@article {Robichauxmol.115.097741, author = {William G. Robichaux and Sukru Sadik Oner and Stephen M. Lanier and Joe B. Blumer}, title = {Direct Coupling of a Seven Transmembrane Span Receptor to a GαiGPR Complex}, elocation-id = {mol.115.097741}, year = {2015}, doi = {10.1124/mol.115.097741}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {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.}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2015/05/12/mol.115.097741}, eprint = {https://molpharm.aspetjournals.org/content/early/2015/05/12/mol.115.097741.full.pdf}, journal = {Molecular Pharmacology} }