Identification of a G protein-coupled receptor for 11(R),12(S)-epoxyeicosatrienoic acid and its effects in the cardiovascular system
Pharmacology
Cell Biology
Final Report Abstract
Epoxyeicosatrienoic acids (EET) are epoxides of arachidonic acid generated by cytochrome P450 (CYP) enzymes that have been attributed anti-inflammatory effects in numerous experimental models. One way to increase cellular and tissue levels of the EETs is to prevent their metabolism to less active diols by the soluble epoxide hydrolase (sEH). While sEH inhibitors have demonstrated some benefit, concerns have been raised about side effects of sEH inhibition in the lung and on tumor development and growth. One potential solution, that could avoid the side effects of general sEH inhibition, is to develop specific agonists of the EET receptor. The only problem with this approach is that a plasma membrane receptor that is specific for EETs and unaffected by other lipid mediators has not been reported. This study was initiated to identify a specific receptor for 11,12-EET in vascular endothelial cells and to link its activation with the rapid and delayed actions of the epoxide in vitro and in vivo. We report here that GPR124 is required for the biological actions of 11,12-EET and appears to react specifically to 11(R),12(S)-EET and not 11(S),12(R)-EET. When stimulated with (±)11,12-EET, GPR124 associated with -arrestin before being internalized and eventually recycled to the plasma membrane. GPR124 was also required for the rapid (within 10 seconds) translocation of TRPC6 channels of the plasma membrane as well as the delayed (hours) effects of 11,12-EET on angiogenesis. 11,12-EET is perhaps best known for its effects on vascular function and its role in responses attributed to the endothelium-derived hyperpolarizing factor (EDHF). In inducible, GPR124 knockout mice the vasodilator response to (±)11,12-EET was abrogated, as was the EDHF-component of the vasodilatation to acetylcholine, and the acetylcholine-induced hyperpolarization of murine endothelial cells. The GPR124 interactome was studied to gain insight into the signalling pathways activated by the binding of 11,12-EET to GPR124. This approach revealed that the catalytic α and subunits of PKA and the transforming growth factor (TGF) type I receptor ALK1 associated with GPR124. Consistent with the clear effects of (±)11,12-EET on endothelial cell proliferation and angiogenesis, the pathways most significantly regulated by (±)11,12-EET via GPR124 were related to the cell cycle, DNA replication and DNA repair, but also included vascular development, response to oxidative stress, cholesterol transport and TGF-/SMAD signalling. Taken together, our data have identified GPR124 as a receptor for 11(R),12(S)-EET on endothelial cells and required for the acute i.e., hyperpolarization, relaxation and TRPC6 channel translocation, as well as chronic i.e. induction of cell proliferation and angiogenesis, actions of the arachidonic acid epoxide. It will be interesting to determine to what extent the activation of GPR124 signalling can also account for the reported anti-inflammatory actions of 11,12-EET in other cell types. Studies are ongoing to demonstrate definitively the binding of 11,12-EET to the GPR124 protein. We see potential for agonists of GPR124 to exploit the anti-inflammatory actions of 11,12-EET without the side effects of the sEH inhibitors. However, without a robust high throughput screening system (the translocation of TRPC6 is likely to prove too difficult to evaluate in such a platform), the search for specific agonists will have to be abandoned. Plans are being made to identify fluorescent tags for the extracellular portion of TRPC6 that may help solve the problem.
