Direct coupling of a seven-transmembrane-span receptor to a Gαi 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 Gαi GDP independent of Gβγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR 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 GαGPR complex in a manner analogous to receptor coupling to the Gαβγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi 2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α 2A adrenergic receptor (α 2A AR-Gαi 2 YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi 2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oβγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of a 2A AR-Gαi 2 YFP C352I :AGS3Rluc and α 2A AR-Gαi 2 YFP C352I :AGS4Rluc BRET was not altered by PT treatment or Gβγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oβγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαβγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.