title: Termination of protease-activated receptor-1 signaling by beta-arrestins is independent of receptor phosphorylation.
abstract: 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 beta-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 beta-arrestin 1 (betaarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas beta-arrestin 2 (betaarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by betaarr1 compared with betaarr2. In contrast, both beta-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated betaarr1 in an agonist-dependent manner, whereas betaarr2 association was virtually undetectable. Remarkably, betaarr1 also interacted with phosphorylation defective PAR1 mutant, whereas betaarr2 did not. Moreover, constitutively active beta-arrestin mutants, betaarr1 R169E and betaarr2 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, beta-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, beta-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.
authors.affiliation: Department of Pharmacology, Cardiovascular Biology Center, Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill 27599-7365, USA.
authors.forename: Chii-Heui
authors.initials: CH
authors.lastname: Chen
authors.forename: May M
authors.initials: MM
authors.lastname: Paing
authors.forename: JoAnn
authors.initials: J
authors.lastname: Trejo
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D019556:COS Cells; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D044463:Receptor, PAR-1 / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; C000605254:ARRB2 protein, human; D019390:Arrestins; D018076:DNA, Complementary; D020033:Protein Isoforms; D044463:Receptor, PAR-1; D000071559:beta-Arrestin 1; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D012701:Serotonin; D013917:Thrombin