β-arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation

Abstract

The widely expressed β-arrestin isoforms 1 and 2 bind phosphorylated G protein-coupled receptors (GPCRs) and mediate desensitization and internalization. Phosphorylation of protease-activated receptor-1 (PAR1 ), a GPCR for thrombin, is important for desensitization and internalization, however, the role of β-arrestins in signaling and trafficking of PAR1 remains unknown. To assess β-arrestin function we examined signaling and trafficking of PAR1 in mouse embryonic fibroblasts (MEFs) derived from β-arrestin (βarr) knockouts. Desensitization of PAR1 signaling was markedly impaired in MEFs lacking both βarr1 and βarr2 isoforms compared with wild-type cells. Strikingly, in cells lacking only βarr1 PAR1 desensitization was also significantly impaired compared with βarr2-lacking or wild-type cells. In wild-type MEFs, activated PAR1 was internalized through a dynamin- and clathrin-dependent pathway and degraded. Surprisingly, in cells lacking both βarr1 and βarr2 activated PAR1 was similarly internalized through a dynamin- and clathrin-dependent pathway and degraded, whereas the β2-adrenergic receptor (β2-AR) failed to internalize. A PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation failed to internalize in both wild-type and β-arrestin knockout cells. Thus, PAR1 appears to utilize a distinct phosphorylation-dependent but β-arrestin-independent pathway for internalization through clathrin-coated pits. Together, these findings strongly suggest that the individual β-arrestin isoforms can differentially regulate GPCR desensitization and further reveal a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of arrestins.