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Identifying Novel Protein Interactors of the Glucagon Superfamily of Receptors

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Identifying Novel Protein Interactors of the Glucagon Superfamily of Receptors Identifying Novel Protein Interactors of the Glucagon Superfamily of Receptors by Gregory Gaisano A thesis submitted in conformity with the requirements for the degree of Masters of Science Graduate Department of Physiology University of Toronto © Copyright by Gregory Gaisano (2009) Identifying Novel Protein Interactors of the Glucagon Superfamily of Receptors Gregory Gaisano Masters of Science Department of Physiology University of Toronto 2009 Abstract G-protein coupled receptors (GPCRs) have been shown to act as part of GPCR associated protein complexes (GAPCs) which are required to appropriately transduce downstream signaling pathways leading to specific cellular actions. I hypothesize that there are distinct molecular effectors that couple to the glucagon superfamily of B-class GPCRs (glucagon, GLP-1, GLP-2, GIP receptors) to effect the myriad of reported actions in numerous target cells including regulation of insulin secretion, intestinal growth and appetite suppression. GLP-1R, GIPR, GLP-2R and GCGR were screened using a newly developed membrane-based split-ubiquitin yeast two-hybrid (MYTH) system to reveal 181 novel candidate protein interactors associated with signal transduction, transport, metabolism and cell survival. Each candidate was validated using yeast two-hybrid prey retransformation tests and by co-purification to confirm coupling to each receptors. The present work is the first demonstration of a split- ubiquitin interaction screen using in situ membrane expressed GPCRs of the secretin- like B class. ii Acknowledgements I would like to express my sincere thanks to my supervisor Dr. Michael Wheeler who provided endless support on multiple levels throughout the project. His guidance on the scientific and personal levels has helped me tremendously throughout the development of this thesis. I would also like to express my heartfelt thanks to Dr. Feihan Dai for all her teachings, encouragements, and caring discussions. Her mentorship has been central to my understanding of the molecular biology field. My deep appreciation goes to Victoria Wong and Saranya Kittanakom for training me to use the membrane-based yeast-two hybrid system and for providing their expertise on all the biochemical aspects of this project. I would also like to thank my student, Kamyar Giglou, for his kind help in all my assays. A sincere thank you for Dr. Igor Stagljar and Dr. Anthony Gramolini and their respective laboratory staff for their guidance in yeast two-hybrid and large-scale proteomic screening techniques. Finally, I would like to express my deepest thanks to God, my family, girlfriend and friends whose endless support have made everything possible. iii TABLE OF CONTENTS ABSTRACT OF THESIS…………………………………………………………….………....ii ACKNOWLEDGEMENTS…………………………………………………………………….. iii TABLE OF CONTENTS………………………………………………..………………………iv LIST OF TABLES………………………………………………………………….……..…….ix LIST OF FIGURES……………….…………………………………………………………… x LIST OF ABBREVIATIONS………………………………………………………….………. xi CHAPTER 1: GENERAL INTRODUCTION……………………...………………………..... 1 1.1 G Protein-Coupled Receptors (GPCR)……………………………………...…..1 1.1.1 Structure……………………..………………………………………… .1 1.1.2 G-proteins…………………………………………………………… ….2 1.1.3 Interactomes………………………………………………………… ….3 1.2 Class-B GPCRs…………………………………………………………………...…5 1.3 Glucagon Superfamily of Receptors…………………………………………… 6 1.4 Glucagon-Like Peptide 1 and GLP-1R………………………………...………...6 1.4.1 Expression……………………………………………………………… 6 1.4.2 Structure………………………………………………………………… 7 1.4.3 Function ……………...………………………………………………… 7 1.4.4 Signaling……………………………….……………………………….. 9 1.4.5 Regulation……………………………………………………………. 10 1.5 Glucose-Dependent Insulinotropic Polypeptide and GIPR…………………11 1.5.1 Expression………………………………………………………………11 1.5.2 Structure………………………………………………………………...11 1.5.3 Function ……………………………...…………………………………12 1.5.4 Signaling …………………………..……………………………………13 1.5.5 Regulation………………………..…………….……………………….13 1.6 Glucagon-Like Peptide 2 and GLP-2R………………………………..………..14 1.6.1 Expression……………………………………………………………...14 1.6.2 Structure……………………………………………………………......14 1.6.3 Function ………………………………………………………………..14 1.6.4 Signaling ……………………………………………………………….16 iv 1.6.5 Regulation ………………………………………………………..…... 16 1.7 Glucagon and GCGR…………………………………………………………......16 1.7.1 Expression……………………………………………………………..16 1.7.2 Structure……………………………..………………………………...17 1.7.3 Function …………………………...……………..……………………17 1.7.4 Signaling ……………………………………………………………… 19 1.7.5 Regulation…………………………………………………………….. 19 RATIONALE AND HYPOTHESIS OF THESIS………………………………………... 20 OBJECTIVES ………………………………………...……………………………….. …..21 CHAPTER 2: ESTABLISHMENT OF MYTH METHOD TO STUDY B-CLASS GPCR……. 22 2.1 Rationale…………………………………………………………………………… 22 2.2 Hypothesis……………………………………………………………………...…. 22 2.3 Introduction…………………………………………………………………..…… 22 2.3.1 Molecular Proteomic Screening Tools………………………..….. 22 2.3.2 Yeast Two-Hybrid Screening………………………………………. 23 2.3.3 Spilt Ubiquitin Membrane-based Yeast Two-Hybrid System….. 23 2.3.4 Variation of YTH systems ………………………………………… 25 2.3.5 Alternative Proteomic Screening Tools…………………………… 25 2.4 Method and Materials……………………………………………………………. 27 2.4.1 Reagents……………………………………………………………… 27 2.4.2 Cell Culture…………………………………………………………… 27 2.4.3 Polymerase Chain Reaction (PCR) and Gel Extraction…………. 27 2.4.4 Yeast Bait Construction …………………….………………………. 27 2.4.5 Immunofluorescence ………………………….…………………….. 28 2.4.6 NubG/NubI Test…………………………………………………....... 29 2.4.7 Receptor Cloning…………………………………………………….. 29 2.4.8 Intracellular cAMP Measurement………………………………….. 29 2.4.9 Statistics………………………………………………………………. 30 2.5 Results……………………………………………………………………………….30 2.5.1 Generating Receptor “Bait” Constructs……………………………. 30 2.5.2 Cub-TF Fused Receptors are Localized to Yeast Plasma Membrane…………………………………………………………….. 31 2.5.3 Cub-TF Fused Receptors are Compatible with MYTH system…. 31 2.5.4 Cloned Receptors can be Expressed in Mammalian Cells……… 32 v 2.5.5 Cloned Receptors are Functional in Mammalian Cells…………. 32 2.6 Summary of Findings……………………………………………………………. 35 2.7 Discussion………………………………………………………………………….36 2.7.1 MYTH Receptor Compatibility……………………………………… 36 2.7.2 GCGR Subcloning…………………………………………………… 36 2.7.3 IF Resolution…………………………………………………………. 37 CHAPTER 3: IDENTIFICATION OF NOVEL INTERACTORS OF RECEPTORS........ 38 3.1 Rationale…………………………………………………………………………… 38 3.2 Hypothesis………………………………………………………………………….38 3.3 Introduction…………………………………………………………………….…..38 3.3.1 Glucagon Receptor Superfamily – Known Interactors…………… 38 3.3.2 Limitation of MTYH System…………………………………………. 39 3.3.2.1 Absence of Receptor Activation…………………….. 39 3.3.2.2 False Negatives………………………………………. 39 3.4 Method and Materials……………………………………………………………. 40 3.4.1 Reagents ………………………………………………………..……. 40 3.4.2 cDNA Synthesis and RT-PCR……………………………………… 40 3.4.3 Large-scale Library Screen…………………………………………. 41 3.4.4 Prey Isolation…………………………………………………………. 41 3.4.5 BLAST Analysis……………………………………………………… 42 3.5 Results……………………………………………………………………………… 42 3.5.1 RT-PCR: GLP-1R is Expressed in Human Fetal Brain cDNA….. 42 3.5.2 GLP-1R Large Scale Library Screen………………………………. 43 3.5.3 GIPR Large Scale Library Screen………………………………….. 45 3.5.4 GLP-2R Large Scale Library Screen……………………………..... 47 3.5.5 GCGR Large Scale Library Screen………………………………… 50 3.6 Summary of Findings…………………………………………………………… 52 3.7 Discussion………………………………………………………………………… 53 3.7.1 Human Fetal Brain Expression……………………………..………. 53 3.7.2 Selection of Yeast Colonies………………………………………… 54 3.7.3 Low Number of Interactors Screened……………………………… 54 3.7.4 List of Putative Interactors…………………………………………... 54 vi CHAPTER 4: VALIDATION OF NOVEL INTERACTORS………………………………. 56 4.1 Rationale…………………………………………………………………………… 56 4.2 Hypothesis……………………………………………………………………….... 56 4.3 Introduction……………………………………………………………………….. 56 4.3.1 False Positives in YTH screens……………….……………………. 56 4.3.2 Alternative Validation Tests…………………………………………. 57 4.4 Method and Materials………………………………………………………….....57 4.4.1 Reagents……………………………………………………………… 57 4.4.2 Cell Culture…………………………………………………………… 57 4.4.3 In Silico Analysis…………………………………..…………………. 57 4.4.4 Prey Retransformation Test …………………..……………………. 58 4.4.5 Interactor Cloning……………………………………………………. 58 4.4.6 Co-purification…………………………………………………………58 4.4.7 Immunoblotting ………………………………………………………. 59 4.5 Results………………………………………………………………………………59 4.5.1 Validation by Prey Retransformation Tests…………………..…… 59 4.5.2 In Silico analysis of Validated Receptor Interactors……………… 64 4.5.3 Validation by Co-purification……………………………................. 64 4.6 Summary of Findings……………………………………………………………. 66 4.7 Discussion………………………………………………………………………….66 4.7.1 Screening out False Positives……………………………………….66 4.7.2 Co-purification…………………………………………..................... 66 4.7.3 Potential Relevance of Validated Interactors …………………….. 67 4.7.3.1 APH1A…………………………………………………. 67 4.7.3.2 APLP1…………………………………………………..68 4.7.3.3 CD81…………………………………………………… 70 4.7.3.4 GABBR2…………………………………….……….....70 4.7.3.5 GPR37………………………………….……………… 71 4.7.3.6 HPN…………………………….……………………….72 4.7.3.7 STMN1………………………………………………….72 CHAPTER 5: GENERAL DISCUSSION……………………………….…………….......... 73 5.1 Summary of Findings……………………………………………………………. 73 5.2 MYTH system……………………………………………………………………… 73 vii 5.3 B-class GPCR Interactomes……………………………………………………. 74 5.4 Pharmacological Relevance……………………………………………………. 75 5.5 Function of Novel Interactors - Preliminary Data…………………………... 76 5.6 Future Directions…………………………………………………………………. 77 5.7 Conclusion………………………………………………………………………… 79 REFERENCE LIST…………………………………………………………………………… 80
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