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J. Biol. Chem., Vol. 279, Issue 11, 10020-10031, March 12, 2004

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Termination of Protease-activated Receptor-1 Signaling by -Arrestins Is Independent of Receptor Phosphorylation^*

Chii-Heui Chen, May M. Paing, and JoAnn Trejo¶

From the Department of Pharmacology, Cardiovascular Biology Center, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7365

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and -arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with -arrestin 1 (arr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas -arrestin 2 (arr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by arr1 compared with arr2. In contrast, both -arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated arr1 in an agonist-dependent manner, whereas arr2 association was virtually undetectable. Remarkably, arr1 also interacted with phosphorylation defective PAR1 mutant, whereas arr2 did not. Moreover, constitutively active -arrestin mutants, arr1 R169E and arr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, -arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine_2A receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, -arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling.

Received for publication, September 24, 2003 , and in revised form, December 16, 2003.

The abbreviations used are: PAR, protease-activated receptor; arr, -arrestin; DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; 5-HT_2A, 5-hydroxytryptamine_2A; IP, inositol phosphate; PI, phosphoinositide; SPR, substance P receptor; GRK, G protein-coupled receptor kinase; S/T A, PAR1 mutant in which all of the serines and threonines in the cytoplasmic tail are converted to alanines; S²⁹⁷SS²⁹⁹, mutant in which serine residues Ser²⁹⁷, Ser²⁹⁸, and Ser²⁹⁹ are converted to alanine.

^* This work was supported in part by National Institutes of Health Grants HL67697 and HL073328 (to J. T.). 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.

Supported by an American Heart Association Predoctoral Fellowship.

^¶ To whom correspondence should be addressed: Dept. of Pharmacology, University of North Carolina, 1106 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. Tel.: 919-843-7691; Fax: 919-966-5640; E-mail: joann_trejo{at}med.unc.edu.

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