TY - JOUR T1 - Direct Coupling of a Seven Transmembrane Span Receptor to a GαiGPR Complex JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.115.097741 SP - mol.115.097741 AU - William G. Robichaux AU - Sukru Sadik Oner AU - Stephen M. Lanier AU - Joe B. Blumer Y1 - 2015/05/13 UR - http://molpharm.aspetjournals.org/content/early/2015/05/12/mol.115.097741.abstract N2 - Group II Activators of G-protein Signaling (AGS) proteins contain one or more G-protein regulatory (GPR) motifs, which serve as docking sites for GαiGDP 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 receptors(7TMR) in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TMR directly couples to the GαGPR complex in a manner analogous to receptor coupling to Gαβγ heterotrimer. As an initial approach to test this hypothesis we utilized BRET to examine 7TMR-mediated regulation of GαGPR in the intact cell when Gαi2YFP was tethered to the carboxyl-terminus of the α2A/D-adrenergic receptor (α2A/DAR-Gαi2YFP). AGS3- and AGS4-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 co-immunoprecipitation and cell fractionation. The tethered Gαi2βγ was rendered pertussis toxin-insensitive by a C352I mutation and receptor coupling to endogenous Gαi/oβγ was subsequently eliminated by cell treatment with PT. Basal and agonist-induced regulation of α2A/DAR-Gαi2YFPC352I:AGS3-Rluc and α2A/DAR-Gαi2YFPC352I:AGS4-Rluc BRET was not altered by PT treatment or Gβγ antagonists. Thus, the localized GαGPR interaction 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 7TMR coupling to Gαβγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications. ER -