beta -Arrestins regulate protease-activated receptor-1 desensitization but not internalization or Down-regulation.
The widely expressed beta-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 beta-arrestins in signaling and trafficking of PAR1 remains unknown. To assess beta-arrestin function we examined signaling and trafficking of PAR1 in mouse embryonic fibroblasts (MEFs) derived from beta-arrestin (betaarr) knockouts. Desensitization of PAR1 signaling was markedly impaired in MEFs lacking both betaarr1 and betaarr2 isoforms compared with wild-type cells. Strikingly, in cells lacking only betaarr1 PAR1 desensitization was also significantly impaired compared with betaarr2-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 betaarr1 and betaarr2 activated PAR1 was similarly internalized through a dynamin- and clathrin-dependent pathway and degraded, whereas the beta(2)-adrenergic receptor (beta(2)-AR) failed to internalize. A PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation failed to internalize in both wild-type and beta-arrestin knockout cells. Thus, PAR1 appears to utilize a distinct phosphorylation-dependent but beta-arrestin-independent pathway for internalization through clathrin-coated pits. Together, these findings strongly suggest that the individual beta-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.
Paing
May M
MM
Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, North Carolina 27599-7365, USA.
Stutts
Amy B
AB
Kohout
Trudy A
TA
Lefkowitz
Robert J
RJ
Trejo
JoAnn
J
D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000378:metabolism; D015536:Down-Regulation; D034281:Dynamins; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D000071557:beta-Arrestins
D019390:Arrestins; D002966:Clathrin; D020033:Protein Isoforms; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D020558:GTP Phosphohydrolases; D034281:Dynamins