The Amino-terminal Domain of G-protein-coupled Receptor Kinase 2 Is a Regulatory Gβγ Binding Site

• Published in: The Journal of biological chemistry 2003-03, Vol.278 (10), p.8052
• Main Authors: Tanja Eichmann, Kristina Lorenz, Michaela Hoffmann, Jörg Brockmann, Cornelius Krasel, Martin J. Lohse, Ursula Quitterer
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M204795200

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 (GRK2 1–185 ) inhibited a Gβγ-stimulated inositol phosphate signal in cells, purified GRK2 1–185 suppressed the Gβγ-stimulated phosphorylation of rhodopsin, and GRK2 1–185 bound directly to purified Gβγ subunits. The amino-terminal Gβγ regulatory site does not overlap with the RGS domain of GRK-2 because GRK2 1–53 with truncated RGS domain inhibited Gβγ-mediated signaling with similar potency and efficacy as did GRK2 1–185 . 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.