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J. Biol. Chem., Vol. 280, Issue 31, 28241-28250, August 5, 2005

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Phosphorylation of GRK1 and GRK7 by cAMP-dependent Protein Kinase Attenuates Their Enzymatic Activities^*

Thierry J. Horner, Shoji Osawa, Michael D. Schaller, and Ellen R. Weiss¶

From the Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, Carolina 27599-7090

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K_m and V_max of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser²¹ and GRK7 at Ser²³ and Ser³⁶ in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca²⁺/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.

Received for publication, May 10, 2005

^* This work was supported by National Institutes of Health Grants GM43582 (to E. R. W.) and EY12224 (to S. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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: Dept. of Cell and Developmental Biology. The University of North Carolina at Chapel Hill, CB# 7090, 108 Taylor Hall, Chapel Hill, NC 27599-7090. Tel.: 919-966-7683; Fax: 919-966-1856; E-mail: erweiss{at}med.unc.edu.

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