Molecular Dissection of G-Protein Coupled Receptor Signaling and Oligomerization
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MOLECULAR DISSECTION OF G-PROTEIN COUPLED RECEPTOR SIGNALING AND OLIGOMERIZATION BY MICHAEL RIZZO A Dissertation Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Biology December, 2019 Winston-Salem, North Carolina Approved By: Erik C. Johnson, Ph.D. Advisor Wayne E. Pratt, Ph.D. Chair Pat C. Lord, Ph.D. Gloria K. Muday, Ph.D. Ke Zhang, Ph.D. ACKNOWLEDGEMENTS I would first like to thank my advisor, Dr. Erik Johnson, for his support, expertise, and leadership during my time in his lab. Without him, the work herein would not be possible. I would also like to thank the members of my committee, Dr. Gloria Muday, Dr. Ke Zhang, Dr. Wayne Pratt, and Dr. Pat Lord, for their guidance and advice that helped improve the quality of the research presented here. I would also like to thank members of the Johnson lab, both past and present, for being valuable colleagues and friends. I would especially like to thank Dr. Jason Braco, Dr. Jon Fisher, Dr. Jake Saunders, and Becky Perry, all of whom spent a great deal of time offering me advice, proofreading grants and manuscripts, and overall supporting me through the ups and downs of the research process. Finally, I would like to thank my family, both for instilling in me a passion for knowledge and education, and for their continued support. In particular, I would like to thank my wife Emerald – I am forever indebted to you for your support throughout this process, and I will never forget the sacrifices you made to help me get to where I am today. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS………………………………………………………………ii TABLE OF CONTENTS………………………………………………………………...iii LIST OF ABBREVIATIONS…………………………………………………………….v LIST OF TABLES………………………………………………………………………..x LIST OF FIGURES………………………………………………………………………xi ABSTRACT……………………………………………………………………………..xii CHAPTER I: G-protein coupled receptors– A review of structure-function relationships critical for receptor signaling ……………………………………..………………………1 REFERENCES…………………………………………………………………..37 CHAPTER II: Unexpected role of a conserved domain in extracellular loop 1 in G protein coupled receptor trafficking……………………………………………………...56 ABSTRACT……………………………………………………………………...57 INTRODUCTION……………………………………………………………….58 METHODS………………………………………………………………………60 RESULTS………………………………………………………………………..63 DISCUSSION……………………………………………………………………68 REFERENCES…………………………………………………………………..72 iii CHAPTER III: Homodimerization of Drosophila Class A neuropeptide GPCRs: Evidence for conservation of GPCR dimerization throughout metazoan evolution…….89 ABSTRACT……………………………………………………………………..90 INTRODUCTION……………………………………………………………….91 METHODS………………………………………………………………………97 RESULTS………………………………………………………………………100 DISCUSSION…………………………………………………………………..104 REFERENCES…………………………………………………………………108 CHAPTER IV: Conclusions and future directions……………….…………………….125 REFERENCES…………………………………………………………………129 CURRICULUM VITAE………………………………………………………………..130 iv LIST OF ABBREVIATIONS 5HT 5 Hydroxytryptophan A2A Adenosine receptor 2A A3 Adenosine Adenosine receptor 3 AC Adenylyl cyclase AKHR AKH receptor AM Adrenomedullin AMP Adenosine monophosphate ANOVA Analysis of variance statistical models AOI Area of interest AstCR2 Drosophila allatostatin C receptor 2 AT1R Angiotensin 1 receptor B1AR Adrenergic receptor beta 1 B2AR Adrenergic receptor beta 2 BiFC Biomolecular fluorescence complementation BK 2R Bradykinin receptor 2 BLAST Basic local alignment search tool BN-PAGE Blue native polyacrylamide gel electrophoresis BRET Bioluminescent resonance energy transfer C5aR Complement component 5a receptor cAMP Cyclic adenosine monophosphate CCR 2b Chemokine receptor type 2b CCR 5 Chemokine receptor type 5 v CFP Cyan flourescent protein cGMP Cyclic guanosine monophosphate CGRP Calcitonin gene-related peptide CHO Chinese hamster ovary cells CLR Calcitonin-like receptor Co-IP Co-immunoprecipitation CPS Counts per second CRD Cysteine-rich domain CRE cAMP response elements CREB cAMP response element-binding protein CRZR Corazonin receptor CXCR4 C-X-C chemokine receptor type 4 D2R Dopamine receptor D2 DAF Abnormal Dauer formation DAG Diacylglycerol DMEM Dulbecco’s modified Eagle medium EL Extracellular loop EPAC Exchange protein activated by cyclic-AMP ER Endoplasmic reticulum FRET Fluorescent resonance energy transfer FSHR Follicle stimulating hormone receptor GABA Gamma aminobutyric acid GALR1 Galanin receptor vi GPCR G protein-coupled receptor GDP Guanosine diphosphate GEF Guanine nucleotide exchange factor GFP Green fluorescent protein GIPs GPCR interacting protein GIRK G protein-gated inwardly rectifying potassium GMP Guanosine monophosphate GnRH Gonadotropin releasing hormone GRKs G protein-coupled receptor kinases GRP Gastrin-releasing peptide GTP Guanosine triphosphate H1R Histamine receptor 1 H2R Histamine receptor 2 HA Hemaglutinin HEK Human embryonic kidney cells IP 3 Inositol triphosphate LH Luteinizing hormone LK Leucokinin M1R Muscarinic acetylcholine receptor 1 M3R Muscarinic acetylcholine receptor 3 MAPK Mitogen activated protein kinase mGlu 2R Metabotropic glutamate receptor 2 NFkB Nuclear factor kappa-light-chain-enhancer of B cells vii NK1R Neurokinin 1 Receptor NK2R Neurokinin 2 Receptor NKA Neurokinin A NMU Neuromedin U NPFR Drosophila NPF receptor NPY Neuropeptide Y ORF Open reading frame OX 1 Orexin receptor 1 PCR Polymerase chain reaction PIP 2 Phosphatidylinositol 4,5-bisphosphate PK1R Drosophila pyrokinin receptor 1 PKA Protein kinase A PKC Protein kinase C PLC Phospholipase C ProcR Proctolin receptor PSD-95 Postsynaptic density protein 95 RAMPs Receptor-activity modifying proteins RCP Receptor component protein RGS Regulators of G-protein signaling SpIDA Spatial intensity distribution analysis SPRINP Single primer reactions in parallel SRE-Luc Serum response element SSTR2 Somatostatin receptor 2 viii T1R1 Taste receptor type 1 receptor 1 T1R3 Taste receptor type 1 receptor 3 T2R Taste receptor type 2 TKR86C Tachykinin receptor at 86C TM Transmembrane domain TR-FRET Time-resolved fluorescence resonance energy transfer TRH Thyrotropin-releasing hormone TSHR Thyroid stimulating hormone receptor VFT Venus fly trap domain WGA Wheat germ agglutinin YFP Yellow fluorescent protein α2b -AR Alpha-2B adrenergic receptor β2AR Beta-2 adrenergic receptor ix LIST OF TABLES Table II.1: Comparison of representative extracellular loop 1 sequences across Class A GPCR subfamilies………………………………………………………………………77 Table III.1: Receptors utilized in FRET dimer screen………………………………...122 Table III.2: List of primers used for directional cloning of receptor cDNA into pcDNA3 CFP or pcDNA3 YFP expression vectors………………………………………………123 x LIST OF FIGURES FIGURE I.1: Two-state model of GPCR activation……………………………………54 FIGURE I.2: Functional importance of GPCR heterodimerization…………………….55 FIGURE II.1: Sequence weighting analysis shows that the WxFG motif’s tryptophan residue exhibits high conservation in Class A GPCR receptor subfamilies…………….79 FIGURE II.2: Mutagenesis of conserved tryptophan residue in LKR ECL1 ablates receptor signaling………………………………………………………………………..80 FIGURE II.3: Leucine substitution for the conserved tryptophan residue in extracellular loop 1 leads to a loss of function in multiple receptor types…………………………….82 FIGURE II.4: Substitution of the conserved tryptophan residue to leucine ablates constitutive activity in a constitutively active AKHR mutant…………………………...84 FIGURE II.5: The WxFG motif is critical for proper receptor trafficking..……………85 FIGURE II.6: Putative tertiary structures of wild type LKR and mutant W101L are superimposed to identify gross changes in receptor topology…………………………...88 FIGURE III.1: Demonstration of acceptor-photobleaching FRET assay……………..116 FIGURE III.2: Verification of experimental system………………………………….117 FIGURE III.3: Multiple Drosophila Class A neuropeptide receptors exhibit FRET responses consistent with homodimerization…………………………………………...119 FIGURE III.S1: Verification of signaling in fluorophore tagged receptors..................124 xi ABSTRACT G protein coupled receptors (GPCRs) are a superfamily of transmembrane proteins responsible for transducing extracellular stimuli into intracellular responses. GPCRs are indispensable to a vast variety of distinct physiologies and behaviors and represent approximately 50% of all human drug targets. However, considerable debate exists as to the structural basis for GPCR activation, with a classical monomeric (two state model) conflicting with a growing number of reports indicating that these receptors form higher order functional oligomers. These receptor-receptor interactions can impact receptor trafficking, ligand sensitivity, desensitization, and strength of effector response. As such, an understanding of GPCR oligomerization is indispensable to our overall understanding of receptor dynamics. Additionally, the specific molecular events underlying receptor activation and signaling remain incompletely understood. Since the initial discovery of the GPCR receptor family, a number of conserved amino acid motifs have been identified that have been shown to play specific and critical roles in GPCR activation, intracellular G-protein coupling, and receptor desensitization. Still, many of these motifs remain incompletely described, with some motifs having only been evaluated in a small subset of receptors, and experimental evidence suggests that in some cases, these conserved motifs may have divergent roles in specific receptor subfamilies.