Characterization of the GRK2 binding site of Gαq

Abstract

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transmit signals from membrane bound G protein-coupled receptors (GPCRs) to intracellular effector proteins. The Gq subfamily of Gα subunits couples GPCR activation to the enzymatic activity of phospholipase C-β (PLC-β). Regulators of G protein signaling (RGS) proteins bind to activated Gα subunits, including Gαq, and regulate Gα signaling by acting as GTPase activating proteins (GAPs), increasing the rate of the intrinsic GTPase activity, or by acting as effector antagonists for Gα subunits. GPCR kinases (GRKs) phosphorylate agonist-bound receptors in the first step of receptor desensitization. The amino termini of all GRKs contain an RGS homology (RH) domain, and binding of the GRK2 RH domain to Gαq attenuates PLC-β activity. The RH domain of GRK2 interacts with Gαq/11 through a novel Gα binding surface termed the "C" site. Here, molecular modeling of the Gαq·GRK2 complex and site-directed mutagenesis of Gαq were used to identify residues in Gαq that interact with GRK2. The model identifies Pro185 in Switch I of Gαq as being at the crux of the interface, and mutation of this residue to lysine disrupts Gαq binding to the GRK2-RH domain. Switch III also appears to play a role in GRK2 binding because the mutations Gαq-V240A, Gαq-D243A, both residues within Switch III, and Gαq-Q152A, a residue that structurally supports Switch III, are defective in binding GRK2. Furthermore, GRK2-mediated inhibition of Gαq-Q152A-R183C-stimulated inositol phosphate release is reduced in comparison to Gαq-R183C. Interestingly, the model also predicts that residues in the helical domain of Gαq interact with GRK2. In fact, the mutants Gαq-K77A, Gαq-L78D, Gαq-Q81A, and Gαq-R92A have reduced binding to the GRK2-RH domain. Finally, although the mutant Gαq-T187K has greatly reduced binding to RGS2 and RGS4, it has little to no effect on binding to GRK2. Thus the RH domain A and C sites for Gαq interaction rely on contacts with distinct regions and different Switch I residues in Gαq.