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J. Biol. Chem., Vol. 279, Issue 9, 7999-8010, February 27, 2004

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The Role of Phosphorylation in D₁ Dopamine Receptor Desensitization

EVIDENCE FOR A NOVEL MECHANISM OF ARRESTIN ASSOCIATION^*

Ok-Jin Kim, Benjamin R. Gardner¶, Daniel B. Williams||, Paul S. Marinec, David M. Cabrera, Jennifer D. Peters**, Chun C. Mak, Kyeong-Man Kim, and David R. Sibley

From the Molecular Neuropharmacology Section, NINDS, National Institutes of Health, Bethesda, Maryland 20892-1406 and College of Pharmacy, Chonnam National University, KwangJu, 500-757, Korea

Homologous desensitization of D₁ dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D₁ receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.

Received for publication, July 29, 2003 , and in revised form, November 5, 2003.

^* 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.

The on-line version of this article (available at http://www.jbc.org) contains movies.

Both authors contributed equally to this work.

^¶ Present address: Pfizer Ltd., Ramsgate Rd., Sandwich, Kent CT13 9NJ, UK.

^|| Present address: Dept. of Natural Sciences, New Mexico Highlands University, Las Vegas, NM 87701.

^** Present address: Dept. of Psychology, Princeton University, Princeton, NJ 08544.

To whom correspondence should be addressed: Molecular Neuropharmacology Section, NINDS/National Institutes of Health, Bldg. 10, Rm. 5C108, 10 Center Dr., MSC 1406, Bethesda, MD 20892-1406. Tel.: 301-496-9316; Fax: 301-496-6609; E-mail: sibley{at}helix.nih.gov.

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