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

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... ese 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.