Interaction between TRPs and GPCRs as a basis for developing TRP biosensors

[Speaker] Richard Leduc:1
[Co-author] Brian Holleran:1, David St-Pierre:1, Audrey Collette:1, Guylain Boulay:1
1:Pharmacology-Physiology, University of Sherbrooke, Canada

Background: GPCRs and transient receptor potential (TRP) channels are both plasma membrane proteins involved in cell signaling. Recent results have revealed that coupling between TRPC3 and activated angiotensin II, AT1 receptor (AT1R) mediates catecholamine secretion. Because another TRP, TRPC6, was shown to be a Gq dependant calcium channel, we believed that developing BRET (bioluminescence resonance energy transfer)-based biosensor would help us better understand the mechanisms by which this coupling mediates calcium signalling.

Methods: We fused GFP10 on the N-terminal extremity of TRPC6 and RlucII at the C-terminal end (GFP10-TRPC6-RlucII). To characterize the biosensor, HEK293 cells were transfected with GFP10-TRPC6-RlucII together with various GPCRs.

Results: Upon co-expression of GFP10-TRPC6-RlucII with AT1R or TPalpha, both Gq coupled receptors, stimulation of the GPCR with AngII and U46619 respectively, caused a decrease in the BRET signal, which was abolished by treatment with UBO-QIC, a Gq inhibitor. Co-expression of GFP10-TRPC6-RlucII with Gi-coupled receptors (MOR, DOR or OPRL1) or stimulation of endogenous VIP receptors (Gs-coupled) did not lead to a BRET response. The GFP10-TRPC6-RlucII biosensor was insensitive to treatment with thapsigargine, which causes release of intracellular Ca2+. Interestingly, when cells co-expressing both AT1R and GFP10-TRPC6-RlucII were treated with [Sar1Ile8]AngII, a beta-arrestin biased ligand unable to activate Gq, the BRET signal was strongly decreased.

Conclusions: These results validate our BRET-based approach to study signaling events mediated by GqPRCs and TRPs.

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