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J. Biol. Chem., Vol. 280, Issue 46, 38346-38354, November 18, 2005

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-Arrestin Mediates Desensitization and Internalization but Does Not Affect Dephosphorylation of the Thyrotropin-releasing Hormone Receptor^*

From the Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to -arrestin after agonist exposure. To define the importance of receptor phosphorylation and -arrestin binding in desensitization, and to determine whether -arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking -arrestin-1 and/or -arrestin-2. Apparent affinity for [³H]MeTRH was increased 8-fold in cells expressing -arrestins, including a -arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of -arrestins, but receptors remained primarily on the plasma membrane without -arrestin. -Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous -arrestins reduced TRH-stimulated inositol phosphate production by 48% (-arrestin-1), 71% (-arrestin-2), and 84% (-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by -arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without -arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking G_q and G₁₁ or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that -arrestin binding is critical for desensitization and internalization.

Received for publication, March 16, 2005 , and in revised form, August 12, 2005.

^* This work was supported by Grant DK19974 from the National Institutes of Health (to P. M. H.) and by a Sproull Fellowship from the University of Rochester and a National Institutes of Health Cardiovascular Research Training Grant (to B. W. J.). 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 Pharmacology and Physiology, University of Rochester Medical Center, Box 711, Rochester, NY 14642. Tel.: 585-275-4933; Fax: 585-273-2652; E-mail: Patricia_Hinkle{at}urmc.rochester.edu.

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