Direct coupling of a seven-transmembrane-span receptor to a G?i G-protein regulatory motif complex

Abstract

Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for G?iGDP independent of G?? and stabilize the GDP-bound conformation of G?i, acting as guanine nucleotide dissociation inhibitors. The G?GPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the G?GPR complex in a manner analogous to receptor coupling to the G??? heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of G?GPR in the intact cell when G?i2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the ?2A adrenergic receptor (?2AAR-G?i2 YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered G?iYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-G?i-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered G?i2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous G?i/o?? was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of a2AAR-G?i2 YFPC352I:AGS3Rluc and ?2AAR-G?i2 YFPC352I:AGS4Rluc BRET was not altered by PT treatment or G?? antagonists. Thus, the localized regulation of G?GPR by receptor activation appears independent of endogenous G?i/o??, suggesting that G?iAGS3 and G?iAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the G??? heterotrimer. The direct coupling of a receptor to the G?iGPR complex provides an unexpected platform for signal propagation with broad implications. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.