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Originally published In Press as doi:10.1074/jbc.M001864200 on July 6, 2000

J. Biol. Chem., Vol. 275, Issue 38, 29724-29730, September 22, 2000
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Phosphorylation of Phosducin and Phosducin-like Protein by G Protein-coupled Receptor Kinase 2*

Ana Ruiz-GómezDagger , Jan Humrich§, Cristina MurgaDagger , Ursula Quitterer§, Martin J. Lohse§, and Federico Mayor Jr.Dagger

From the Dagger  Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, E-28049 Madrid, Spain and the § Institut für Pharmakologie und Toxikologie der Universität Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbeta gamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 ± 0.1 and 0.83 ± 0.09 mol of Pi/mol of protein, respectively). The phosphorylation reactions exhibit apparent Km values in the range of 40-100 nM, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with Gbeta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its Gbeta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

* This work was supported by Grants PM95-0033 and PM98-0020 from the Ministerio de Educación y Cultura, Grant 8.4/6/98 from Comunidad de Madrid (to F. M., Jr.), Grant BMH4-98-3566 from the European Union (to F. M., Jr. and M. J. L.), and the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to M. J. L.). The Centro de Biología Molecular holds an institutional grant from the Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 34-91-3974865; Fax: 34-91-3974799; E-mail: fmayor@cbm.uam.es.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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