showing the efficacy of DA on oocytes co-expressing D2R and 2AR (yellow, Emax=2.15) is increased by ~100% compared with oocytes expressing D2R alone (black, Emax=1.09). Gi activity (GIRK4* current activation) was measured relative to the basal currents and was normalized relative to the maximal activation of D2R by DA (100% or 1) (Mean ± SEM; N=6-7/condition). RGS2 was co-injected to inhibit Gq signaling.

Figure 2.5 Co-expression of D2R with 2AR enables 5-HT induced Gi signaling A. Representative barium-sensitive traces of GIRK4* currents obtained in response to sequential addition of 1 nM, 10 nM, 100 nM, 1 µM, 10 µM, 100 µM 5-HT and 10 µM DA in oocytes co- expressing D2R and 2AR. (orange: outward current; blue: inward current) B. Dose-response curves showing 5-HT induces robust Gi trans-signaling in the absence of DA on oocytes co-expressing D2R and 2AR (yellow, Emax=1.47), but not on oocytes expressing D2R alone (gray solid, Emax=0.26) or 2AR alone (gray dash, Emax=0.07). Gi activity was measured and normalized as Figure 2.4B (Mean± SEM; N=4-6/condition). RGS2 was co-injected to inhibit Gq signaling.

Figure 2.6 Integrated Gi signaling of D2R-2AR in response to DA and 5-HT A. Dose-response curves showing the addition of 0.5 µM 5-HT with DA further increases the efficacy of DA on oocytes co-expressing D2R and 2AR (orange, Emax=2.79). Gi activity was measured and normalized as Figure 2.3B (Mean± SEM; N=4-7/condition). RGS2 was co- injected to inhibit Gq signaling. B. Same as Figure 2.5B

A B

Figure 2.7 Co-expression of D2R with 2AR potentiates 5-HT induced Gq signaling Dose-response curves showing the efficacy of 5-HT on HEK293 cells co-expressing D2R and 2AR (red, Emax=126.7) is increased compared with cells expressing 2AR alone (black, Emax=101.8). Gq activity was measured relative to the basal florescence and was normalized relative to the Emax of 2AR by 5-HT (100). (N=6/condition)

Figure 2.8 Co-expression of D2R with 2AR enables DA induced Gq signaling Dose-response curves showing DA generates robust Gq trans-signaling in the absence of 5-HT on HEK293 cells co-expressing D2R and 2AR (red, Emax=110.6), but not on cells expressing 2AR alone (gray solid, Emax=25.4) or D2R alone (gray dash, Emax=12.7). Gq activity was measured and normalized as Figure 2.7. (N=6/condition)

Figure 2.9 Integrated Gq signaling of D2R-2AR in response to DA and 5-HT A. Dose-response curves showing the addition of 0.75 µM DA increases the efficacy and the potency of 5-HT on HEK293 cells co-expressing D2R and 2AR (orange, Emax=158.1). Gq activity was measured and normalized as Figure 2.7. (Mean± SEM; N=6/condition) B. Same as Figure 2.7

A B

Figure 2.10 Co-expression of D2R with 2AR potentiates Gi and Gq signaling

A. Schematic Gi signaling of D2R and D2R+2AR showing the presence of 2AR increases the Gi signaling of D2R in response to either DA (cis-signaling) or 5-HT (trans-signaling); when both ligands are applied, the Gi signaling is further increased (combination of cis and trans-signaling). B. Schematic Gq signaling of 2AR and D2R+2AR showing the presence of D2R increases the Gq signaling of 2AR in response to either 5-HT (cis-signaling) or DA(trans-signaling); co-addition of both ligands further augmented Gq signaling (combination of cis and trans-signaling).

Gi Gi Gi Gi

Gq Gq Gq Gq

Figure 2.11 D2R synthetic ligands cross-talk to 2AR A. Dose-response curves showing quinpirol cross-talks to activate 2AR. B. Dose-response curves showing haloperidol cross-talks to antagonize 5-HT at 2AR. Gq activity was measured relative to the basal florescence. (Mean± SEM; N=6/condition)

57 Figure 2.12 D2R ligands cross-talk to allosterically modulate 2AR agonist induced Gq signaling A. Dose-response curves showing the addition of 0.1 µM QP increases both the efficacy and potency of 5-HT on HEK293E cells co-expressing D2R and 2AR (orange, Emax=121.8), while the addition of 15 µM HLP decreases the potency of 5-HT (purple, Emax=94.4). B. Dose-response curves showing the addition of 0.1 µM QP increases both the efficacy and potency of DOI on HEK293E cells co-expressing D2R and 2AR (orange, Emax=115.1), while the addition of 10 µM HLP decreases the potency of 5-HT (purple, Emax=100). Gq activity was measured relative to the basal florescence and was normalized relative to the maximal activation of D2R-2AR by 5-HT or DOI in the absence of D2R ligand (100). (Mean± SEM; N=6/condition) A

58 2.4 References

1. Fribourg, M., Moreno, J. L., Holloway, T., Provasi, D., Baki, L., Mahajan, R., ... & Ruta, J. D. (2011). Decoding the signaling of a GPCR heteromeric complex reveals a unifying mechanism of action of antipsychotic drugs. Cell, 147(5), 1011-1023.

2. Hatcher-Solis, C., Fribourg, M., Spyridaki, K., Younkin, J., Ellaithy, A., Xiang, G., ... & E Logothetis, D. (2014). G protein-coupled receptor signaling to Kir channels in Xenopus oocytes. Current pharmaceutical biotechnology, 15(10), 987-995.

3. Nagai, T., Sawano, A., Park, E. S., & Miyawaki, A. (2001). Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proceedings of the National Academy of Sciences, 98(6), 3197-3202.

4. Bhattacharyya, S., Raote, I., Bhattacharya, A., Miledi, R., & Panicker, M. M. (2006). Activation, internalization, and recycling of the serotonin 2A receptor by dopamine. Proceedings of the National Academy of Sciences, 103(41), 15248-15253.

5. Bhattacharyya, S., Raote, I., Bhattacharya, A., Miledi, R., & Panicker, M. M. (2006). Activation, internalization, and recycling of the serotonin 2A receptor by dopamine. Proceedings of the National Academy of Sciences, 103(41), 15248-15253.

6. Levant, B. E. T. H., Grigoriadis, D. E., & DeSOUZA, E. B. (1993). [3H] quinpirole binding to putative D2 and D3 dopamine receptors in rat brain and pituitary gland: a quantitative autoradiographic study. Journal of Pharmacology and Experimental Therapeutics, 264(2), 991- 1001.

7. Brown, R. W., Maple, A. M., Perna, M. K., Sheppard, A. B., Cope, Z. A., & Kostrzewa, R. M. (2012). Schizophrenia and substance abuse comorbidity: nicotine addiction and the neonatal quinpirole model. Developmental neuroscience, 34(2-3), 140-151.

8. Kostrzewa, R. M., Nowak, P., Brus, R., & Brown, R. W. (2016). Perinatal treatments with the dopamine D 2-receptor agonist quinpirole produces permanent D 2-receptor supersensitization: a model of schizophrenia. Neurochemical research, 41(1-2), 183-192.

9. Zarifian, E., Scatton, B., Bianchetti, G., Cuche, H., Loo, H., & Morselli, P. L. (1982). High doses of haloperidol in schizophrenia: a clinical, biochemical, and pharmacokinetic study. Archives of general psychiatry, 39(2), 212-215.

10. Kroeze, W. K., Hufeisen, S. J., Popadak, B. A., Renock, S. M., Steinberg, S., Ernsberger, P., ... & Roth, B. L. (2003). H1-histamine receptor affinity predicts short-term weight gain for typical and atypical antipsychotic drugs. Neuropsychopharmacology, 28(3), 519.

11. Tyler, M. W., Zaldivar-Diez, J., & Haggarty, S. J. (2017). Classics in chemical neuroscience: haloperidol. ACS chemical neuroscience, 8(3), 444-453.

59 Chapter 3. DEVELOPING TOOLS TO DISRUPT THE FORMATION AND FUNCTION OF

D2R-2AR IN HETEROLOGOUS SYSTEM AND NATIVE TISSUE

3.1 Background introduction

3.1.1 Use of cell-penetrating peptides as tools to disrupt GPCR heteromers

To verify the signaling change upon D2R and 2AR co-expression is due to formation of

D2R-2AR heteromeric complex, we need tools that disrupt D2R-2AR heteromer formation and attenuate D2R-2AR heteromer-induced signaling, thereby confirming the integrated signaling we monitored through D2R and 2AR co-expression displays the signaling properties of D2R-2AR heteromer. Since 1988 Frankel et al. discovered that the Human Immunodeficiency Virus (HIV)

Trans-activator of transcription (TAT) protein possessed the ability to permeate cell membranes

,1 the cell-penetrating peptide (CPP) has been used as a tool in therapeutic delivery2-5 and GPCR heteromer studies.6 In the use of CPP to disrupt GPCR heteromer, CPP such as TAT peptide is fused to synthetic peptides mimicking and targeting the heteromer interfaces to facilitate their translocation to cell membrane.6 Synthetic peptides delivered on cell membrane disrupt heteromer formation through competition binding. Here, we use three examples to represent the use of CPP as tools to disrupt GPCR heteromers in vitro, ex vivo and in vivo, respectively.

Bonaventura et al. applied TAT- tagged synthetic peptides mimicking the TM5/7 of D2R or

2AR in HEK293 cells co-expressing D2R and A2AR to assess their ability to disrupt the D2R-

7 A2AR heteromer (Figure 3.1). Their fluorescence complementation assay results showed that the D2R TM5 peptide and A2AR TM5 peptide significantly decreased the fluorescence between

D2R and A2AR, suggesting D2R TM5 and A2AR TM5 peptides possess the ability to disrupt D2R-

A2AR heteromer.

60 Navarro et al. treated rat striatum neurons with TAT-tagged synthetic peptide mimicking

TM4, 5, 6, 7 of D2R and TM4, 5, 6, 7 of A2AR, and detected D2R-A2AR heteromers by Ploximity

8 Ligation Assay (PLA) (Figure 3.2). Their results showed D2R TM4 and TM5 peptides and

A2AR TM4 and TM5 peptides disrupted endogenous D2R-A2AR heteromer in striatum samples, indicating D2R and A2AR heteromerize through a symmetric TM4,5-TM4,5 interface.

Liu et al. injected TAT-tagged synthetic peptide into the spinal cord of mice to assess the function of the mu opioid receptor and gastrin releasing peptide receptor MOR1D-GRPR (Figure

9 3.3). This synthetic peptide contained a RNEEPSS motif in the MOR1D C terminus which was found necessary for MOR1D-GRPR heteromerizaiton. Their animal behavioral results showed that MOR1DCT peptide blocked morphine-induced scratching without affecting GRP-induced scratching or morphine-induced analgesia, suggesting opioid- induced itch was independent of opioid analgesia through the function of MOR1D-GRPR heteromer.

3.1.2 The role of the pre-frontal cortex (PFC) in schizophrenia

The PFC mediates cognitive and executive functions required for goal-directed behavior, such as working memory, decision-making and selective attention.10-13 Dysfunction of the PFC is a key component of the pathophysiology underlying the negative and cognitive symptoms of schizophrenia.14,15 Neuroimaging studies in schizophrenic patients and drug addicts have shown

“task-related hypofrontality” --- decreased basal and working memory task-induced regional cerebral blood flow specific to PFC.16-19 Changes in the intrinsic properties of layer V pyramidal neurons have been found in cocaine treated rats.20,21 Alteration of neuronal excitability and short- term synaptic plasticity in PFC are found in mouse models of schizophrenia.22,23 Deficits in LTD are found both in patients and MK-801models of schizophrenia.24,25 Taken together, abnormal

61 neuronal excitability and impaired synaptic plasticity in PFC are associated with the pathophysiology of schizophrenia.

Both 2AR and D2R have been found in the rodent PFC and are abundantly expressed in

26-28 pyramidal neurons and interneurons, especially in layer V. D2R activation results in decreased neuronal excitability and inhibitory postsynaptic potentials.29 VTA DA neurons are found to be under excitatory control of 2AR in medial PFC.30, 31

3.13 The role of the ventral tegmental area (VTA) in schizophrenia

The VTA is closely related to the pathogenesis of schizophrenia.32 DA neurons in the VTA project to the striatum nucleus accumbens and the prefrontal cortex.32,33 Dysfunction of VTA underlies hyperdopaminergic states in the striatum and hypodopaminergic states in the PFC, characteristic of schizophrenia.32,33 MRI studies showed significantly decreased signal intensity of the VTA in patients with schizophrenia.34 Morphology studies showed hypoplasticity and reduced number and small size of VTA DA neurons in drug-naïve patients with schizophrenia.35-

37. In vivo electrophysiology studies showed antipsychotic drug effectiveness correlated with normalization of VTA function.38,39

D2R and 2AR are both expressed in VTA and control VTA neuronal activity. 2AR is

40-42 natively resident within VTA DA neurons. Interaction between D2R and 2AR in the VTA controls D2 receptor trafficking, determining the sensitivity of DA neurons to autoreceptor regulation.43 In animal model studies, cocaine-evoked locomotor activity has been found increased by viral-mediated overexpression of 2AR in VTA and blocked by intra-VTA microinjection of a selective 2AR antagonist, suggesting a role of VTA 2AR in modulating psychotic behavior.44,45

62 3.14 Aims

In this study, we adapt the CPP strategy to develop tools to manipulate the formation and function of D2R-2AR heteromer, as this strategy has been successfully used to disrupt multiple

GPCR heteromers in vitro, ex vivo and in vivo. We first aim to assess the ability of CPPs in disrupting the heteromeric formation and signaling of D2R-2AR in oocytes and HEK cells (in vitro), then apply them in brain slice recordings to determine whether the D2R-2AR heteromer exists and has a function in PFC and VTA neurons (ex vivo). Finally, we will apply these peptides in animal behavioral studies to investigate the function of the the D2R-2AR heteromer on mediating psychotic behavior (hyperlocomotor activity) and on the action of antipsychotic drugs (in vivo).

3.2 Results and discussion

3.2.1 CPPs mimicking TM5/6 of D2R and 2AR disrupt the formation of D2R-2AR heteromer

To assess the ability of peptides disrupting D2R-2AR heteromer, our collaborating Ferré lab at the NIH synthesized eight peptides that mimic TM4-7 of D2R and 2AR respectively. The TAT peptide was fused to the N-terminus of TM5 and TM7 peptides, and the C-terminus of TM4 and

TM6 peptides to facilitate their translocation and correct orientation in the cell membrane.

Robust fluorescence signals induced by complementation of YFP were detected in HEK293 cells co-expressing D2R-nYFP and 2AR-cYFP but not in cells co-expressing negative control combinations, suggesting the formation of a D2R-2AR heteromeric complex in HEK cells

(Figure 3.4). Cells co-expressing D2R and 2AR were incubated 4 h with 4 µM of each peptide.

D2R TM5 and TM6 peptides as well as 2AR TM5 and TM6 peptides eliminated the fluorescence

63 signals between D2R and 2AR, suggesting that these peptides possessed the ability to disrupt the formation of D2R-2AR heteromeric complex.

3.2.2 D2R TM5 peptide disrupts D2R-2AR heteromeric signaling

We used the D2R TM5 peptide from the Ferré lab to assess its ability to disrupt D2R-2AR heteromeric signaling. Oocytes expressing D2R alone or D2R with 2AR were incubated 4 h with

10 uM D2R TM5 peptide (Figure 3.5). D2R TM5 peptide had no impact on D2R homomeric Gi signaling, but abolished the Gi cis-signaling enhancement in response to DA in oocytes co- expressing D2R and 2AR that is observed without peptide treatment. More strikingly, the Gi signaling augmentation in response to co-addition of DA and 5-HT was also taken away after treatment with the TM5 peptide. These results suggest the TM5 peptide disrupts the heteromeric

Gi signaling of D2R -2AR heteromer (the negative control experiment with the D2R TM7 peptide is underway). In the calcium mobilization assay, D2R TM5 but not TM7 peptide significantly reduced the DA-induced Gq trans-signaling in HEK293 cells co-expressing D2R and 2AR without inhibiting the Gq signaling of 2AR homomer, indicating the TM5 peptide disrupts the heteromeric Gq signaling of the D2R -2AR heteromer (Figure 3.6). Taken together, we conclude from these results that D2R TM5 peptide disrupts the heteromeric signaling of D2R -2AR heteromer.

3.2.3 D2R TM5 peptide reverses the enhancement of field Excitatory postsynaptic potential

(fEPSP) in PFC neurons

The collaborating Sidiropoulou lab at the University of Crete, Greece applied the D2R

TM5 and TM7 peptides in brain slice recordings to assess whether disruption of D2R and 2AR

64 heteromers in PFC affected PFC synaptic activity (Figure 3.7). In their previous study, the field excitatory post-synaptic potential (fEPSP) of PFC neurons in brain slices was found to be significantly enhanced in response to co-addition of DA and 5-HT but not DA or 5-HT alone.

This effect was hypothesized to be a function of D2R-2AR heteromer in PFC neurons.

Application of D2R TM5 or TM7 peptide in field recordings showed the enhancement of fEPSP upon 5-HT and DA stimulation was reversed by the TM5 but not TM7 peptide. Considering that the D2R TM5 peptide disrupts the formation and function of D2R-2AR heteromer, these results suggest D2R and 2AR form heteromeric complex in the PFC and modulate the postsynaptic activity of PFC neurons.

3.2.4 D2R TM5 peptide disrupts DA desensitization in VTA neurons

It is known that DA inhibits excitation of VTA dopaminergic neurons and reduces their firing rate.43 Prolonged exposure to DA reverses the DA-induced neuronal inhibition, a phenomenon called DA desensitization or Dopamine Inhibition Reversal (DIR) (Figure 3.8 A).46

Brodie et al. found this desensitization is dependent on Gq, phospholipase C, protein kinase C,

G-protein receptor kinase and dynamin.47-52 Given that Gq signaling pathway is required for DA desensitization, it is hypothesized that D2R-2AR heteromer is involved in and is necessary for

DA desensitization in VTA neurons. To test this hypothesis, our collaborating Brodie lab at the

University of Illinois in Chicago applied the D2R TM5 peptide in the DA desensitization assay.

The results that DA desensitization is removed after treatment of the TM5 peptide supports this hypothesis and suggests a role of D2R-2AR heteromer in modulating the activity of VTA neurons (the negative control experiment with the D2R TM7 peptide is underway).

65 3.2.5 A mini gene method to disrupt the D2R-2AR heteromer

Besides the disruptive peptide method, I sought to adopt a parallel unique strategy that could systematically and readily identify different regions of the interacting receptors critical for heteromerization (including non-TM regions). I used the bifunctional vector pXoom (expresses both in Xenopus oocytes and mammalian cells) to generate a construct that includes the region of interest (e.g., the D2R TM5) preceded by the first 45 aa of the rat somatostatin receptor 3 (sstr3), which contains a plasma membrane (PM) localization signal and has been used to localize proteins to the PM that otherwise show poor cell surface localization.53 Digestion with specific restriction enzymes allows replacement of TM5 with any other region of D2R that could be critical in its interactions with 2AR. The first preliminary tests with the TM5 mini gene expression in Xenopus oocytes (Figure 3.9) have been promising enough, showing that the TM5 mini gene abolished the heteromeric Gi signaling. Along with optimizing the experimental conditions (e.g. level of mini gene expression) I have started to construct additional D2R TM mini genes (positive control: TM6, negative controls: TM4 and TM7) to disrupt D2R-2AR heteromerization. Once this assay is fully established, it will enable us not only to test for the involvement of all TMs but also any part of the protein that may be involved in enabling heteromerization. Moreover, identification of the key protein regions involved in heteromerization will provide important constraints for structural models of D2R-2AR heteromers. The mini gene approach will be an efficient and cost effective method to identify interacting surfaces between GPCRs. Any region of a receptor could be tested for effects on heteromer formation before ordering the appropriate peptides for ex vivo and in vivo studies.

66 3.3 Summary

In this series of experiments, we successfully developed TAT-tagged synthetic peptide as tools to disrupt D2R-2AR putative heteromer in heterologous systems and in ex vivo brain slices.

In vivo animal behavior experiments using the disruptive peptides are ongoing in the collaborating Gonzalez-Maeso lab at Virginia Commonwelath University.

We summarize our results and conclusions as follows:

1. D2R and 2AR form a heteromeric complex in heterologous systems.

2. TAT-tagged synthetic peptides mimicking TM5 and TM6 of D2R or 2AR disrupt D2R-

2AR heteromeric complex in heterologous systems.

3. TM5 and TM6 of D2R and 2AR are involved in D2R-2AR heteromeric interface.

4. D2R TM5 peptide disrupts the heteromeric signaling of D2R-2AR in heterologous systems.

5. D2R and 2AR form a heteromeric complex in PFC and VTA neurons.

6. The D2R TM5 peptide disrupts the formation and function of D2R-2AR heteromer in PFC and VTA neurons.

67 Figure 3.1 Cell-penetrating peptides mimicking D2R TM5 and A2AR TM5 but not D2R TM7 and A2AR TM7 disrupt A2AR R-D2R heteromerization in HEK293 cells A. Fluorescence (AU) induced by complementation of YFP Venus in HEK-293 cells co- expressing A2AR-nYFP and A2AR-cYFP, D2R-nYFP and D2R-cYFP, or A2AR-nYFP and D2R- cYFP treated or not treated with the indicated HIV TAT peptides (4 µM) for 4 h. B. BRET (mBU) induced by bimolecular luminescence and fluorescence complementation in cells expressing A2AR-nRluc, D2R- cRluc, A2AR-nYFP and D2R-cYFP, and negative controls. Adapted from Bonaventura et al., PNAS (2015)

68 Figure 3.2 A2AR and D2R TM4-TAT and TAT-TM5 peptides disrupt A2AR-D2R heteromerization and function in striatal neurons a. and c. A2AR-D2R heteromer detected by proximity ligation assay (PLA) in rat striatal neurons. A2AR-D2R heteromers appeared as red dots in confocal microscopy images. Samples were treated with medium (a) or indicated TM peptides of A2AR or D2R (4 µM) for 4 h (c). b. Quantification from a and c: values are expressed as the ratio between the number of red spots and the number of cells showing spots (r); % values represent the percentage of cells showing one or more red spots as compared to control. d. and e. cAMP production determined in rat striatal neurons incubated 4 h with 4 µM of indicated TM peptides of A2AR (d) or D2R (e) and exposed to indicated ligands. Values are expressed as percentage of cAMP accumulation in non-treated cells (basal). Adapted from Navarro et al., Nature Communication (2018)

69 Figure 3.3 TAT-MOR1DCT peptide disrupts MOR1D-GRPR heteromerization and function in mice A. Tat-MOR1DCT peptide contains TAT domain and the RNEEPSS motif in MOR1D C terminus. Control peptide contains Tat domain and scrambled sequence of MOR1DCT. B. and C. Tat-MOR1DCT blocked morphine-induced scratching without affecting GRP-induced scratching (B) or morphine-induced analgesia (C). *p < 0.05. D. Co-IP by anti-MOR1D. E. Quantified O.D. ratio of GRPR and MOR1D from (D). Adapted from Liu et al., Cell (2011)

70 Figure 3.4 Cell-penetrating peptides mimicking D2R or 2AR TM5 and TM6 disrupt D2R- 2AR heteromerization in HEK293 cells (In collaboration with Ferre lab at NIH)

Fluorescence induced by complementation of YFP Venus in HEK-293 cells co-expressing D2R- nYFP and 2AR-cYFP, not treated or treated 4 h with indicated HIV TAT peptide (4 µM). (**** p<0.0001, ANOVA with Dunnet’s multiple comparisons; n=6-8)

71 Figure 3.5 D2R TAT-TM5 disrupts the heteromeric Gi signaling of D2R-2AR in oocytes Bar graph summary of Gi activity measured in oocytes expressing D2R alone, D2R with 2AR, not incubated or incubated 4 h with 10 µM D2R TAT-TM5 peptide, in response to 1 µM DA or 1 µM DA with 1 µM 5-HT. Incubation with D2R TAT-TM5 peptide abolishes the Gi cis- signaling enhancement and the Gi trans-signaling, without affecting D2R homomeric Gi signaling. (Mean ± SEM; NS P>0.05, ** P<0.005, * P<0.05; N=5-7/condition)

72 Figure 3.6 D2R TAT-TM5 disrupts the heteromeric Gq signaling of D2R-2AR in HEK293 cells A. Bar graph summary of Gq activity measured in HEK293 cells expressing 2AR, incubated 4h with medium (vehicle) or 10 µM D2R TAT-TM5 peptide, in response to 0.1 µM 5-HT. B. Bar graph summary of Gq activity measured in HEK293 cells expressing 2AR and D2R, incubated 4 h with medium (vehicle), 10 µM D2R TAT-TM5 peptide, and 10 µM D2R TAT- TM7 peptide, respectively, in response to 10 µM DA. D2R TAT-TM5 peptide significantly reduces the Gq trans-signaling in response to DA (B) without inhibiting 2AR homomeric Gq signaling in response to 5-HT (A). (Mean ± SEM; *** P<0.0005; N=8/condition)

A B

73 Figure 3.7 D2R TAT-TM5 disrupts field Excitatory postsynaptic potential (fEPSP) in PFC neurons (in collaboration with Sidiropoulou lab) A. Representative fEPSP trace in response to no ligand (control), 5-HT, 5-HT and DA, respectively. B. Bar graph summary showing the fEPSP of PFC neurons is significantly increased in response to co-addition of DA and 5-HT, which is reversed by D2R TM5 but not TM7 peptide.

74 Figure 3.8 D2R TAT-TM5 disrupts DA desensitization in VTA neurons (In collaboration with Brodie lab) A. Dopamine administration inhibits the neuron firing rate. Prolonged exposure to DA (25 min wash with DA plus water) reversed the inhibition of neuron firing rate (Dopamine Inhibition Reversal) upon DA administration at 30 min and 45 min. B. The change of neuron firing rate upon DA administration is decreased with time (blue), which is abolished by treatment of D2R TM5 peptide (red).

75 Figure 3.9 A mini gene method to disrupt the D2R-2AR heteromer A. Construct map of the D2R TM5 inserted in pXoom vector, preceded by the first 45 aa of the rat somatostatin receptor 3 (sstr3). B. Co-expression of the D2R TM5 with D2R and 2AR abolished the Gi potentiation in response to DA.

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81 Chapter 4. STRUCTURE MODEL OF D2R-2AR DEMONSTRATING ITS COUPLING MECHANISM

4.1 Background introduction

4.1.1 GPCR homodimer interface

Since 2000 Palczewsk et al. solved the crystal structure of rhodopsin,1 over 200 crystal structures of more than 50 GPCRs have been reported.2 Crystal structures of GPCRs have provided the structural basis and protein folding topology for GPCR high-order oligomerization.

Analysis of crystallography of GPCRs by Katritch et al., Stevens et al., and Shang et al. suggests that all 7 TMs as well as helix H8 could be found in GPCR homodimer interface,3,4,5 among which two types of dimerization interfaces are predominant: one involves TM1, TM2 and H8, the other involves TM4, TM5 and TM6.6 Most frequently homodimerization interfaces are found to be symmetric TM1,2, H8-TM1,2, H8, TM4,5-TM4,5 and TM5,6-TM5,6.7 Meng et al. and

Kaczor et al. used computational approaches to investigate the stability and dynamics of putative

GPCR homodimers inferred by existing GPCR crystal structures.8.9 Their results are consistent with these major types of GPCR homodimer interfaces.

4.1.2 Putative GPCR heteromer interface

Up to date, although 183 class A, B and C GPCRs were found to form heteromers,10 no crystal structure of GPCR heteromers has been solved. To investigate the dimerization interface

82 of GPCR heteromers, multiple methods have been applied, including disruptive peptides, genetic mutations and computational simulations. The results inferred by experimental and computational methods suggest two types of interaction upon GPCR heterodimerization. One is the transmembrane helix-helix interactions, the other is intracellular electrostatic interactions

(Table 4.1).

The helix-helix interfaces of several GPCR heteromers have been determined using a disruptive peptide method, including 5HT2A-CB1, A2A-D2, AT1a-SCT, CRF1–OX1, D1-D3, and

11-17 DOR-MOR (Table 4.1). The dimerization interface of mGlu2-5HT2A was identified by

18 genetic mutation (chimera). A computational model was built for A2A-D2 and mGlu2-5HT2A based on the experimental results (Figure 4.2), and for A1A-A2A, T1R2-T1R3, CXCR4-CCR2 and CXCR4-CCR5 based on MD simulation.13,19-22 These results suggest that TM1, TM4, TM5 and TM6 are hot spots for GPCR heterodimerization (Table 4.1).

In 2005, Woods et al. proposed a role for electrostatic interactions in GPCR heteromerization, in which positively charged residues of one protomer (usually a cluster of

Arginine-rich (R-rich) epitope at IL3) interacts with negatively charged residue(s) of the other

(could be a phosphorylated Ser (pS) or Thr (pT), or two adjacent Glu (EE) or Asp (DD) at the C- terminus or IL3).23 Using mutagenesis methods combined with mass spectrometry and bioluminescence resonance energy transfer (BRET), Ciruela et al., Fernández-Dueñas et al., and

Borroto-Escuela et al. showed that the interaction between positively charged R-rich residues at the IL3 of D2R and the negatively charged pS or DD residues at the C terminus of A2AR was

24-26 necessary to the A2AR-D2R heteromer formation (Figure 4.3). This type of electrostatic interaction has also been found necessary to the heteromerization of A2A-CB1, D1-D2, CB1-D2 and

GRPR-MOR (Table 4.1).23,27-29

83 4.1.3 The R-rich motif at IL3 of D2R is necessary for the co-localization of D2R and 2AR

The heteromeric dimerization interface of D2R-2AR putative heteromer has not been reported. In 2010, using confocal microscopy and mutagenesis methods, Borroto-Escuela et al.

217 222 investigated the role of positively charged R-rich residues ( RRRRKR ) at IL3 of D2R and negatively charged glutamate residues (454EE455) at the C-terminus of 2AR in the formation of

30 heteromeric complex between D2R and 2AR. Their results showed that in cells co-expressing

D2R and 2AR, these two receptors were found to co-localize at the plasma membrane and within the cells, whereas mutating the R-rich residues of D2R to alanine significantly decreased the co- localization between D2Rmut and WT 2AR, indicating that the R-rich motif at IL3 of D2R is necessary to the co-localization and possibly heteromeric formation of D2R and 2AR (Figure

4.4).

4.14 Aims

The bimolecular fluorescence complementation (BiFC) data with peptides from our collaborating Ferré lab indicate TM5 and TM6 of D2R or 2AR are involved in the D2R-2AR heteromeric interface. In this study, we aim to present both functional data and evidence towards the structural basis of the coupling mechanism of the D2R-2AR heteromer. We first aim to identify residues on the heteromeric interface and provide direct evidence that these residues are necessary to the heteromeric signaling of D2R-2AR heteromer. We further aim to build a structural model of D2R-2AR that recapitulates the functional cross-talk between D2R and 2AR and offers insights to the structural coupling mechanism between these two receptors.

84 4.2 Results and discussion

4.2.1 Pi-Pi stacking interaction among conserved aromatic residues 5.47, 5.48, 6.51 and 6.52

To identify the residues involved in helix-helix interaction of D2R-2AR, I aligned the TM5 and TM6 of all dopamine receptors, serotonin type 2 receptors, and five class A GPCR receptors that form heteromers with D2R. The sequence alignment shows that residues at position 5.47,

5.48, 6.51 and 6.52 are conserved aromatic residues, either phenylalanine (F) or tyrosine (Y)

(Figure 4.5A). Crystal structures have been reported for D2R (PDB ID 6C38) and 2CR (or 5-

31,32 HT2CR) (PDB ID 6BQH) in their inactive states. The conformation of the four conserved aromatic residues in the structures of D2R and 2CR indicates edge-to-face Pi-Pi stacking interactions between residues 5.48 and 5.47, 6.52 and 6.51, and face-to-face Pi-Pi stacking interactions between residues 5.47 and 6.52 (Figure 4.5B). Measurement in Pymol shows that the distance between the closest carbon atoms of two adjacent aromatic rings are within the 3.4

Å-4.1Å range (Figure 4.5B), which allows the Pi-Pi stacking interaction to form between two proximal aromatic rings. This conformation information extracted from the crystal structures of

D2R and 2CR suggests a network of Pi-Pi stacking interaction among residues 5.48, 5.47, 6.52 and 6.51; if the conformation of any residue changes, the conformation of the others may change accordingly. Considering 2AR and 2CR are in the same subfamily and have high similarity, presumably it is the same case for residues 5.48, 5.47, 6.52 and 6.51 of 2AR.

4.2.2 The role of residues 6.51 and 6.52 in agonist binding and receptor activation

Wacker et al. solved the crystal structure of 2CR binding to the agonist ergotamine and inverse agonist ritanserin.32 According to their results, the ergoline ring of the ergotamine stacks

85 against and forms Pi-Pi stacking interactions with F6.51 and F6.52 of 2CR (Figure 4.6). Given that dopamine and serotonin share the same backbone with the ergoline core and based on published pharmacology results, F6.51 and F6.52 of D2R and 2AR form Pi-Pi stacking interactions with dopamine and serotonin respectively as well.33-35 Upon agonist binding, the conformation change of F6.51 and F6.52 leads to the conformation changes of nearby P6.50.36

The rotation of the conserved proline further triggers the global rotamer toggle switch, which is the most common mechanism of GPCR activation, with W6.48 (in the CWxxP motif) moving down, and the F6.44 and I3.40 (in the W-I-F motif) switching their upper and lower positions

(Figure 4.6), resulting in the breakage of the ionic lock (between R3.50 and E6.30) and receptor activation.32,36,37

4.2.3 The 5.48 residue is localized at the outer surface of the helical bundle

The crystal structures of D2R and 2CR show that the four conserved aromatic residues are at the same height as the orthosteric ligand-binding sites. Among them, only the residues at positions 5.48 are localized at the outer surface of the helical bundle, the other three residues are all embedded in the binding pocket inside the helical bundle (Figure 4.7). Snapshots of D2R and

2CR crystal structures from a top view indicate that Y5.48 of D2R and F5.48 of 2CR could form face-to-face Pi-Pi stacking, if D2R and 2CR co-localize and interact through the TM5,6-TM5,6 interface, forming a network of Pi-Pi stacking interaction among eight aromatic residues. When agonist binds to one receptor, the conformation of residues 6.51 and 6.52 will change. The network of Pi-Pi stacking interactions connected by residues 5.48 would enable this

86 conformational change to transfer to residues 6.51 and 6.52 of the partner receptor, thus resulting in the activation of the partner receptor.

4.2.4 Hypothesis of the coupling mechanism of D2R-2AR heteromer

Based on the structural conformation of the four conserved aromatic residues and the role of residues 6.51 and 6.52 in agonist binding and receptor activation, I proposed the hypothesis for the coupling mechanism of D2R-2AR putative heteromer: D2R and 2AR heteromerize through symmetric TM5,6-TM5,6 interface with the 5.48 residues forming Pi-Pi stacking interactions. The 5.48 residues would enable the cross-talk between D2R and 2AR by transmitting energy and conformation change from one receptor to the other through the network of Pi-Pi stacking interactions.

5.48 To test this hypothesis, I mutated the 5.48 residues to make D2R(Y199 L) and

2AR(F2445.48L) mutants, and expressed them alone or with the WT partner receptor to assess their role in D2R-2AR functional cross-talk.

5.48 4.2.5 D2R(Y199 L) abolishes the Gi signaling functional cross-talk

5.48 D2R(Y199 L) was expressed alone or with WT 2AR in oocytes (Figure 4.8). The Gi

5.48 activity of D2R(Y199 L) is comparable with that of the WT D2R, indicating this mutant is

5.48 functional. The Gi activity in response to 1 µM DA measured in D2R(Y199 L) and 2AR co- expression is significantly decreased compared to D2R and 2AR co-expression but similar to

5.48 D2R alone, suggesting that D2R(Y199 L) abolished the Gi cis-signaling enhancement shown in

87 the D2R-2AR heteromer. More strikingly, remarkable Gi activity reduction was observed in

5.48 response to 10 µM 5-HT in oocytes co-expressing D2R(Y199 L) and 2AR, indicating the Gi trans-signaling is also abolished. These results suggest that the single mutation of

5.48 D2R(Y199 L) abolishes the Gi signaling functional cross-talk between D2R and 2AR.

4.2.6 2AR(F2445.48L) abolishes the Gi signaling functional cross-talk

5.48 2AR(F244 L) was expressed alone or with WT D2R in HEK293E cells (Figure 4.9).

2AR(F2445.48L) gave comparable Gi activity comparable with WT 2AR, indicating this mutation does not affect the function. The Gq activity in response to 0.1 µM 5-HT obtained in cells co-

5.48 expressing 2AR(F244 L) and D2R is significantly decreased compared with cells co- expressing WT 2AR and D2R but similar with cells expressing 2AR alone, suggesting that

5.48 2AR(F244 L) abolished the Gq cis-signaling enhancement shown in D2R-2AR heteromer.

Remarkable Gq activity reduction was observed in response to 10 µM DA cells co-expressing

5.48 2AR(F244 L) and D2R, indicating the Gq trans-signaling is also abolished. These results suggest that the single mutation of 2AR(F2445.48L) abolishes the Gq signaling functional cross- talk between D2R and 2AR.

4.2.7 Computational model of D2R-2AR

The fluorescence complementation data from Dr. Ferré lab and our mutagenesis data suggest the involvement of TM5 and TM6 in the D2R-2AR heteromeric interface. Based on these results, Dr. Meng Cui in the Logothetis lab built a computational model of the D2R-2AR

88 heteromer recapitulating its functional cross-talk. In 2012, Manglik et al. reported the crystal structure of the Mu opioid receptor (MOR), and demonstrated that MOR homomer dimerizes

38 through symmetric TM5,6-TM5,6 interface. Cui et al. superimposed the structure of D2R and

2AR into the structure of MOR dimer and obtained the model of D2R-2AR heteromer (Figure

4.10). The structures of D2R and 2AR fit well into the structure of MOR dimer without showing collision at TM5 or TM6. Measurement in PyMol shows that the most stable conformation of

D2R(Y1995.48) and 2AR(F2445.48) is observed when their aromatic rings are positioned parallel, with the distance of 4.7 Å between the closest carbon atoms of the two rings. According to the

PDB protein database, the maximal distance for parallel Pi-Pi stacking is 5.99 Å. Thus, in the

D2R-2AR model, the 5.48 residues of D2R and 2AR form face-to-face Pi-Pi stacking interaction.

In the Molecular Dynamic simulation of the D2R-2AR model, the conformation change of the eight conserved aromatic residues of D2R and 2AR are synergized, clearly showing the network of Pi-Pi stacking interaction among these residues. MD simulation is underway to monitor whether a receptor protomer will be activated after adding the agonist of the partner receptor.

4.2.8 The positively charged R-rich motif is necessary to the heteromer formation between dopamine receptor and 2AR

Sequence alignment of mouse Dopamine D2 Receptor (mD2R), human Dopamine D3

Receptor (hD3R) and human Dopamine D4 Receptor (hD4R) shows that hD3R has a R-rich motif at the same position as mD2R (at the beginning of IL3) whereas the R-rich motif of hD4R is removed to a different location (at the C-terminal end of IL3). D3R with 2AR and D4R with 2AR were co-expressed in oocytes respectively to assess whether they can form heteromers by

89 exhibiting functional cross-talk (Figure 4.11). The Gi activity enhancement was observed in oocytes co-expressing D3R and 2AR in response to 1 µM DA and co-addition of 1 µM DA and

1 µM 5-HT, but not in oocytes co-expressing D4R and 2AR, indicating heteromer formation between D3R and 2AR but not D4R and 2AR. Introducing the R-rich motif to the N-terminal end of IL3 at D4R enabled the Gi signaling functional cross-talk between D4R(RKRRKR) and 2AR, indicating the R-rich motif is necessary to the heteromer formation between dopamine receptor and 2AR.

4.2.9 The electrostatic interaction between the R-rich residues of D2R and the EE residues of

2AR is necessary for the D2R-2AR heteromeric signaling

To determine whether the R-rich residues of D2R form electrostatic interaction with the EE

217 222 267 269 residues of 2AR, the RKRRKR and three additional RRR at IL3 of D2R were mutated

454 455 to alanine to make the D2R(9A) mutant. Additionally, the EE at the C-terminus of 2AR were mutated to alanine to make the 2AR(2A) mutant (Figure 4.12). Co-expression of D2R(9A) and 2AR showed no Gi activity enhancement in response to either DA or co-addition of DA and

5-HT; similarly, co-expression of D2R and 2AR(2A) showed no Gq activity enhancement in response to either 5-HT or co-addition of DA and 5-HT, suggesting the functional cross-talk between D2R and 2AR is abolished by either D2R(9A) or 2AR(2A) mutation. These results indicate that likely electrostatic interactions involving the positively charged cluster of R-rich residues at the IL3 of D2R and the negatively charged EE residues at the C terminus of 2AR are critical for D2R-2AR heteromer cross signaling.

90 4.3 Summary

In the experiments summarized in this chapter, we identified residues on TM5 of D2R and 2AR that are necessary to D2R-2AR heteromeric signaling. We showed intracellular residues important for the heteromeric formation of D2R-2AR. We proposed a coupling mechanism for

D2R and 2AR and built a computational model of D2R-2AR heteromer that recapitulates its functional cross-talk. We summarize the results and conclusions as follows:

5.48 5.48 1. D2R(Y199 ) and 2AR(F244 ) are necessary to D2R-2AR heteromeric signaling.

2. D2R and 2AR heteromerize through a symmetric TM5,6-TM5,6 interface.

3. The network of Pi-Pi stacking interactions among residues 5.47, 5.48, 6.51 and 6.52 of

D2R and 2AR may underlie the mechanism for the functional cross-talk between D2R and 2AR.

4. Electrostatic interactions involving the R-rich motif at IL3 of D2R and EE residues at

C-terminus of 2AR are important for D2R-2AR heteromer signaling.

Figure 4.1 Putative GPCR homodimer interfaces inferred by existing crystal structures Dimers are boxed (gray color) according to interface type. Protein name and interface type are reported under each dimeric structure. PDB ID and interface area are included in parenthesis. Adapted from Shang et al., Eur J Pharmacol (2015)

92 Table 4.1 Putative GPCR heterodimer interface inferred by disruptive peptides, genetic mutation, and computational methods P=disruptive peptide M=genetic mutation or chimera C=computational simulation

Transmembrane helix-helix interaction

Complex Helical interface Method Reference 1. Viñals et al., 2014 2. Navarro et al., 2016 5HT -CB TM5,6-TM5,6 P 1 2A 1 3. Borroto-Escuela et al., 2010

A1A-A2A TM4,5-TM4,5 C 2 4. Bonaventura et al., 2015 5. Navarro et al., 2018 A2A-D2 TM4,5-TM4,5 P, C 3, 4, 5 6. Lee et al., 2014 7. Navarro et al., 2015 AT1a-SCT AT1a TM1 P 6 8. Guitart et al., 2014 9. He et al., 2011 CRF1–OX1 OX1 TM1,5 P 7 10. Moreno et al., 2012 D1-D3 D1 TM5,6 P 8 11. Moreno et al., 2016 12. Kim et al., 2017 DOR-MOR MOR TM1 P 9 13. Gahbauer et al., 2018 mGlu -5HT TM4-TM4 M, C 10, 11 14. Ciruela et al., 2004 2 2A 15. Azdad et al., 2009 T1R2-T1R3 TM5,6-TM5,6 C 12 16. Navarro et al., 2010 17. Fernández-Dueñas et al., 2012 CXCR4-CCR2 TM1-TM1 C 13 18. Pei et al., 2010 19. Hasbi et al., 2014 CXCR4-CCR5 TM1-TM4,5 C 13 20. Liu et al., 2011

Intracellular electrostatic interaction

Complex Positively Negative Method Reference

A2A-CB1 A2A IL3 RR CB1 CT pTpS M 14

A2A-D2 D2 IL3 RRRRKR or RRR A2A CT pS or DD M, P 3, 14, 15, 16, 17

D1-D2 D2 IL3 RRRRKR or RRR D1 CT EE P 18, 19

CB1-D2 D2 IL3 RRRRKR CB1 IL3 pTpS M 14

GRPR-MOR1D GRPR IL3 RKR MOR1D CT EE P 20

93 Figure 4.2 Putative heterodimer helical interface of A2AR-D2R and mGlu2-2AR A. Computational model of A2AR-D2R showing A2AR and D2R dimerize through TM4,5-TM4,5 interface, based on results obtained using disruptive peptide (Figure 2.3). Adapted from Navarro et al., Nature Communication (2018) B. Computational model of mGlu2-2AR showing mGlu2 and 2AR dimerize through TM4-TM4 interface, based on Co-IP results obtained with chimera. Adapted from Moreno et al., Science Signaling (2014)

A B

94 Figure 4.3 The electrostatic interaction between the positively charged cluster of R-rich residues at the IL3 of D2R and the negatively charged pS or DD residues at the C terminus of A2AR is necessary to the A2AR-D2R heteromer formation a. Schematic showing the D2R mutation locates at the third intracellular loop 3 (IL3). b. BRET saturation curves of A2ARRluc/D2RYFP (red) and A2ARRluc/ D2RmutYFP (blue) c. Bar graph summary of (b) and negative control. Values represent percentages of maximal saturable BRET responses (BRETmax). *** p < 0.001, +++ p < 0.001. Adapted from Fernández-Dueñas V et al., J Neurochem (2012) d. BRET saturation curves of A2AR and D2R compared to indicated D2R–A2AR mutant heterooligomers. *** P < 0.001, * P < 0.05. Adapted from Borroto-Escuela et al., BBRC (2010) e. Mass spectrometry results showing the formation of peptide-peptide complex. Adapted from Ciruela et al., Analytic Chemistry (2004).

(d)

(e)

95 Figure 4.4 The R-rich motif at IL3 of D2R is necessary to D2R and 2AR co-localization A. Confocal microscopy images of HEK293 cells co-transfected with either 5-HT2A-CFP or 5- HT2AMUT-CFP and either D2-YFP, D2R1-YFP, D2R2-YFP, D2R3-YFP or 5-HT2A-YFP (green and red). B. Bar graph of Pearson's correlation coefficient calculated for HEK293 cells co-transfected with indicated serotonin 5-HT2A and dopamine D2 receptor construct combination. *p<0.05 Adapted from Borroto-Escuela et al., BBA (2010)

D2R1: RRRRKRàAARRKR D2R2: RRRRKRàAAAAKR D2R3: RRRRKRàAAAAAA 5-HT2A MUT: EEàAA

96 Figure 4.5 Pi-Pi stacking interaction among conserved aromatic residues 5.47, 5.48, 6.51 and 6.52 A. and B. TM5 and TM6 alignment of dopamine receptors, serotonin type 2 receptors and five receptors that form heteromer with D2R. The 5.47, 5.48, 6.51 and 6.52 residues are conserved aromatic residues (highlighted in pink). The residue positions are numbered according to the Ballesteros-Weinstein numbering scheme. C. and D. Crystal structure of D2R and 2CR showing the four conserved aromatic residues (shown as sticks) form either edge-to-face or face-to-face Pi-Pi stacking interaction (helical structure of the receptor is hidden for better visibility of the aromatic residues). The distance between the closest carbon atoms of two adjacent aromatic rings are shown and measured in PyMOL. PDB ID is included in parenthesis.

A B

C D 5.48 5.47 6.52 6.51 5.48 5.47 6.52 6.51 Y ---F ---F ---F F ---F ---F ---F

D R (6C38) 2CR (6BQH) 2

97 Figure 4.6 The role of residues 6.51 and 6.52 in agonist binding and receptor activation A. The ergoline core of ergotamine in the structures of 5-HT2C (orange), 5-HT1B (light blue; PDB: 4IAR), and 5-HT2B (yellow; PDB: 4IB4). Key residues that recognize and interact with the ergoline core are labeled and shown as sticks. Residues 6.51 and 6.51 (circled in red) form Pi-Pi stacking interaction with the ergoline ring. B. The conformational changes of F3376.51 and F3386.52 upon agonist binding potentiates the conformational changes of W3246.48, I1423.40 and F3206.44 in the W-I-F motif (rotamer toggle switch), leading to the opening of ionic lock and receptor activation. Adapted from Peng et al., Cell (2018) C. Chemical structures of ergotamine, DA and 5-HT showing DA and 5-HT have the same backbones as ergotamine (highlighted in blue). Adapted from Wacker et al., Cell (2017)

A B

98 Figure 4.7 The 5.48 residues are localized at the outer surface of the helical bundle A. and B. Side view of D2R (A) and 2CR (B). The 5.47, 6.51 and 6.52 residues are embedded in the binding pocket in the helical bundle. 5.48 residue (labelled) locates at the out surface of the helical bundle and points toward TM6. C. Top view of D2R and 2CR (horizontally flipped). The 5.48 residues of D2R and 2CR could form face-to-face Pi-Pi stacking if D2R and 2CR co-localize and interact through the TM5-TM6 interface, forming a network of Pi-Pi stacking interaction among eight aromatic residues. The four conserved aromatic residues are shown as sticks. Structure of receptor is shown as cartoon. PDB ID is included in parenthesis.

5.48 5.47 6.52 6.51 5.48 5.47 6.52 6.51 A Y ---F ---F ---F B F ---F ---F ---F

5.48 5.48 Y F

D R (6C38) 2 2CR (6BQH)

5.48 Y

5.48 F

99 5.48 Figure 4.8 D2R(Y199 L) abolishes the Gi signaling functional cross-talk Bar graph summary of Gi activity showing co-expression of D2R(Y199L) with 2AR abolishes the Gi cis-signaling potentiation in response to 1 µM DA and Gi trans-signaling in response to 10 µM 5-HT which is observed in co-expression of WT D2R with 2AR, while the Gi activity of D2R(Y199L) is comparable to WT D2R. (Mean ± SEM; ** P<0.005; N=6-7/condition)

100 Figure 4.9 2AR(F2445.48L) abolishes the Gq signaling functional cross-talk Bar graph summary of Gq activity showing co-expression of 2AR(F244L) with D2R abolishes the Gq cis-signaling potentiation in response to 0.1 µM 5-HT and Gq trans-signaling in response to 10 µM DA which is shown when WT 2AR is co-expressed with D2R, while 2AR(F244L) gave comparable response to 5-HT as WT 2AR. (Mean ± SEM; NS P>0.05, *** P<0.0005; N=8- 16/condition)

101 Figure 4.10 The TM5,6-TM5,6 dimerization interface of Mu-opioid receptor a. Schematic structure of the TM5,6–TM5,6 four-helix bundle interface. b. The tight association between the ligand (green sticks) in the bind pocket and residues that are involved in forming the dimeric interface (blue spheres) in the four-helix bundle. c. The interacting residues in the four-helix bundle (shown as sticks). d. The high surface complementarity within the four-helix bundle interface along the dimer interface (as indicated in panel c). Adapted from Manglik et al., Nature (2012)

102 Figure 4.11 Computational model of D2R-2AR recapitulating its functional cross-talk A. Crystal structure of MOR dimer. PDB ID 4DKL B. and C. Side view (B) and top view (C) of the D2R-2AR MD simulation model. The structure of D2R and 2AR is superimposed into the structure of MOR dimer. D2R and 2AR dimerize through the TM5-TM6 interface. The distance between the closest carbon atoms of the 5.48 residues is 4.7A° (at the beginning stage of simulation, measured in PyMOL).

A B

MOR dimer D2R-2AR

TM6

TM5

D2R 2AR

TM5 TM6

103 Figure 4.12 The positively charged R-rich motif is necessary to DxR-2AR heteromer formation Bar graph summary of Gi activity showing functional cross-talk between D3R (with R-rich motif at the same position as D2R) and 2AR (A), and introducing the R-rich motif to the corresponding position of D4R enables the functional cross-talk between D4R and 2AR (B and C). (Mean ± SEM; NS P>0.05, * P<0.05, ** P<0.005, *** P<0.0005; N=5-12/condition)

A B C

104 Figure 4.13 Electrostatic interactions involving the positively charged cluster of R-rich residues at the IL3 of D2R and the negatively charged EE residues at the C terminus of 2AR are critical for D2R-2AR heteromeric signaling Bar graph summary of Gi and Gq activity showing co-expression of D2R(9A) with 2AR, or D2R with 2AR(2A) abolished the Gi and Gq functional cross-talk observed in co-expression of WT D2R and 2AR. The 9A mutation locates at IL3 of D2R; The 2A mutation locates at C terminus of 2AR. (Mean ± SEM; NS P>0.05, * P<0.05, ** P<0.005, *** P<0.0005; N=5-12/condition)

D R+2AR D2R(9A)+2AR 2

X X X X

D R+2AR(2A) D2R+2AR 2

D2R(9A): RKRRKRàAAAAAA (ICL3) + RRRàAAA (ICL3); 2AR(2A): EEàAA (C-tail)

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29. Liu, X. Y., Liu, Z. C., Sun, Y. G., Ross, M., Kim, S., Tsai, F. F., ... & Chen, Z. F. (2011). Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids. Cell, 147(2), 447-458.

30. Łukasiewicz, S., Polit, A., Kędracka-Krok, S., Wędzony, K., Maćkowiak, M., & Dziedzicka- Wasylewska, M. (2010). Hetero-dimerization of serotonin 5-HT2A and dopamine D2 receptors. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1803(12), 1347-1358.

31. Wang, S., Che, T., Levit, A., Shoichet, B. K., Wacker, D., & Roth, B. L. (2018). Structure of the D2 dopamine receptor bound to the atypical antipsychotic drug risperidone. Nature, 555(7695), 269.

32. Peng, Y., McCorvy, J. D., Harpsøe, K., Lansu, K., Yuan, S., Popov, P., ... & Huang, X. P. (2018). 5-HT2C receptor structures reveal the structural basis of GPCR polypharmacology. Cell, 172(4), 719-730.

33. Cho, W., Taylor, L. P., Mansour, A., & Akil, H. (1995). Hydrophobic residues of the D2 dopamine receptor are important for binding and signal transduction. Journal of neurochemistry, 65(5), 2105-2115.

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108 ergolines require an aromatic residue at position 340 for high affinity binding. Molecular pharmacology, 47(3), 450-457.

35. Shi, L., & Javitch, J. A. (2002). The binding site of aminergic G protein–coupled receptors: the transmembrane segments and second extracellular loop. Annual review of pharmacology and toxicology, 42(1), 437-467.

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109 Chapter 5. EFFECTIVENESS OF ANTIPSYCHOTIC DRUGS ON THE FUNCTION OF THE

D2R-2AR HETEROMER

5.1 Background introduction

5.1.1 2AR is necessary to haloperidol’s full antipsychotic effectiveness

In an animal behavioral study, Albizu et al. used MK-801, an NMDA receptor antagonist to induce psychotic behavior in mice (Figure 1.6).1 The locomotor activity of mice was significantly increased following MK-801 injection, an effect that was reversed by administration of typical antipsychotic haloperidol (HLP). However, the effect of HLP to reduce

MK-801 induced psychotic behavior was significantly attenuated in 2AR KO mice. It is known

2 that HLP’s antipsychotic action is through D2R antagonism and it has low affinity to 2AR. The fact that HLP could still antagonize D2R in 2AR KO mice but is less efficacious suggests that

2AR is necessary for HLP to exert its full antipsychotic effect.

5.1.2 Combination of antipsychotic drugs (APDs) exerts enhanced antipsychotic effect and reduced side effect

Combinations of typical and atypical APDs are regularly prescribed for treatment of schizophrenia.3 The clinical benefits include: improvement in positive and negative symptoms, decreased total doses of medications, reduced extra-pyramidal side effects, weight gain and hyperprolactinemia.4

In a meta-analysis, Correl et al. (2008) evaluated antipsychotic combinations versus monotherapy in schizophrenia.5 11 Combination antipsychotics, including the use of 2 typical

APDs, 2 atypical APDs, or 1 of each, were found to be more efficacious in producing a clinically

110 significant response than monotherapy. Among them combination of a typical and an atypical

APD was found to be especially efficacious.

Pimavanserin, an atypical APD with high affinity for 2AR and no affinity for D2R was found to reduce psychotic behaviors (head twitches, hallucinations and delusions) and has completed Phase II trials for treatment of schizophrenia.6-11 In experimental model studies, combining high levels of PIMA and low levels of HLP or risperidone improved the antipsychotic efficacy and side effect profile of HLP and risperidone.12 In a large randomized controlled study of 423 patients, combining PIMA with low dose of risperidone or HLP augmented the efficacy of risperidone to a standard dose and reduced the side effects of risperidone and HLP.13

5.1.3 Typical and atypical APDs potentiate the heteromerization of D2R-2AR

14,15 Lukasiewicz et al. assessed the role of APDs in D2R-2AR heteromerization. They applied the typical APD butaclamol and the atypical APD ketanserin to HEK cells co-expressing

D2R and 2AR, and detected the heteromerization of D2R-2AR by FRET assay. Fluorescence signal detected from co-expression of D2R and 2AR was significantly increased with addition of butaclamol and ketanserin, indicating these APDs potentiate the heteromerization of D2R-2AR.

Interestingly, Lukasiewicz et al. also showed the typical APD haloperidol and the atypical

APD ketanserin both decreased the homodimerization of 2AR.15 Although it has not been studied whether haloperidol or ketanserin decreases the homodimerization of D2R, Marsango et al. found that the typical APDs haloperidol and spiperone increased the distance between carbon atoms at

16 D3R homomeric interface thus disrupted the homodimerization of D3R. These results indicate a potential feature of typical and atypical APDs in potentiating the heterodimerization of D2R-2AR and disrupting the homodimerization of D2R and 2AR.

111 5.1.4 Aims

In this study, we aim to investigate the effectiveness of antipsychotic drugs on the function of the D2R-2AR heteromer. We will compare the action of typical and atypical APDs on the D2R and 2AR homomers versus D2R-2AR heteromer, explore whether the effectiveness of APDs on

D2R-2AR heteromer is dependent on the cross-talk between D2R and 2AR, and assess whether a combination of typical and atypical APDs on the D2R-2AR heteromer potentiates their effectiveness.

5.2 Results and discussion

5.2.1 The potency of HLP antagonizing DA is enhanced by the D2R-2AR heteromer

To assess whether the D2R-2AR heteromer plays a role in the action of typical antipsychotic drug, six doses of HLP were applied with 1 µM DA to oocytes expressing D2R alone or D2R with 2AR (Figure 5.1). Previous results showed that the potency of DA is similar on the D2R homomer and the D2R-2AR heteromer (Figure 2.4). Here, the dose-response curve of D2R+2AR is significantly left shifted compared with D2R, indicating HLP is more potent in antagonizing DA acting at the D2R-2AR hoteromer than the D2R homomer. The IC50 values showed that the potency of HLP antagonizing DA acting at D2R+2AR was 11-fold greater than that on D2R. At 0.01 nM, HLP’s antagonism on D2R and D2R+2AR was negligible, thus the DA induced Gi signaling of D2R+2AR was two-fold of D2R. At 1 nM, HLP blocked half of the Gi signaling of D2R+2AR, whereas it showed little antagonism on D2R, resulting in similar Gi signaling in response to DA. Between the 1 nM to 1 µM range, with HLP antagonizing DA, the

Gi signaling of D2R+2AR was significantly lower than D2R. The negative values of Gi activity in response to high doses of HLP exhibits HLP’s function as inverse agonist.

112 The results that HLP is more potent in antagonizing DA acting at the D2R-2AR heteromer could lead to a greater relative potency of HLP in vivo, which may explain the behavioral results of Albizu et al. that 2AR is necessary for HLP’s full antipsychotic effect. A rational reasoning of these results is that HLP has higher binding affinity for the D2R-2AR heteromer than the D2R homomer, thus a lower dosage of HLP is needed to achieve the antipsychotic effect through the function of the D2R-2AR heteromer.

5.2.2 The enhanced potency of HLP antagonizing DA depends on the cross-talk between D2R and 2AR through the 5.48 residue

Because HLP is more potent in antagonizing DA acting at the D2R-2AR heteromer, the

Gi signaling of D2R+2AR in response to 100nM HLP+1uM DA is markedly lower than D2R. To assess whether the enhanced potency of HLP is dependent on the cross-talk between D2R and

2AR, 100nM HLP+1uM DA was applied to oocytes co-expressing D2R(Y199L) and 2AR

(Figure 5.2). The results show the Gi signaling of D2R(Y199L)+2AR is significantly higher than

D2R+2AR and relatively close to D2R, suggesting that the enhanced potency of HLP antagonizing DA is attenuated when the cross-talk between D2R and 2AR is abolished. Control experiments with D2R(Y199L) homomer and D2R+2AR(F244L) are pending.

5.2.3 The potency of HLP antagonizing Gq signaling is enhanced in the D2R-2AR heteromer

In Figure 2.11B we showed HLP decreased Gq signaling of D2R+2AR. We further assess whether HLP antagonizes 5-HT acting on the 2AR homomer by applying HLP doses with

0.1 µM 5-HT to HEK cells expressing 2AR alone (Figure 5.3). The results show HLP decreases

113 Gq signaling of 2AR in a dose-dependent manner, but the potency is halved compared with

D2R+2AR, as the IC50 values indicate. At 1 mM, the highest soluble concentration, HLP blocked almost 90% of the Gq signaling of D2R+2AR whereas it only reduced 60% of the Gq signaling of 2AR homomer. These results suggest HLP above micromolar concentrations acts as an antagonist to both 2AR homomer and D2R-2AR heteromer, and it is more potent in antagonizing the heteromer. Given the fact that HLP has markedly higher affinity to D2R than 2AR, it is possible that HLP decreases the Gq signaling of D2R-2AR through antagonism on D2R rather than compete binding with 5-HT.

5.2.4 The enhanced potency of HLP antagonizing Gq signaling depends on the cross-talk between D2R and 2AR through the 5.48 residue

Because HLP is more potent in antagonizing D2R-2AR heteromer, the Gq signaling of

D2R+2AR in response to 100 µM HLP+0.1 µM 5-HT is significantly lower than 2AR. To assess whether the enhanced potency of HLP antagonizing Gq signaing is dependent on the cross-talk between D2R and 2AR, 100 µM HLP+0.1 µM 5-HT was applied to HEK cells co-expression

D2R and 2AR(F244L) (Figure 5.4). The results show Gq signaling of D2R+2AR(F244L) is significantly higher than D2R+2AR but comparable to 2AR, suggesting the enhanced potency of

HLP antagonizing Gq signaling is reversed when the cross-talk between D2R and 2AR is abolished. Control experiments with 2AR(F244L) homomer and D2R(Y199L)+2AR are pending.

5.2.5 Pimavanserin cross-talks to antagonize D2R

Previous work by Younkin et al. showed PIMA antagonized the Gq signaling of 2AR homomer and had no effect on antagonizing the Gi signaling of D2R homomer (Figure 5.5A). In

114 oocytes co-expressing D2R and 2AR, 5 µM PIMA reversed the Gi signaling enhancement in response to 1 µM DA (Figure 5.5B). Considering that typical and atypical APDs such as butaclamol and ketanserin potentiate the formation of D2R-2AR heteromer, we anticipate that

PIMA also potentiates the D2R-2AR heteromer rather than disrupting the heteromer. Thus, we conclude from these results that the reversed Gi signaling of D2R+2AR is due to PIMA cross- talks to antagonize D2R. We will further perform full-dose responses of PIMA antagonizing the

Gi signaling of D2R+2AR.

To assess whether PIMA’s antagonism on D2R is dependent on the cross-talk between D2R and 2AR, we will apply 5 µM PIMA with 1 µM DA to oocytes expressing D2R(Y199L) and

D2R(Y199L)+2AR. Furthermore, we will assess whether PIMA is more potent in antagonizing

D2R-2AR heteromer than 2AR homomer by applying PIMA doses with 0.1 µM 5-HT in HEK cells expressing 2AR and 2AR+D2R.

The results showing PIMA cross-talks to antagonize D2R (Figure 5.5) and HLP cross-talks to antagonize 2AR (Figure 5.3) bring up the possibility that the effectiveness of these two APDs may be altered or potentiated upon co-administration. Clinical use of antipsychotic combinations also suggests augmentation and possible interplay of APDs. It would be interesting to know whether PIMA would increase the potency of HLP antagonizing the D2R-2AR heteromer and vice versa. To meet this aim, we will consider concurrently applying low concentrations of

PIMA with HLP doses and DA to oocytes co-expressing D2R and 2AR and low concentration of

HLP with PIMA doses and 5-HT to HEK cells co-expressing D2R and 2AR, to assess the effect of combining typical and atypical APDs on the D2R-2AR heteromer.

115 5.3 Summary

In the experiments discussed in the chapter, we obtained dose-response curves of HLP acting on the D2R homomer, the 2AR homomer, and the co-expression of D2R and 2AR. We assessed the dependency of HLP’s effectiveness on the cross-talk between D2R and 2AR. We showed PIMA’s antagonism on the Gi signaling of D2R-2AR. We summarize the results and conclusions as follows:

1. HLP is more potent in antagonizing DA acting on the D2R-2AR heteromer than the D2R homomer.

2. HLP’s enhanced antagonism on Gi signaling is dependent on the cross-talk between D2R and 2AR.

3. HLP is more potent in antagonizing the Gq signaling of D2R-2AR heteromer than 2AR homomer.

4. HLP’s enhanced antagonism on Gq signaling is dependent on the cross-talk between

D2R and 2AR.

5. PIMA cross-talks to antagonize the Gi signaling of D2R-2AR.

116 Figure 5.1 The potency of HLP antagonizing DA is increased on the D2R-2AR heteromer A. Representative barium-sensitive traces of GIRK4* currents obtained in response to addition of 10 nM HLP (5 min or till current stabilizes), and 1 µM DA in oocytes expressing D2R alone (left panel), D2R with 2AR (right panel). (orange: outward current; blue: inward current) B. Dose-response curves showing the potency of HLP antagonizing DA at oocytes co-expressing D2R and 2AR (yellow) is increased by 11-fold compared with oocytes expressing D2R alone (black). Gi activity (GIRK4* current activation) was measured relative to the basal currents and was normalized relative to the Gi activity of D2R in response to 1 µM DA in the absence of HLP (100% or 1). RGS2 was co-injected to inhibit Gq signaling. (Mean ± SEM; N=6-12/condition)

117 Figure 5.2 The increased potency of HLP antagonizing DA depends on the cross-talk between D2R and 2AR through 5.48 residue A. Representative barium-sensitive traces of GIRK4* currents obtained in response to 10 nM HLP, and 1 µM DA in oocytes expressing D2R(Y199L) with 2AR. (orange: outward current; blue: inward current) B. Bar graph summary showing the Gi activity obtained in response to 1 µM DA after addition of 10 nM HLP in oocytes expressing D2R alone, D2R with 2AR, and D2R(Y199L) with 2AR, respectively. Gi activity (GIRK4* current activation) was measured and normalized as Figure 5.1. RGS2 was co-injected to inhibit Gq signaling. (Mean ± SEM; * P<0.05, **** P<0.0001; N=9-15/condition)

118 Figure 5.3 The potency of HLP antagonizing Gq signaling is increased on the D2R-2AR heteromer Dose-response curves showing the potency of HLP antagonizing 5-HT at HEK293E cells co- expressing D2R and 2AR (red) is enhanced compared with cells expressing 2AR alone (black). Gq activity was measured relative to the basal fluorescence and was normalized relative to the Gq activity of 2AR in response to 0.1 µM 5-HT in the absence of HLP (100). (Mean ± SEM; N=4-9/condition)

119 Figure 5.4 The increased potency of HLP antagonizing Gq signaling depends on the cross- talk between D2R and 2AR through 5.48 residue Bar graph summary showing the Gq activity obtained in response to co-addition of 0.1 µM 5-HT and 100 µM HLP in HEK293E cells expressing 2AR alone, D2R with 2AR, and D2R(Y199L) with 2AR, respectively. Gq activity was measured and normalized as Figure 5.3. (Mean ± SEM; ** P<0.005, **** P<0.0001; N=4-14/condition)

120 Figure 5.5 Pimavanserin cross-talks to antagonize D2R A. Gi signaling (yellow) in response to co-addition of PIMA doses with 1 µM DA in oocytes expressing D2R alone and Gq signaling (red) in response to co-addtion of PIMA doses with 1uM 5-HT in oocytes expressing 2AR alone. Dash line represents Gi and Gq activity in response to 1 µM DA or 5-HT. B. Bar graph summary showing 5 µM PIMA reversed the Gi signaling enhancement in response to 1 µM DA in oocytes co-expressing D2R and 2AR whereas the D2R homomeric Gi signaling is not affected.

A B

121 5.4 References

1. Albizu, L., Holloway, T., González-Maeso, J., & Sealfon, S. C. (2011). Functional crosstalk and heteromerization of serotonin 5-HT2A and dopamine D2 receptors. Neuropharmacology, 61(4), 770-777.

2. Kroeze, W. K., Hufeisen, S. J., Popadak, B. A., Renock, S. M., Steinberg, S., Ernsberger, P., ... & Roth, B. L. (2003). H1-histamine receptor affinity predicts short-term weight gain for typical and atypical antipsychotic drugs. Neuropsychopharmacology, 28(3), 519.

3. Maayan, N., Soares-Weiser, K., Xia, J., & Adams, C. E. (2011). Antipsychotic combinations for schizophrenia. The Cochrane database of systematic reviews, (2).

4. Abbas, A., & Roth, B. L. (2008). Pimavanserin tartrate: a 5-HT2A inverse agonist with potential for treating various neuropsychiatric disorders. Expert opinion on pharmacotherapy, 9(18), 3251-3259.

5. Correll, C. U., Rummel-Kluge, C., Corves, C., Kane, J. M., & Leucht, S. (2008). Antipsychotic combinations vs monotherapy in schizophrenia: a meta-analysis of randomized controlled trials. Schizophrenia bulletin, 35(2), 443-457.

6. Abbas, A., & Roth, B. L. (2008). Pimavanserin tartrate: a 5-HT2A inverse agonist with potential for treating various neuropsychiatric disorders. Expert opinion on pharmacotherapy, 9(18), 3251-3259.

7. McFarland, K., Price, D. L., & Bonhaus, D. W. (2011). Pimavanserin, a 5-HT2A inverse agonist, reverses psychosis-like behaviors in a rodent model of Parkinson's disease. Behavioural pharmacology, 22(7), 681-692.

8. Press Announcements — FDA approves first drug to treat hallucinations and delusions associated with Parkinson's disease". U.S. Food and Drug Administration. Retrieved 1 May 2016.

9. Cruz, M. P. (2017). Pimavanserin (Nuplazid): A Treatment for Hallucinations and Delusions Associated With Parkinson’s Disease. Pharmacy and Therapeutics, 42(6), 368.

10. Ebdrup, B. H., Rasmussen, H., Arnt, J., & Glenthøj, B. (2011). Serotonin 2A receptor antagonists for treatment of schizophrenia. Expert opinion on investigational drugs, 20(9), 1211- 1223.

11. ACADIA Announces Positive Results From ACP-103 Phase II Schizophrenia Co-Therapy Trial"(Press release). ACADIA Pharmaceuticals. 2007-03-19. Retrieved 2009-04-11.

12. Gardell, L. R., Vanover, K. E., Pounds, L., Johnson, R. W., Barido, R., Anderson, G. T., ... & Davis, R. E. (2007). ACP-103, a 5-hydroxytryptamine 2A receptor inverse agonist, improves the

122 antipsychotic efficacy and side-effect profile of haloperidol and risperidone in experimental models. Journal of Pharmacology and Experimental Therapeutics, 322(2), 862-870.

13. Meltzer, H. Y., Elkis, H., Vanover, K., Weiner, D. M., van Kammen, D. P., Peters, P., & Hacksell, U. (2012). Pimavanserin, a selective serotonin (5-HT) 2A-inverse agonist, enhances the efficacy and safety of risperidone, 2 mg/day, but does not enhance efficacy of haloperidol, 2 mg/day: comparison with reference dose risperidone, 6 mg/day. Schizophrenia research, 141(2- 3), 144-152.

14. Łukasiewicz, S., Polit, A., Kędracka-Krok, S., Wędzony, K., Maćkowiak, M., & Dziedzicka- Wasylewska, M. (2010). Hetero-dimerization of serotonin 5-HT2A and dopamine D2 receptors. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1803(12), 1347-1358.

15. Łukasiewicz, S., Faron-Górecka, A., Kędracka-Krok, S., & Dziedzicka-Wasylewska, M. (2011). Effect of clozapine on the dimerization of serotonin 5-HT2A receptor and its genetic variant 5-HT2AH425Y with dopamine D2 receptor. European journal of pharmacology, 659(2- 3), 114-123.

16. Marsango, S., Caltabiano, G., Jiménez-Rosés, M., Millan, M. J., Pediani, J. D., Ward, R. J., & Milligan, G. (2017). A molecular basis for selective antagonist destabilization of dopamine D 3 receptor quaternary organization. Scientific reports, 7(1), 2134.

123 Chapter 6. DISCUSSION AND CONCLUDING REMARKS

6.1 Is D2R-2AR officially a heteromer?

Over the last decade, many GPCR receptors have been reported to form receptor heteromers.1-4 Ferré at al. defined GPCR heteromer as a “macromolecular complex composed of at least two (functional) receptor units with biochemical properties that are demonstrably different from those of its individual components”.5 To fulfil this definition, evidence is needed that the receptors exist as a macromolecular complex and possess unique properties compared to the protomers. Many studies have assessed the formation of GPCR heteromers using recombinant receptors expressed in heterologous systems; however, only a few have demonstrated novel biochemical or physiological properties of these complexes in native tissues.

In an annual review, Gomes et al. proposed the criteria to establish GPCR heteromers: “Criterion

1: Heteromer components should co-localize and physically interact. Criterion 2: Heteromers should exhibit properties distinct from those of the protomers. Criterion 3: Heteromer disruption should lead to a loss of heteromer-specific properties.” (Figure 6.1 A)6 Reports on DOR-MOR,

7-12 13-21 22-26 27 28,29 A2AR-D2R, D1R-D2R, α1A/BAR-CXCR4, and M1-M2 have fulfilled all three criteria to establish these heteromers in native tissue.

Our studies on D2R-2AR have fulfilled all three criteria to establish the D2R-2AR heteromers in heterologous systems. The bimolecular fluorescence complementation (BiFC) data from our collaborators (Ferré et al.) showed heteromerization of D2R-2AR in HEK293 cells. Our

124 oocyte and calcium mobilization data demonstrated unique pharmacological properties of D2R-

2AR distinct from each protomer. Applying disruptive peptides and genetic mutation methods, we showed disruption of D2R-2AR heteromeric complex and removal of D2R-2AR heteromeric signaling.

Published reports 30 and studies from our collaborators (Sidiropoulou and Brodie labs) have fulfilled criterion 1 and 2 to establish D2R-2AR heteromer in native tissues. Using in situ

30 proximity ligation assay, Borroto-Escuela et al. revealed the existence of an endogenous D2R-

2AR complex in rat striatum. Brain slice data from our collaborating Sidiropoulou and Brodie labs exhibited the function of D2R-2AR in controlling the electrophysiological behavior of PFC and VTA neurons, respectively. Regarding the third criterion, our collaborating Gonzalez-Maeso lab is applying a disruptive peptide in an animal behavioral study to explore the effectiveness of antipsychotic drugs on the function of the putative native D2R-2AR heteromer. Thus, fulfilling all three criteria to establish the existence of the D2R-2AR heteromer in native tissue is pending on the animal behavioral results (Figure 6.1 B).

125 6.2 A common coupling mechanism for aminergic GPCR heteromers?

The studies in Chapter 4 suggest that a network of Pi-Pi stacking interaction among residues 5.47, 5.48, 6.51 and 6.52 may underlie the coupling mechanism for the D2R-2AR heteromer. Alignment of all aminergic receptors reveals high conservation in residues 5.47,

5.48, 6.51 and 6.52 (Figure 6.2). 31 out 44 receptors possess aromatic residues at these positions, including all dopaminergic receptors, serotoninergic receptors, adrenergic receptors, 2 histaminergic receptors and 2 trace amine receptors. The high conservation of aromatic residues in aminergic receptors raises the question: could the coupling mechanism of the D2R-2AR heteromer be a common coupling mechanism for aminergic GPCR heteromers?

My speculation is based on the following reasons. TM5 and TM6 of aminergic GPCRs undergo significant conformational changes upon receptor activation and are key components for aminergic GPCRs activation.31-33 The residues 6.51 and 6.52 have been found to play important roles in agonist binding and receptor activation not only for dopaminergic and serotoninergic receptors but for all aminergic receptors.31-33 Among the 33 receptors with four conserved

34 35 36 aromatic residues, 9 have been reported with solved crystal structure, including D2, D3, D4,

37 38-42 43 44-52 53-67 68 5HT1B, 5HT2B, 5HT2C, β1A, β2B, and H1. Examination of the crystal structures of these receptors reveals the formation of Pi-Pi stacking interaction among the residues 5.47, 5,48,

6,51 and 6.52 of all 9 receptors in either active or inactive states, indicating that this Pi-Pi stacking in the cluster of aromatic residues is a common feature for aminergic GPCRs.

Aminergic GPCRs are of critical importance as they are associated with various diseases and are the targets for 25% of current drugs.69 Many aminergic GPCR heteromers have been

70-74 75-76 77- found to play an important role in psychiatric disorders, including D1-D2, D1-D3, D2-D3,

126 79 80 81,82 83 84 84 D2s-D4, D1-H3, D2-H3, α1B-D4, , and β1A-D4 . Determining the coupling mechanism of these heteromers and other potential aminergic GPCR heteromers is of great significance:

1. It will help to understand and predict the integrated signaling of aminergic GPCR heteromers.

2. It will motivate mutation at residues 5.48 as potential negative controls to assess the pharmacological and biochemical properties of aminergic GPCR heteromers.

3. It will provide TM5 and TM6 as potential targets for the design of disruptive peptides to manipulate the formation and assess the physiological properties of aminergic GPCR heteromers.

4. It will facilitate the design of heteromer-selective drugs specifically targeting the heteromeric interface of aminergic GPCR heteromers, therefore eliminating the side-effects of antipsychotic drugs, which in many cases are due to promiscuous interactions with proteins other than the intended target(s).86-90

127 6.3 Unifying model of D2R-2AR’s dual role in psychosis

Psychosis associated with schizophrenia shows alterations in the level of Gi signaling and

91 Gq signaling. Postmortem studies have shown up-regulated D2R in the striatum and 2AR in cortical regions, leading to potentiation of both Gi and Gq signaling in psychosis compared to the physiological state.91-97

Our studies exhibit a dual role of the D2R-2AR heteromer in psychosis (Figure 6.3). Both

Gi and Gq signaling are potentiated due to the functional cross-talk of D2R-2AR, thus shifting the balance from the physiological state to a psychotic state. In this sense, the D2R-2AR heteromer could be considered as a “bad” heteromer, as it promotes psychosis. On the other hand, antagonists of either D2R or 2AR could inhibit both receptors through the cross-talk between D2R and 2AR, and the effectiveness of both D2R and 2AR antagonists (typical and atypical antipsychotic drugs) is potentiated through the function of the D2R-2AR heteromer, thus reversing both Gi and Gq signaling potentiation and shifting the balance from a psychotic state to the physiological state. In this sense, the D2R-2AR heteromer could be considered as a “good” heteromer, as it would enhance the antipsychotic effect of APDs and reduce psychosis.

128 6.4 Putative bivalent ligands of the D2R-2AR heteromer

Since 1986 with the first report of a bivalent ligand targeting opioid receptor dimers, the bivalent ligand approach has received increasing attention as new methods for validation of physical interactions and novel pharmacological and physiological properties of GPCR heterodimers.98,99 Moreover, the bivalent ligand approach has been widely used in therapeutic development for GPCR heteromers associated with drug tolerance and psychiatric diseases.99

A number of bivalent ligands have been designed for GPCR heteromers, including µOR-

100,101 102 103 104 105 106 δOR, µOR-CB1, µOR-mGlu5, µOR-CCR5, δOR-κOR, µOR-NK1, MT1-

107 108 109 MT2, A2AR-D2R, and D2R-NTS1R.

Bivalent ligands are defined as compounds that contain two pharmacophore units linked by a spacer (Figure 6.4 A and B). The two pharmacophores can be either identical

(homobivalent) or different (heterobivalent). The length of the spacer is adjustable to achieve optimal pharmacological properties. Bivalent ligands are designed to span across homo- or heterodimeric GPCRs and target the two receptor protomers simultaneously. An advantage of bivalent ligands is the increase in affinity (relative to the individual binding of each pharmacophore unit), which is an inherent thermodynamic feature of a cooperative binding system.98 In this way, the binding of the first pharmacophore facilitates the binding of the second pharmacophore by having it localized in the vicinity of its respective binding pocket, thereby significantly lowing the overall entropy of the ligand-receptor complex.98

Significant pharmacological and therapeutic advantages have been reported for administration of bivalent ligands targeting µOR-δOR, µOR-CB1, and µOR-mGlu5, including enhanced antinociception compared to morphine and reduced side effects such as tolerance, dependence and respiratory depression.101-103 Bivalent ligands simultaneously antagonizing both

129 receptor protomers in GPCR heteromers have been found to be more efficacious on targeting

µOR-CCR5 and µOR-κOR.104,105 Interestingly, two bivalent ligands MMG22 and MCC22, have been found to induce co-localized receptors (µOR, mGlu5 and CCR5) to form heteromers (µOR- mGlu5 and µOR-CCR5 respectively) that do not occur naturally.110 Overall, the use of bivalent ligands has exhibited great potential in optimizing and manipulating the desired pharmacological and therapeutic effects.

Homobivalent ligands have been reported for both the D2R homomer and the 2AR homomer. Novel bivalent ligands of the D2R homomer exhibit markedly enhanced affinity with

111,112 EC50 in the subnanomolar or picomolar range. Two homobivalent ligands of the antipsychotic clozapine showed significant gains in affinity (75- and 79-fold) and activity (9- and

5-fold) relative to the original pharmacophore, clozapine.113 M100907, a novel bivalent antagonist of the 2AR homomer, exhibits high affinity and potency in vitro and efficacy in vivo.114,115

Heterobivalent ligands with the two pharmacophores targeting D2R and 2AR respectively have not been reported in literature. Considering the pharmacological advantages of bivalent

98 ligands, the enhanced effectiveness of APDs on the function of the D2R-2AR heteromer, and

116 the clinical benefits of APDs combinations, I propose the utility of putative D2R-2AR heterobivalent ligands that could antagonize D2R and 2AR simultaneously (with one pharmacophore unit being a typical APD moiety and the other an atypical APD moiety) (Figure

6.4 C). I anticipate that this type of bivalent ligands would have superior advantages compared to normal APDs:

130 1. Such bivalent ligands would have significantly higher affinity compared to typical or atypical APDs, leading to enhanced improvement of schizophrenia symptoms and reduced application of doses.

2. Antipsychotic effects through antagonism on 2AR of the D2R-2AR heteromer would reduce the side effects of typical APDs such as extrapyramidal symptoms and weight gain,

117,118 hyperprolactinemia due to D2R antagonism.

3. Heteromerization between D2R and 2AR would be promoted upon application of the heterobivalent ligands. Subsequently, the potency of the two pharmacophores antagonizing D2R and 2AR would be potentiated through the function of the D2R-2AR heteromer.

4. The effectiveness of each APD pharmacophore unit would be augmented through the functional cross-talk of the D2R-2AR heteromer.

131 Figure 6.1 Criteria to establish GPCR heteromers A. Criteria to establish GPCR heteromers in native tissue Adapted from Gomes et al., Annu. Rev. Pharmacol. Toxicol. (2016) B. Methods used to assess D2R-2AR heteromer in heterologous system and native tissue. Fulfilled criterion with established evidence are highlighted in green.

B Heterologous system Native tissue

Criterion 1 BiFC PLA in striatum

Criterion 2 oocyte assay fEPSP assay in PFC Ca assay DA desensitization assay in VTA

Criterion 3 disruptive peptide disruptive peptide mutagenesis AMPH model in mice

132 Figure 6.2 Aromatic residues are highly conserved at positions of 5.47, 5.48, 6.51 and 6.52 in aminergic receptors Alignment of the 5.47, 5.48, 6.51 and 6.52 residues of all 44 human aminergic receptors. Receptors are grouped by subtype. Receptors with four conserved aromatic residues are highlighted in green. Receptors with solved crystal structure are highlighted in bold. Non- conserved (non-aromatic) residues are highlighted in red.

133 Figure 6.3 The dual role of D2R-2AR in psychosis Schematic model showing both Gi and Gq signaling is increased in a psychotic state and through the functional cross-talk of D2R-2AR heteromer compared to the physiological state. Application of antagonist to either D2R or 2AR reverses the Gi and Gq signaling through the functional cross-talk of D2R-2AR.

Complex

Antipsychotics

134 Figure 6.4 Putative D2R-2AR bivalent ligand potentiating D2R-2AR heteromerization and antagonism on each protomer A. Computational model of bivalent ligand binding to the orthosteric binding sites of GPCR heterodimer. The two pharmacophore units (magenta) are linked by spacer (grey). B. The linker length is optimizable and shortest along the receptors van der walls surfaces. Adapted from Pérez-Benito et al., Bioinformatic (2018) C. Schematic model of putative bivalent ligands of D2R-2AR with one pharmacophore antagonizing D2R and the other antagonizing 2AR. These bivalent ligands switch the equilibrium between homomers, monomers and heteromers toward the heteromers and potentiate the antagonism on each protomer.

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146 A. MATERIALS AND METHODS

A1. Molecular biology

For expression in Xenopus oocytes, the pGEMHE vector was used for GIRK4*, PTX and

D2R; the pXoom vector was used for 2AR and RGS2. All DNAs were human except for mouse

D2R. Point mutations were introduced using standard Pfu-based mutagenesis techniques according to the QuikChange protocol (Agilent Technologies) and mutations were verified by sequencing (Macrogen). Plasmids were linearized and purified. In vitro transcription was performed using a commercially available T7 mRNA transcription kit followed by a final purification.

A2. Drugs and chemicals

Dopamine hydrochloride, serotonin hydrochloride, and 2,5-Dimethoxy-4- iodoamphetamine (DOI) hydrochloride, were purchased from Sigma. R-quinpirole hydrochloride, and haloperidol were purchased from Tocris. Pimavanserin was purchased from

MedChem Express.

A3. Cells and transfections

HEK-293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM,

Invitrogen, Carlsbad, CA) supplemented with 10 % (v/v) fetal bovine serum at 37 °C in a 5 %

CO2 humidified atmosphere. Transfections were performed using PEI according to the manufacturer’s instructions.

147 A4. Oocyte preparation and injection

Oocytes from Xenopus laevis were surgically removed and subjected to collagenase treatment according to standard protocols. Once defolliculated, oocytes were incubated in OR2

2+ solution supplemented with Ca and Penicillin/Streptomycin antibiotics. Stage V-VI oocytes were selected and injected with 50 nL of cRNA resuspended in DEPC water.

A5. Two-electrode voltage clamp

Borosilicate glass electrodes were pulled using a Flaming-Brown micropipette puller and filled with a 3M KCl solution containing 1% agarose. Resistances were kept between 0.1 and

1.0 MW. Oocyte currents were measured by two-electrode voltage clamp (TEVC) with a

GeneClamp 500 amplifier (Axon Instruments, Union City, CA). A high-potassium (HK) solution

(96 mM KCl, 1 mM NaCl, 1 mM MgCl2, 5 mM KOH/HEPES, pH 7.4) was used to perfuse cRNA-injected oocytes expressing the appropriate proteins to obtain basal currents. 3mM BaCl solution was used to block GIRK4* channel. Inwardly rectifying potassium currents through

GIRK4* were obtained by clamping the cells at a voltage ramp from -80 to +80 mV.

A6. Statistics

All error bars represent the standard error of the mean (SEM). Statistical significance was assessed using t test (unpaired and nonparametric) in Prism. Statistical significance was set at p <

0.05, 0.005, 0.0005 or 0.0001 denoted in figures by *, **, ***, **** asterisks, respectively.

Unless otherwise noted, all experiments were repeated in at least two separate batches of oocytes or cells.

148