Direct Coupling of a Seven-Transmembrane-Span Receptor to a G alpha i G-Protein Regulatory Motif Complex

作者：Robichaux William G III; Oner Sukru S; Lanier Stephen M; Blumer Joe B^*

来源：Molecular Pharmacology, 2015, 88(2): 231-237.

DOI：10.1124/mol.115.097741

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 alpha i(GDP) independent of G beta gamma and stabilize the GDP-bound conformation of G alpha i, acting as guanine nucleotide dissociation inhibitors. The G alpha 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 alpha GPR complex in a manner analogous to receptor coupling to the G alpha beta gamma heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of G alpha GPR in the intact cell when G alpha i(2) yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the alpha(2A) adrenergic receptor (alpha(2A)AR-G alpha i(2)YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered G alpha iYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-G alpha i-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered G alpha i(2) was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous G alpha i/o beta gamma was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of alpha(2A)AR-G alpha i(2)YFP(C352I):AGS3Rluc and alpha(2A)AR-G alpha i(2)YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or G beta gamma antagonists. Thus, the localized regulation of G alpha GPR by receptor activation appears independent of endogenous G alpha i/o beta gamma, suggesting that G alpha iAGS3 and G alpha iAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the G alpha beta gamma heterotrimer. The direct coupling of a receptor to the G alpha iGPR complex provides an unexpected platform for signal propagation with broad implications.

• 出版日期2015-8