Identification of a G protein-coupled receptor for 11(R),12(S)-epoxyeicosatrienoic acid and its effects in the cardiovascular system

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Epoxyeicosatrienoic acids (EETs) are generated from arachidonic acid by cytochrome P450 (CYP) enzymes as different regioisomers and play a central role in cellular processes including Ca2+ signalling, hyperpolarization, intracellular communication, angiogenesis, and the prevention or resolution of inflammation as well as the control of vascular tone and blood pressure. Work from our group has demonstrated that 11,12-EET seems to be the biologically more active of the regioisomers and able to elicit rapid cellular responses, the fastest in endothelial cells being the translocation of transient receptor (TRP) C3/C6 channels to caveolae. Exactly how 11,12-EET initiates cell signalling has yet to be resolved. Despite this there is a considerable amount of circumstantial evidence to indicate that a specific G protein coupled membrane receptor for 11,12-EET; more specifically 11(R),12(S)-EET, exists: (1) high affinity EET binding sites have been detected on the surface of some cells, (2) different EET stereoisomers, and regioisomers show distinct biological effects, (3) stable and specific EET agonists and antagonists have been generated to mimic or prevent the actions of 11,12-EET, (4) 11,12-EET induced cellular responses; such as cell proliferation, gap junctional communication or TRP channel translocation, are absolutely reliant on the activation of protein kinase A (PKA), moreover, (5) the downregulation of Gs but not Gq/11 proteins abolishes endothelial cell sensitivity to 11,12-EET. Identifying a receptor for 11(R),12(S)-EET receptor is not straight forward given that the agonist is a fatty acid epoxide, this means that many classical binding studies and labelling approaches used to identify peptide receptors are not easily applicable. Also although many of the effects of the EETs rely on PKA activation, the EET-induced changes in cyclic AMP tend to be small and inconsistent, particularly in endothelial cells.In preliminary studies it has been possible to demonstrate GPR124 as a candidate 11,12-EET receptor in endothelial cells. Indeed, the downregulation of GPR124 abolishes the 11,12-EET-induced translocation of TRPC6 channels as well as 11,12-EET-induced endothelial cell proliferation (responses to VEGF were unaffected) and endothelial cell sprouting. These effects correlated with changes in the cellular distribution of the receptor. Our hypothesis is that GPR124 is the receptor for 11(R),12(S)-EET and is required for the biological actions of the epoxide in endothelial cells. The aims of this project are to (1) determine whether GPR124 displays characteristics of a receptor for 11,12-EET by assessing its sensitivity to EET analogues (so called EET agonists and antagonists), as well as receptor desensitization, internalization and recycling, (2) determine the downstream signals activated by GPR124 in the presence of 11,12-EET, and (3) to determine whether the deletion of GPR124 in adult mice alters biological responses to 11,12-EET.