Investigation of the function, pharmacology and oligomerisation of GPR40, GPR41 and GPR43

Stoddart, Leigh Ann (2007) Investigation of the function, pharmacology and oligomerisation of GPR40, GPR41 and GPR43. PhD thesis, University of Glasgow.

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GPR40, GPR41 and GPR43 are a small family of GPCRs. Fatty acids were identified as the ligands for these receptors in 2003. High levels of circulating fatty acids have been linked to a variety of diseases, including type 2 diabetes mellitus, cancer, and high levels of fatty acids are produced by anaerobic bacteria at the site of infection. High levels of GPR40 have been detected in the beta cells of the pancreas and all three receptors have been shown to be expressed in immune cells. Due to the recent identification of the ligand for these receptors, their pharmacology, function and oligomerisation was investigated during this study. A [35S]GTPgammaS binding assay was developed to monitor the activation of GPR41 using a GPR41-Gαi3 Cys351Ile fusion protein. Using the fusion protein improved the signal to background ratio and allowed the potency of a variety of short chain fatty acids to be determined. A GPR40-Gαq fusion protein was also generated, but basal levels of [35S]GTPgammaS binding in Gαq immunoprecipitates was high and did not increase significantly upon the addition of long chain fatty acids. Treatment of membranes expressing GPR40-Gαq with fatty acid free BSA reduced the basal [35S]GTPgammaS binding in a concentration dependent manner and allowed the responsiveness of GPR40 to fatty acids to be uncovered. This also allowed the potency of a variety of thiazolidinediones and small molecule agonists to be determined. It was found that troglitazone was the most potent thiazolidinedione tested. Rosiglitazone also acted as an agonist of GPR40, albeit with lower potency than troglitazone, whereas ciglitazone and pioglitazone displayed little potency. Using clones of Flp-In TREx HEK293 cells where GPR40-eYFP was cloned into the Flp-In locus, the expression of GPR40-eYFP could be controlled by the addition of doxycycline. Using this cell line confirmed that GPR40 mediated the rise in [Ca2+]i induced by the addition of troglitazone. The expression of GPR40 was detected in the rat beta cell line, INS-1E. Addition of lauric acid or troglitazone to these cells induced a large, transient rise of [Ca2+]i. Membranes prepared from INS-1E cells also displayed high basal [35S]GTPgammaS binding, which could be reduced by fatty acid free BSA. The combination of fatty acid free BSA and lauric acid or troglitazone increased [35S]GTPgammaS binding. The high levels of [35S]GTPgammaS binding observed in membranes expressing GPR40 may reflect the binding of an endogenous ligand to GPR40. Homology models of all three receptors based on the crystal structure of rhodopsin indicated that a conserved Arg residue in TM5 may co-ordinate the carboxylate group of the fatty acid. To confirm this observation a series of mutant receptors were generated, with the Arg residue in TM5 mutated to alanine. Neutralisation of the charge in TM5 in GPR41 and GPR43 resulted in receptors that were unable to respond to short chain fatty acids. Further mutants of GPR43 were generated in which the Arg in TM5 was replaced by Lys, Leu or Ser. None of these mutants were able mediate a rise in [Ca2+]i in response to short chain fatty acids. The equivalent mutation in GPR40 did not abolish the receptors ability to respond to long chain fatty acids. There are two further conserved basic residues in TM regions of the receptors; an Arg/Lys in TM2 and a His in TM4. These residues were also mutated to alanine. In GPR41 and GPR43, both mutants had similar function to the wild type receptors. Both GPR40 mutants were also able to respond to a variety of fatty acids measured by their ability to mediate a rise in [Ca2+]i in a FLIPR based assay system. A series of further GPR40 mutants were generated where two of the three basic TM residues were mutated. The most striking observation was found with the mutant in which the His in TM4 and Arg in TM5 mutated to alanine, this mutant was unable to mediate a rise in [Ca2+]i in response to a variety of saturated fatty acids. This may indicate that upon loss of charge in TM5 the His in TM4 compensates and vice versa. None of the small molecule agonists or thiazolidinediones were able to activate the TM4 or TM5 mutants. This indicated that the synthetic agonists of GPR40 require a more conserved binding pocket which may be due to their more rigid structure. The ability of GPR40 and GPR43 to form homo-oligmers was also investigated. It was found that GPR40 and GPR43 formed homo-oligomers as monitored by co-immunoprecipitation, FRET in single, living cells and by time resolved FRET.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: orphan GPCR, GRP40, GPR41, GPR43, mutagenesis, oligmerisation
Subjects: R Medicine > RM Therapeutics. Pharmacology
Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Supervisor's Name: Graeme, Professor Milligan
Date of Award: 2007
Depositing User: Morag Greig
Unique ID: glathesis:2007-1
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 03 Oct 2007
Last Modified: 10 Dec 2012 13:14

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