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J. Biol. Chem., Vol. 281, Issue 11, 7684-7692, March 17, 2006

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Role of the G Protein-coupled Receptor Kinase Site Serine Cluster in ₂-Adrenergic Receptor Internalization, Desensitization, and -Arrestin Translocation^*

David J. Vaughan, Ellen E. Millman, Veronica Godines, Jacqueline Friedman, Tuan M. Tran, Wenping Dai^¶, Brian J. Knoll^¶, Richard B. Clark, and Robert H. Moore¹

From the Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, Department of Integrative Biology and Pharmacology, University of Texas Health Sciences Center, Houston, Texas 77030, and ^¶Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, Texas 77204

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of ₂-adrenergic receptors (₂ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in -arrestin-dependent ₂AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nM isoproterenol for 5 min induced 2-fold increases in the EC₅₀ for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 µM isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)--arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP--Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent -arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced -arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, -arrestin binding, and internalization of ₂ARs.

Received for publication, January 10, 2005 , and in revised form, December 29, 2005.

^* This work was supported by National Institutes of Health Grants HL64934 (to R. H. M.) and GM031208 (to R. B. C.) and by American Heart Association, Texas affiliate Grant 0455072Y (to B. J. K). 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 Pediatrics, Baylor College of Medicine, 6621 Fannin, CCC1040, Houston, TX 77030. Tel.: 832-824-3312; Fax: 832-825-4255; E-mail: rmoore{at}bcm.tmc.edu.

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