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J. Biol. Chem., Vol. 280, Issue 41, 35051-35061, October 14, 2005

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Chemical Genetic Engineering of G Protein-coupled Receptor Kinase 2^*

Denise M. Kenski, Chao Zhang, Mark von Zastrow¶, and Kevan M. Shokat1

From the Chemistry and Chemical Biology Graduate Program, ^¶Department of Psychiatry, and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143

G protein-coupled receptor kinases (GRKs) play a pivotal role in receptor regulation. Efforts to study the acute effects of GRKs in intact cells have been limited by a lack of specific inhibitors. In the present study we have developed an engineered version of GRK2 that is specifically and reversibly inhibited by the substituted nucleotide analog 1-naphthyl-PP1 (1Na-PP1), and we explored GRK2 function in regulated internalization of the µ-opioid receptor (µOR). A previously described method that conferred analog sensitivity on various kinases, by introducing a space-creating mutation in the conserved active site, failed when applied to GRK2 because the corresponding mutation (L271G) rendered the mutant kinase (GRK2-as1) catalytically inactive. A sequence homology-based approach was used to design second-site suppressor mutations. A C221V second-site mutation produced a mutant kinase (GRK2-as5) with full functional activity and analog sensitivity as compared with wild-type GRK2 in vitro and in intact cells. The role of GRK2-as5 activity in the membrane trafficking of the µOR was also characterized. Morphine-induced internalization was completely blocked when GRK2-as5 activity was inhibited before morphine application. However, inhibition of GRK2-as5 during recycling and reinternalization of the µOR did not attenuate these processes. These results suggest there is a difference in the GRK requirement for initial ligand-induced internalization of a G protein-coupled receptor compared with subsequent rounds of reinternalization.

Received for publication, July 13, 2005 , and in revised form, August 3, 2005.

^* This work was supported by National Institutes of Health Grant AI44009. 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.

¹ To whom correspondence should be addressed: Dept. of Cellular and Molecular Pharmacology, University of California, 600 16th St., San Francisco, CA 94143. Tel.: 415-514-0472; Fax: 415-514-0822; E-mail: shokat{at}cmp.ucsf.edu.

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