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Pharmacological Characterisation of the Fatty Acid Receptors GPR120

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Pharmacological Characterisation of the Fatty Acid Receptors GPR120 Pharmacological characterisation of the fatty acid receptors GPR120 and FFA1 Sarah-Jane Watson, BSc. Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy NOVEMBER 2013 Abstract In recent years, two G protein coupled receptors have been de-orphanised which respond to long chain free fatty acids (FFAs), and so are able to mediate the signalling of these important nutrient molecules. FFA1 (GPR40) is predominantly expressed in pancreatic -cells, while the expression profile of GPR120 includes gut endocrine cells and adipose tissue. These distributions, together with the potential of both receptors to stimulate insulin and incretin hormone secretion, singled them out as potential drug targets for type 2 diabetes and obesity. The aim of this thesis was to evaluate the pharmacology of these receptors and their signalling properties, including the development of fluorescent FFA receptor ligands to evaluate agonist binding using imaging techniques. GPR120 has been identified to exist as two splice isoforms in humans, differing by a short insertion in the third intracellular loop, but no full isoform specific characterisation of receptor signalling and trafficking had been undertaken. This work therefore studied the GPR120S and GPR120L isoforms in terms of both G protein dependent and arrestin dependent signalling, and trafficking. It was found that the long GPR120L isoform exhibited reduced G protein signalling, but similar -arrestin recruitment and lysosomal intracellular trafficking profiles as GPR120S. Potentially, expression of the long GPR120 isoform provides a mechanism to direct signalling to the -arrestin pathway, for example to produce anti-inflammatory effects in macrophages. As the expression profile of GPR120 overlaps with that of FFA1, for example in i colonic endocrine cells, a series of constrained GPR120 homo-dimers and GPR120:FFA1 heterodimers were created using irreversible bimolecular fluorescence complementation, and the potential for novel pharmacology was investigated by monitoring dimer internalisation. However, there was no evidence that such dimerisation altered the pharmacology of the ligands tested. Second, a model of the GPR120S ligand binding site was tested using point mutagenesis of the receptor. This mutagenesis validated key features of the model, including the role of Arg99 in co-ordinating the agonist carboxylate group, and interactions of the agonists with the conserved transmembrane VI Trp “toggle-switch” involved in receptor activation. Another mutation (Asn215) provided evidence for ligand-specific binding modes within the pocket. This study showed the complexity of testing mutants designed to interfere with ligand binding indirectly through signalling assays and highlighted the requirement for a FFA receptor binding assay to measure ligand affinity directly. In the absence of radioligands of suitable selectivity and affinity, a novel fluorescent ligand, based on the FFA1/GPR120 agonist GW9508, was used to successfully develop a whole cell FFA1 competition binding assay for the first time, obtaining FFA1 affinity estimates for a range of synthetic ligands. Fluorescent ligand binding was further investigated using fluorescence correlation spectroscopy and photon counting histogram analysis, defining the diffusion characteristics of FFA1 receptors in the membrane of single living cells, and providing preliminary evidence for their dimerisation. ii Acknowledgements I would like to thank Dr Nick Holliday and Dr Steve Hill for the great opportunity to do this PhD project. I am especially grateful to Nick, for letting me briefly escape to New Zealand when I should have been writing my thesis! I would also like to thank CellAura, Nottingham for synthesis of 40Ag-Cy5; and both the EPSRC and AstraZeneca for financial support. Thanks also to my industrial supervisor Alastair Brown for hosting my times at Alderley Park, Graeme Robb for his model of the GPR120 binding site and both for helpful discussions of my data. I have especially enjoyed my time being part of the Institute of Cell Signalling, not least for the many cakes over the years! Thanks to all the ICS members, past and present, for scientific support and for the great times had at home and away at conferences overseas. Special thank-yous go to Laura Kilpatrick for answering every little question I have ever had; Dr Leigh Stoddart for being my point of reference for FFA receptors; Dr Rachel Thomas for her supportive pep talks; Marleen Groenen for her assistance, Dr Steve Briddon for his help with FCS and Tim Self for his training and expertise when using the microscopes. I would also like to thank my family and my friends outside of science, who still have no idea what I do, but are supportive nonetheless. Avslutningsvis vill jag tacka mina svenska vänner Josie Gilbert, Lars Bergström, och speciellt Jens Haapalahti… tack så mycket! iii Papers Watson SJ, Brown AJ, Holliday ND (2012) Differential signaling by splice variants of the human free fatty acid receptor GPR120 Mol Pharmacol. 81(5):631-42 Holliday ND, Watson SJ, Brown AJ (2012) Drug discovery opportunities and challenges at G protein-coupled receptors for long chain free Fatty acids Front Endocrinol (Lausanne) 3;2:112 iv Abbreviations 2-APB 2-aminoethoxydiphenyl borate AF AlexaFluor β2AR β2 adrenoceptor BG benzyl guanine BRET bioluminescence resonance energy transfer BSA bovine serum albumin CCK cholecystokinin cAMP adenosine-3',5' cyclic monophosphate DHA docosahexaenoic acid DMEM Dulbecco's modified Eagle's medium DMSO dimethyl sulphoxide EC50 concentration for 50% maximal response ECL extracellular loop ERK extracellular signal-related protein kinase FBS foetal bovine serum FCS fluorescence correlation spectroscopy FFA free fatty acid FRET fluorescence resonance energy transfer GDP guanosine diphosphate GEF guanine nucleotide exchange factor GFP green fluorescent protein GI gastrointestinal v GIP glucose-dependent insulinotropic peptide GLP-1 glucagon-like peptide-1 GPCR G protein-coupled receptor GRK GPCR related kinase GSIS glucose stimulated insulin secretion GTP guanosine 5’ triphosphate HBSS Hepes buffered saline solution HEK human embryonic kidney IC50 concentration for 50% inhibition ICL intracellular loop IP3 inositol 1,4,5-trisphosphate JNK c-Jun N-terminal kinase Ki equilibrium dissociation constant LB Luria Bertani MAPK mitogen activated protein kinase Met36 Metabolex compound 36 PP2A protein phosphatase 2A PPAR perioxisome proliferator-activated receptor PBS phosphate buffered saline PCH photon counting histogram PDE phosphodiesterase PIP2 phosphatidylinositol 4,5-bisphosphate PLC phospholipase C vi PTx Pertussis toxin PUFA polyunsaturated fatty acid PKA/C Protein kinase A or C S1P sphingosine-1-phosphate SOCE store operated calcium entry TM transmembrane helix TZD thiazolidinedione vYFP venus yellow fluorescent protein vYC carboxyl fragment (173-238) venus yellow fluorescent protein vYN amino fragment (2-172) venus yellow fluorescent protein WT wild type vii Table of contents Chapter One: Introduction .......................................................................................................... 1 1.1 Fatty acids ......................................................................................................................... 1 1.2 Types of FFA signalling ...................................................................................................... 2 1.2.1 Effects via metabolism and as signalling precursors .................................................. 2 1.2.2 The influence of FFAs on cellular membranes ........................................................... 3 1.2.3 The PPAR family of nuclear receptors ........................................................................ 6 1.2.4 Fatty acid receptor family of GPCRs ........................................................................... 7 1.3 G protein-coupled receptors ........................................................................................... 12 1.3.1 Classification ............................................................................................................ 12 1.4 Long chain fatty acid GPCRs, FFA1 and GPR120 ............................................................. 15 1.4.1 FFA GPCR pharmacology .......................................................................................... 15 1.4.2 Polymorphisms found in FFA1 and GPR120 ............................................................. 16 1.4.3 Physiological roles of FFA1 and GPR120 .................................................................. 19 1.4.4 FFA1 and GPR120 as targets for diabetes and obesity ............................................ 24 1.4.5 Evaluation of FFA1 and GPR120 as therapeutic targets........................................... 28 1.5 The general molecular pharmacology of GPCRs ............................................................. 33 1.5.1 Structure and activation of class A GPCRs ............................................................... 33 1.6 Signalling & Trafficking .................................................................................................... 45 1.6.1 G proteins ................................................................................................................. 45 1.6.2 β-arrestin .................................................................................................................. 53 1.6.3 β-arrestin dependent signalling ..............................................................................
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