title: Phosphorylation of protease-activated receptor-2 differentially regulates desensitization and internalization.
abstract: Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor irreversibly activated by extracellular proteases. Activated PAR2 couples to multiple heterotrimeric G-protein subtypes including G alpha(q), G alpha(i), and G alpha(12/13). Most activated G protein-coupled receptors are rapidly desensitized and internalized following phosphorylation and beta-arrestin binding. However, the role of phosphorylation in regulation of PAR2 signaling and trafficking is not known. To investigate the function of phosphorylation, we generated a PAR2 mutant in which all serines and threonines in the C-tail were converted to alanines and designated it PAR2 0P. In mammalian cells, the addition of agonist induced a rapid and robust increase in phosphorylation of wild-type PAR2 but not the 0P mutant, suggesting that the major sites of phosphorylation occur within the C-tail domain. Moreover, desensitization of PAR2 0P signaling was markedly impaired compared with the wild-type receptor. Wild-type phosphorylated PAR2 internalized through a canonical dynamin, clathrin- and beta-arrestin-dependent pathway. Strikingly, PAR2 0P mutant internalization proceeded through a dynamin-dependent but clathrin- and beta-arrestin-independent pathway in both a constitutive and agonist-dependent manner. Collectively, our studies show that PAR2 phosphorylation is essential for beta-arrestin binding and uncoupling from heterotrimeric G-protein signaling and that the presence of serine and threonine residues in the PAR2 C-tail hinder constitutive internalization through a non-canonical pathway. Thus, our studies reveal a novel function for phosphorylation that differentially regulates PAR2 desensitization and endocytic trafficking.
authors.affiliation: Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA.
authors.forename: Tiffany K
authors.initials: TK
authors.lastname: Ricks
authors.forename: JoAnn
authors.initials: J
authors.lastname: Trejo
mesh_terms: D000818:Animals; D019390:Arrestins / Q000737:chemistry; D002460:Cell Line; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D044464:Receptor, PAR-2 / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D044464:Receptor, PAR-2; D000071557:beta-Arrestins; D013912:Threonine; D012694:Serine; D019204:GTP-Binding Proteins