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Characterization of Novel Glucagon Receptor Interactors That Modify Receptor Activity

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Characterization of Novel Glucagon Receptor Interactors That Modify Receptor Activity Characterization of Novel Glucagon Receptor Interactors that Modify Receptor Activity by Sean Froese A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Physiology University of Toronto © Copyright by Sean Froese 2015 Characterization of Novel Glucagon Receptor Interactors that Modify Receptor Activity Sean Froese Master of Science Department of Physiology University of Toronto 2015 Abstract Glucagon helps maintain blood glucose homeostasis by stimulating gluconeogenesis and glycogenolysis. Elevated glucagon levels have been reported in type 2 diabetics, and may be in part responsible for their abnormally high blood glucose. A comprehensive understanding of glucagon receptor regulators is currently unavailable. Members of my lab performed a mass spectrometry screen to detect glucagon receptor interactors, 5 of which were validated to be true interactors by co-immunoprecipitation and Western blotting. The project outlined in this thesis began with the functional characterization of these interactors. Two interactors, low-density lipoprotein receptor (LDLR) and transmembrane emp24 domain trafficking protein 2 (TMED2) significantly enhanced glucagon stimulated glucose production, while tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein, beta (YWHAB) lowered glucagon stimulated glucose production in primary hepatocytes. Because of YWHAB’s ability to lower glucose production, the underlying mechanism was explored and evidence suggests YWHAB enhances endocytosis of the glucagon receptor into the cell following glucagon stimulation. ii Acknowledgments This thesis would not have been possible on my own. The support and guidance from everyone in the lab, my family, and my friends, has been invaluable throughout these past two years. I would like to thank first and foremost Dr. Wheeler for taking a chance on an inexperienced undergraduate two and a half years ago, for his guidance and insights, and his constant patience during my time in the lab. I have learned more about science, and myself, in these past two years than any other time and my life, and this is in no small part due to Dr. Wheeler. Thank you to my committee members, Drs. Adeli, Brubaker and Heximer. Your patience with planning committee meetings and willingness to accommodate my schedule was incredibly appreciated. More importantly, your insights and challenges were highly motivating and incredibly valuable to my education. I would like to thank all of my co-authors, particularly Junfeng Han and Ming Zhang whose work formed the basis for this thesis. I would also like to thank my fellow lab members for all of their amazing work their constant helpful advice and proofing. Special mention to Kacey Prentice and Andrea Eversley for listening to me vent for hours on end. Your scientific insight and friendship has been vital for my education, and my sanity. I would like to thank my girlfriend Sabrina Parrotta for her unwavering support, her constant encouragement, and her understanding when I can’t go to the movies because my cells need to be split. Last, I would like to thank my family, especially my parents Patricia and Daryl Froese for their encouragement, support, and dedication to providing me with an education many would envy. Most say they would do anything for their children, my parents have proved it. iii Table of Contents Abstract……………………………………………………………………………………..........ii Acknowledgments……………………………………………………………………………….iii Table of Contents………………………………………………………………………………..iv List of Figures………………………………………………………………………………......vii Abbreviations………………………………………………………...…………...…………......ix Chapter 1: General Introduction 1.1 Glucagon 1.1.1 Blood glucose homeostasis………………………………………………………………1 1.1.2 Glucagon expression and processing……………………………………………………2 1.1.3 Secretion…………………………………………………………………………………2 1.1.4 The effects of glucagon 1.1.4.1 Stimulatory effects…………………………………………………………………...5 1.1.4.2 Inhibitory effects……………………………………………………………………..6 1.2 Glucagon Receptor 1.2.1 Regulation of receptor expression………………………………………………………..6 1.2.2 Structure………………………………………………………………………………….7 1.2.3 Signalling 1.2.3.1 Activation and transduction…………………………………………………………..8 1.2.3.2 Glucagon receptor regulation………………………………………………………....9 1.3 Type 2 Diabetes and the Role of Glucagon 1.3.1 Pathogenic glucagon secretion………………………………………………………….11 1.3.2 Glucagon receptor antagonists………………………………………………………….12 1.4 Receptor Interactomes 1.4.1 Effects of accessory proteins on G-protein coupled receptors…………………………..13 1.4.2 Experimental approaches to receptor interactome identification 1.4.2.1 Membrane yeast two hybrid system…………………………………………………15 1.4.2.2 Fluorescence resonance energy transfer……………………………………………..16 iv 1.4.2.3 Mass spectrometry…………………………………………………………………..16 1.5 Rationale and Hypothesis………………………………………………………………….…17 Chapter 2: Screening and Characterization of Glucagon Receptor Interactors 2.1 Introduction…………………………………………………………………………..………21 2.2 Materials and Methods 2.2.1 Animals and cell culture…………………………………………………………………22 2.2.2 Isolation of primary mouse hepatocytes…………………………………………………22 2.2.3 Plasmid preparation and transfection……………………………………………………23 2.2.4 Western blotting………………………………………………………………………....23 2.2.5 Glucose production assay………………………………………………………………..23 2.2.6 cAMP assay……………………………………………………………………………..23 2.2.7 Quantitative real-time PCR……………………………………………………………...24 2.2.8 Statistics and Bioinformatics…………………………………………………………….24 2.3 Results 2.3.1 Glucagon receptor interactors affect glucose production in primary mouse hepatocytes………………………………………………………………….25 2.3.2 Changes to cAMP accumulation mediated by select glucagon receptor interactors 2.3.2.1 Primary mouse hepatocytes………………………………………………………….27 2.3.2.2 CHO and HepG2-GCGR cells………………………………………………………28 2.3.4 Expression of key gluconeogenic genes in primary mouse hepatocytes with glucagon receptor interactor over expression……………………………………………………………..29 2.3.5 YWHAB does not alter cellular proliferation……………………………………………30 2.3.6 YWHAB overexpression decreases cell surface expression of GCGR in HepG2-GCGR cells……………………………………………………………….31 2.3.7 siRNA knockdown of YWHAB enhances cAMP production and decreases GCGR gene expression………………………………………………………….32 Chapter 3: Discussion 3.1 Summary 3.1.1 Effects of select glucagon receptor interactors on receptor function…………………….35 v 3.1.2 Effects of interactor overexpression on expression of key gluconeogenic genes……….................................................................................................36 3.1.3 Potential mechanism of YWHAB mediate reduction in cAMP production……………………………………………………………………………37 Chapter 4: Future Direction and Conclusions 4.1 Future Directions…………………………………………………………………………..…39 4.2 Conclusions…………………………………………………………………………………..40 References……………………………………………………………………………………….41 vi List of Figures Figure 1. Diagram of proglucagon products that result from tissue specific processing ……………….………………………………………………………………………………….....2 Figure 2. Diagram of the glucagon secretory pathway in pancreatic α-cells……………….……...3 Figure 3. Crystal structure of the human glucagon receptor………………….…………….……...7 Figure 4. Schematic of the glucagon receptor signalling pathway in hepatocytes………….……...8 Figure 5. Diagram of the fate of internalized glucagon receptor…………......…………….……………………………………………………………...10 Figure 6. GLP-1R interactome identified using the MYTH screening system……………..…….17 Figure 7. GLP-1R interactome identified using the affinity purification tandem mass spectrometry approach……………….………………………………...……….….18 Figure 8. Co-immunoprecipitation validation of GCGR accessory protein interaction.……...….19 Figure 9. Glucose production in hepatocytes overexpressing interactors of interest……………..26 Figure 10. cAMP production in hepatocytes overexpressing interactors in interest………………………………………………………………………………......….…….27 Figure 11. cAMP production in two cells lines overexpressing interactors of interest……..…….28 Figure 12. mRNA expression of gluconeogenic genes in interactor overexpressing hepatocytes……………………………………………………………………………………….30 Figure 13. Cell proliferation in CHO cells overexpressing YWHAB……………...…...….…….31 Figure 14. Cell surface expression of the GCGR in YWHAB overexpressing vii HepG2-GCGR cells……………………………………………………………………………...32 Figure 15. mRNA expression of endogenous YWHAB and GCGR in primary hepatocytes…….33 Figure 16. cAMP production in hepatocytes following siRNA knockdown of YWHAB….…….33 Figure 17. mRNA expression of GCGR in primary hepatocytes following siRNA knockdown of YWHAB…………………………………………………………………………………………34 Figure 18. Schematic of the proposed mechanism of YWHAB’s inhibition of glucose production……………………………………………………………………...….…….38 viii Abbreviations CAMK Ca2+/calmodulin-dependant protein kinase CAV1 Caveolin 1 cAMP cyclic adenosine monophosphate CHO Chinese hamster ovary CREB cAMP-response element binding protein DMEM Dulbecco's Modified Eagle Medium FRET Fluorescence resonance energy transfer G-6-P Glucose-6-phosphate G6Pase Glucose-6-phosphatase GABA Gamma-amminobutyric acid GALK1 Galactokinase 1 GASP G-protein-coupled receptor associated sorting protein GCGR Glucagon receptor GFP Green fluorescent protein GLP-1/2 Glucagon-like peptide 1/2 GLP-1R Glucagon-like peptide-1 receptor GPCR G-protein-coupled receptor GRK G-protein-coupled receptor kinase IBMX 3-isobutyl-1-methylxanthine Katp ATP regulated potassium channel LDLR Low density lipoprotein receptor MPGF Major proglucagon fragment MYTH Membrane
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