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2017Raihanphd (2).Pdf Raihan, Sheikh Zahir (2017) Desensitisation of the human long chain fatty acid receptors FFA1 and FFA4. PhD thesis. http://theses.gla.ac.uk/8144/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten:Theses http://theses.gla.ac.uk/ [email protected] Desensitisation of the human long chain fatty acid receptors FFA1 and FFA4 Sheikh Zahir Raihan M.Pharm, B.Pharm (Hons) A thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy February 2017 ii Abstract G protein-coupled receptors (GPCRs) constitute the largest, most ubiquitous and most versatile family of membrane proteins encoded by the human genome. Due to diverse ligands and multiple physiological activities, this set of receptors has frequently been explored as potential drug targets. Deorphanisation of GPCRs successfully identified FFA1 and FFA4 (previously named GPR40 and GPR120) as long chain free fatty acid receptors. With diverse expression patterns and close association to pathophysiology of metabolic diseases, both receptors are being studied to understand both receptor pharmacology and their potential for drug development. Due to the overlap in the activation of FFA1 and FFA4 by endogenous fatty acid ligands, selective synthetic ligands have been developed for these receptors. Using a number of biochemical and biophysical assays, I have characterised TUG-770, TUG-905 and GW-1100 as FFA1 ligands and TUG- 891, TUG-1197 and TUG-1275 as FFA4 ligands. TUG-905 was found to be most potent and selective FFA1 agonist and GW-1100 showed insurmountable antagonism at FFA1. At FFA4, TUG-1197 was found to be a highly potent and selective agonist. TUG-1275 showed insurmountable antagonism at FFA4 in β- arrestin2 recruitment, receptor internalisation and inositol monophosphate accumulation studies and showed complete selectivity for hFFA4. Agonist exposure rapidly phosphorylated FFA4 in an agonist-concentration-dependent fashion which was totally blocked by TUG-1275. The protein kinase C activator PMA was also noted to phosphorylate FFAA in a concentration-dependent manner. Thus both homologous and heterologous phosphorylation is involved in FFA4 regulation. The FFA4-agonist TUG-891 produced robust internalisation of FFA4 as detected by each of confocal microscopy, and both cell surface ELISA and biotinylation. PMA was able to internalise FFA4 although it was unable to recruit β-arrestin2 to FFA4 suggesting that this internalisation might not be β- arrestin2-dependent. Constitutive internalisation was seen for FFA1, where the selective FFA1 antagonist GW-1100 had no effect. Repeated agonist-exposure desensitised both FFA1 and FF4 as revealed in single-cell calcium imaging studies. Although there was a small reduction of FFA4-internalisation and a slight elevation of total calcium levels from a single-chronic exposure of agonist, elimination of β-arrestin1/2 from HEK293 cells by genome editing did not significantly change the desensitisation of FFA4 to repeated exposure of agonist iii and did not prevent agonist-promoted internalisation. These studies indicate that β-arrestins are not the sole factors in desensitisation of human FFA4. Gαq/11 elimination by genome editing completely blocked intracellular calcium mobilisation and accumulation of inositol monophosphates mediated by both FFA1 and FFA4 indicating that Gαq/11 coupling to agonist-activated receptors is essential for this functional signalling outcome via both receptors. FFA4 expressed in Gαq/11-null cells was found to be phosphorylated by agonist, indicating that phosphorylation-mediated desensitisation of this receptor is not dependent on Gαq/11 proteins. FRET and BRET experiments revealed for the first time homo and hetero-oligomerisation of both FFA1 and FFA4. Although ligand regulation of oligomerisation was not investigated these preliminary observations of oligomerisation may help in the future to answer many questions of regulation and desensitisation of these receptors. The selective FFA1 and FFA4 ligands characterised here in this project might be used as tool compounds to further explore the physiology and pharmacology of these therapeutically important receptors. iv Contents Abstract ............................................................................................................. ii Contents ............................................................................................................ iv List of Tables .................................................................................................... viii List of Figures...................................................................................................... ix Acknowledgement .............................................................................................. xiii Author’s Declaration ........................................................................................... xiv Abbreviations ..................................................................................................... xv 1 Introduction .............................................................................................. 1 1.1 G protein-coupled receptors .......................................................................... 1 1.1.1 Structural feature of GPCRs ........................................................................... 1 1.1.2 GPCR activation ......................................................................................... 4 1.1.3 GPCR signalling .......................................................................................... 4 1.1.4 Classification of GPCRs ................................................................................. 8 1.1.5 Heterotrimeric G proteins and their effectors ..................................................... 8 1.1.6 GPCR biased signalling ................................................................................. 9 1.1.7 GPCR oligomerisation ................................................................................. 12 1.1.8 GPCR deorphanisation ................................................................................. 17 1.2 Free fatty acid receptors ............................................................................. 18 1.3 Short chain fatty acid receptors ..................................................................... 20 1.4 Long chain fatty acid receptors ..................................................................... 23 1.4.1 Free fatty acid receptor 1 (FFA1) ................................................................... 23 1.4.2 Free fatty acid receptor 4 ............................................................................ 31 1.5 Receptor desensitisation .............................................................................. 44 1.5.1 Receptor phosphorylation ............................................................................ 48 1.5.2 Receptor internalisation .............................................................................. 49 1.5.3 Receptor recycling and resensitisation ............................................................. 51 1.5.4 Receptor down regulation ............................................................................ 51 1.5.5 β-arrestin and GPCR trafficking ..................................................................... 52 1.6 Aims of the project .................................................................................... 53 2 Materials and Methodology ........................................................................... 55 2.1 Materials ................................................................................................. 55 2.1.1 General Reagents, Enzymes and Kits ............................................................... 55 2.1.2 Antibodies and antisera ............................................................................... 58 2.1.3 Tissue culture ........................................................................................... 58 2.1.4 Buffers and solutions .................................................................................. 60 2.1.5 Pharmacological test compounds ................................................................... 62 2.2 Molecular Biology Protocols .......................................................................... 63 2.2.1 Preparation of Luria Bertani borth .................................................................. 63 2.2.2 Preparation of LB plates .............................................................................. 63 2.2.3 Preparation of competent bacterial cells ......................................................... 63 v 2.2.4 Transformation of competent bacterial cells ..................................................... 64 2.2.5 Preparation of plasmid DNA .......................................................................... 65 2.2.6 Quantification of DNA ................................................................................. 66 2.2.7 Polymerase chain reaction
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