Phosphorylation of protease-activated receptor-2 differentially regulates desensitization and internalization.

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.