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The Activation of the Glucagon-Like Peptide-1 (GLP-1) Receptor by Peptide and Non-Peptide Ligands

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The Activation of the Glucagon-Like Peptide-1 (GLP-1) Receptor by Peptide and Non-Peptide Ligands The Activation of the Glucagon-Like Peptide-1 (GLP-1) Receptor by Peptide and Non-Peptide Ligands Clare Louise Wishart Submitted in accordance with the requirements for the degree of Doctor of Philosophy of Science University of Leeds School of Biomedical Sciences Faculty of Biological Sciences September 2013 I Intellectual Property and Publication Statements The candidate confirms that the work submitted is her own and that appropriate credit has been given where reference has been made to the work of others. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Clare Louise Wishart to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. © 2013 The University of Leeds and Clare Louise Wishart. II Acknowledgments Firstly I would like to offer my sincerest thanks and gratitude to my supervisor, Dr. Dan Donnelly, who has been nothing but encouraging and engaging from day one. I have thoroughly enjoyed every moment of working alongside him and learning from his guidance and wisdom. My thanks go to my academic assessor Professor Paul Milner whom I have known for several years, and during my time at the University of Leeds he has offered me invaluable advice and inspiration. Additionally I would like to thank my academic project advisor Dr. Michael Harrison for his friendship, help and advice. I would like to thank Dr. Rosalind Mann and Dr. Elsayed Nasr for welcoming me into the lab as a new PhD student and sharing their experimental techniques with me, these techniques have helped me no end in my time as a research student. Additionally I would like to thank Dr. Natalie Keat for her support whilst I was researching in the LIGHT laboratories. I am grateful to have collaborated with Dr. Colin Fishwick and Dr. Marco Migliore (University of Leeds, School of Chemistry) who provided me with the ‘Pm compounds’ and were constantly enthusiastic about their biological consequences. Special thanks go to Dr. Gareth Rosbrook and Dr. Sue Whittle for their help and support throughout my time at the University of Leeds. To my future husband Chris, it’s been such an experience for us to both do a PhD at the same time. We have shared joy upon each other’s success, and felt each other’s pain when things have gone wrong in the lab. I feel I wouldn’t have done as well as I did if I didn’t know you were supporting me throughout the whole process, and you always seem to know the right things to say. You are my rock; I am looking forward to spending the rest of my days with you. Rachel, I have only known you for a short amount of time but you have brought such light and warmth into my life. It is not often in life that special friendships like ours are forged. I wish you the best of luck to you in the future for your PhD. I hope you enjoy the experience and meeting the variety of interesting characters that academia offers. To you, I gift the PCR staff. May it bring you good luck and success in your future thermal cycling experiments. Caroline, Benjamin and Dave, it has been brilliant working alongside you in the Garstang laboratories these past two years. Caroline, you are an inspiration and I wish you all the success you deserve. Dave, your wit and quick quips have made me chuckle and I wish you luck in your new postdoc position. Benjamin, I have thoroughly enjoyed your autoclaving III escapades and I hope you manage to publish your ‘will it autoclave?’ paper. Sorry for all the pranks. I would like to give special thanks to my Mum. You have been so very supportive throughout my entire education, which is now coming to a close. I feel 20 years of learning should tide me over sufficiently. You are the best Mum a girl could wish for, and this thesis is dedicated to you. I would like to thank my laptop at home for putting up with such high levels of usage. And finally, I would like to thank the BBSRC for funding this PhD. IV Abstract The glucagon-like peptide-1 receptor (GLP-1R) potentiates glucose-stimulated insulin release from pancreatic β cells and promotes correct β cell function, as such it is a validated target for the treatment of type 2 diabetes (T2D). GLP-1R is a Family B GPCR, activated by the cognate ligand GLP-1(7-36), a 30 residue peptide hormone secreted after eating, and Exendin4 (Ex4), a 39-residue synthetic peptide. Peptide ligands interact with both the large extracellular domain and core domain of GLP-1R. Core domain interaction is thought to activate the receptor. Whilst the interaction between the receptor extracellular domain and ligand is well characterised, the ligand-core domain interaction and subsequent activation is not fully understood. Herein, a combination of mutant peptides and non-peptide ligands based on a pyrimidine scaffold (Pm compounds) are used in HTR-FRET cAMP accumulation assays, using recombinant FlpIn-HEK293 cells expressing human GLP-1R, to characterise the activation profiles of these ligands to decipher the underlying activation mechanism at the GLP-1R core domain. Structure-function studies of Pm compounds showed a trifluoromethyl and sulphur dioxide group are essential for GLP-1R activation, and that they allosterically enhance GLP- 1(9-36) and Ex4(9-39) cAMP signalling profiles independently from their own cAMP response. Insulin secretion assays showed Pm compounds potentiate insulin release from INS-1 832/13 cells in combination with truncated GLP-1(9-36), implicating the use of allosteric modulators as treatment for T2D. Truncated GLP-1(15-36) was capable of binding and activating GLP-1R with low affinity and low potency, yet analogously truncated Ex4(9-39) was an antagonist with high affinity. Previous studies had demonstrated GLP-1(15-36) was an antagonist, and peptide- mediated activity had been attributed to the amino-terminus. Furthermore, the Pm compound- mediated cAMP response at GLP-1R was potentiated by Ex4(9-39). Mutant peptide activation data suggest activating residues D15, V16 and S17 are situated more centrally within the peptide ligand, and an extension to the currently accepted GLP-1R activation model is proposed. V Contents Intellectual Property and Publication Statements I Acknowledgments II Abstract IV Contents V Abbreviations XI List of figures XV List of tables XIX Chapter 1: General Introduction 1 1.0. Preface 2 1.1. G protein-coupled receptors 2 1.2. GPCR Classification 4 1.2.1 The A-F Classification system 4 1.2.2 The GRAFS Classification System 4 1.2.3 Glutamate Family of GPCRs 6 1.2.4 Rhodopsin Family of GPCRs 7 1.2.5 Adhesion Family of GPCRs 8 1.2.6 Frizzled Family of GPCRs 9 1.2.7 Secretin Family of GPCRs 10 1.3. Secretin Family Receptor Crystal Structures-New Data 14 1.4. G Protein Coupling and Signalling Cascades 16 1.5. The Development of Receptor Theory 19 1.6. Allosteric Ligands 23 1.7. GPCR Oligomerisation 26 1.8. Receptor Desensitisation and Internalisation 28 1.9. Glucagon-Like Peptide-1 29 1.10. The Incretin Effect 30 1.11. Physiological Effectors of GLP-1 32 VI 1.11.1. PKA-Mediated Insulin Release 32 1.11.2. Epac2-Mediated Insulin Release 34 1.12. GLP-1-Mediated Glucose Homeostasis 35 1.13. Extra-Pancreatic Effects of GLP-1 36 1.14. GLP-1R Agonists are Therapeutic Agents for Type II Diabetes 37 1.15. The GLP-1 Receptor 39 1.1.6. GLP-1R Peptide-Ligand Interactions 42 1.1.6.1. GLP-1R Extracellular Domain Interaction 42 1.1.6.2. Exendin-4 Specific Receptor Extracellular Domain Interaction 46 1.1.6.3. GLP-1 Receptor Core Domain Interaction 47 1.1.7. Current GLP-1R Activation Models 51 1.1.8. Non-Peptide Ligand Agonists of GLP-1R 57 1.1.9. Aims of This Study 58 Chapter 2: Methods and Materials 60 2.1. Materials 61 2.1.1. General Materials 61 2.1.2. Cell Culture Reagents 61 2.1.2.1. HEK-293 Cell Culture Reagents 61 2.1.2.2. INS-1 832/13 Cell Culture Reagents 61 2.1.3. Molecular Cloning Reagents 62 2.1.4. LANCE® Reagents 62 2.1.5. Radioligand Assay Reagents 63 2.1.6. GLP-1R Ligands 63 2.1.7. Plasmids and DNA 64 2.1.8. Bacterial Media Components 64 2.1.9. Bacterial Growth Medium 65 2.1.10. Antibiotic Supplement Concentrations 65 VII 2.2. Methods 66 2.2.1. Mammalian Cell Culture 66 2.2.1.1. HEK-293 Cell Growth Media 66 2.2.1.2. INS-1 832/13 Cell Growth Media 66 2.2.1.3. Propagation & Passage of HEK-293 & INS-1 832/13 Cells 66 2.2.1.4. Long Term Storage of Cells 67 2.2.1.5. Transient Transfection of FlpIn-HEK293 Cells 67 2.2.1.6. Stable Transfection of FlpIn-HEK293 Cells 68 2.2.1.7. Crude Membrane Preparations 69 2.2.1.8. Bicinchoninic Acid Assay 70 2.2.2. Molecular Biology and Cloning 70 2.2.2.1. Preparation of Chemically Competent E.coli 70 2.2.2.2. Transformation of Chemically Competent E.coli 71 2.2.2.3. Small Scale Alkaline Lysis (Miniprep) 72 2.2.2.4.
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