The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gβγ binding site

Abstract

G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gβγ subunits. A Gβγ binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gβγ binding site of GRK2 also regulates Gβγ-stimulated signaling by sequestering free Gβγ subunits. We report here that truncation of the carboxyl-terminal Gβγ binding site of GRK2 did not abolish the Gβγ regulatory activity of GRK2 as determined by the inhibition of a Gβγ-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gβγ binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK21-185) inhibited a Gβγ-stimulated inositol phosphate signal in cells, purified GRK21-185 suppressed the Gβγ-stimulated phosphorylation of rhodopsin, and GRK21-185 bound directly to purified Gβγ subunits. The amino-terminal Gβγ regulatory site does not overlap with the RGS domain of GRK-2 because GRK21-53 with truncated RGS domain inhibited Gβγ-mediated signaling with similar potency and efficacy as did GRK21-185. In addition to the Gβγ regulatory activity, the amino-terminal Gβγ binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified Gβγ subunits, strongly suggesting that Gβγ binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gβγ binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gβγ-stimulated signaling.