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• W2073037108 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. 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. protease-activated receptor-1 β2-adrenergic receptor bovine serum albumin Dulbecco's modified Eagle's medium enzyme-linked immunosorbent assay G protein-coupled receptor carboxyl tail green fluorescence protein hemagglutinin phosphate-buffered saline G protein-coupled kinase adaptor protein complex-2 mouse embryonic fibroblasts β-arrestin monodansylcadaverine angiotensin type 1A receptor Protease-activated receptor-1 (PAR11), a G protein-coupled receptor (GPCR) for thrombin, is the prototype member of a family of protease-activated receptors. PAR1 couples to Gq, Gi, and G12/13 to elicit a variety of signaling events important for hemostasis, thrombosis, and embryonic development (1Coughlin S.R. Nature. 2000; 407: 258-264Google Scholar, 2Griffin C.T. Srinivasan Y. Zheng Y.-W. Huang W. Coughlin S.R. Science. 2001; 293: 1666-1670Google Scholar). PAR1 is activated by thrombin through an unusual proteolytic mechanism. Thrombin, a serine protease, binds to and cleaves the extracellular amino terminus of PAR1 (3Vu T.-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Google Scholar). The newly formed amino terminus of PAR1 then functions as a tethered peptide ligand by interacting with the receptor to trigger signaling (3Vu T.-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Google Scholar, 4Vu T.-K.H. Wheaton V.I. Hung D.T. Coughlin S.R. Nature. 1991; 353: 674-677Google Scholar, 5Chen J. Ishii M. Wang L. Ishii K. Coughlin S.R. J. Biol. Chem. 1994; 269: 16041-16045Google Scholar). Despite PAR1's irreversible proteolytic mechanism of activation and the generation of a tethered ligand that cannot diffuse away, signaling by the receptor is rapidly terminated at the plasma membrane. The molecular mechanisms responsible for termination of PAR1 signaling are not clearly understood.The molecular mechanisms responsible for GPCR desensitization and resensitization have been extensively studied for the β2-adrenergic receptor (β2-AR) (6Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Google Scholar, 7Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Google Scholar). In this model GPCRs are initially desensitized by rapid phosphorylation of the activated form of the receptor by G protein-coupled kinases (GRKs). Phosphorylated receptor then binds arrestin, which impedes interaction with G proteins. Arrestin also facilitates GPCR internalization by interacting with clathrin and the adaptor protein complex-2 (AP-2), components of the endocytic machinery (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). Once internalized into endosomes, receptor dissociates from the ligand, becomes dephosphorylated, and is then recycled back to the plasma membrane ready for activation again.Phosphorylation of activated PAR1 initiates rapid desensitization and internalization from the plasma membrane. Overexpression of GRK3 and GRK5 enhances PAR1 phosphorylation and markedly inhibits PAR1 signaling (10Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Google Scholar, 11Tiruppathi C. Yan W. Sandoval R. Naqvi T. Pronin A.N. Benovic J.L. Malik A.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7440-7445Google Scholar). Activation of PAR1 cytoplasmic tail mutants that were not phosphorylated signaled more robustly than wild-type PAR1 (12Nanevicz T. Wang L. Chen M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: 702-706Google Scholar, 13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar). Moreover, these same mutants also failed to exhibit agonist-triggered internalization. Interestingly, a cytoplasmic tail mutant of PAR1 with limited alanine substitution for serines between residues 391 and 406 is phosphorylated but defective in its ability to uncouple from signaling (14Hammes S.R. Shapiro M.J. Coughlin S.R. Biochemistry. 1999; 38: 2486-2493Google Scholar). This same receptor mutant is internalized following activation like wild-type PAR1. Together, these findings suggest that phosphorylation of PAR1's cytoplasmic tail is important for both rapid desensitization and internalization, however, distinct mechanisms may regulate these processes.Internalization and lysosomal sorting of activated PAR1 is also important for termination of receptor signaling. Activated PAR1 is internalized through a dynamin- and clathrin-dependent pathway, like many recycling receptors (15Hoxie J.A. Ahuja M. Belmonte E. Pizarro S. Parton R. Brass L.F. J. Biol. Chem. 1993; 268: 13756-13763Google Scholar, 16Trejo J. Altschuler Y. Fu H.-W. Mostov K.E. Coughlin S.R. J. Biol. Chem. 2000; 275: 31255-31265Google Scholar). Once internalized, PAR1 is sorted away from recycling receptors and targeted to lysosomes for degradation; an event critical for termination of receptor signaling (17Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Google Scholar, 18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). In transfected fibroblasts, a mutant PAR1 able to internalize and recycle to the cell surface signaled persistently after activation by thrombin (19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). This prolonged signaling is apparently due to recycling and continued signaling by receptors that return to the plasma membrane with their tethered ligands intact. Thus, phosphorylation of activated PAR1 promotes rapid desensitization at the plasma membrane while internalization and lysosomal sorting prevents the receptor from recycling and continuing to signal on the cell surface.The β-arrestin isoforms 1 and 2 are widely expressed and bind to phosphorylated GPCRs to mediate desensitization and internalization (20Krupnick J.G. Benovic J.L. Annu. Rev. Pharm. Toxicol. 1998; 38: 289-319Google Scholar). Phosphorylation of PAR1 is important for desensitization and internalization, however, the role of arrestins in signaling and trafficking of PAR1 is not known. In this study, we investigate the function of arrestin in PAR1 signaling and trafficking through the use of mouse embryonic fibroblasts (MEFs) derived from β-arrestin (βarr) knockouts (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Our findings strongly suggest that βarr1 functions as the predominant regulator of PAR1 desensitization. Moreover, these studies reveal a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of β-arrestins.DISCUSSIONPAR1's proteolytic mechanism of activation is clearly distinct from that of most GPCRs and raises questions regarding the molecular mechanisms responsible for termination of PAR1 signaling. Phosphorylation of activated PAR1 is important for both rapid termination of receptor signaling and internalization from the plasma membrane (12Nanevicz T. Wang L. Chen M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: 702-706Google Scholar, 13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar). Many phosphorylated GPCRs bind arrestins, which uncouple the receptor from signaling and facilitate receptor internalization (20Krupnick J.G. Benovic J.L. Annu. Rev. Pharm. Toxicol. 1998; 38: 289-319Google Scholar). The role of arrestins in the regulation of PAR1 signaling and trafficking has not previously been determined. Prior studies have used heterologous overexpression of wild-type and dominant-negative forms of arrestin to assess function; however, such studies are often complicated by the expression of endogenous protein. Our recent generation of mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockouts offered an opportunity to assess function in cells in which the expression of the individual arrestin isoforms was eliminated genetically by gene knockout (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). We therefore assessed signaling and trafficking of PAR1 in cells that lack either one or both isoforms of β-arrestin.PAR1 is endogenously expressed in MEFs, and thrombin signaling is completely abolished in MEFs derived from PAR1 knockout mice, thus PAR1 is the predominant mediator of thrombin signaling in these cells (38Connolly A.J. Ishihara H. Kahn M.L. Farese R.V. Coughlin S.R. Nature. 1996; 381: 516-519Google Scholar). In cells lacking both β-arrestin isoforms (βarr1,2−/−) endogenous PAR1 signaling was significantly more robust than wild-type controls. Consistent with these results, in transfected MEFs expressing similar amounts of PAR1, the rate of PAR1 desensitization was significantly slowed, resulting in a greater accumulation of inositol phosphates in β-arrestin knockouts compared with wild-type control cells. Thus, in the absence of β-arrestins, desensitization of PAR1 signaling is significantly impaired. To our knowledge these findings are the first to demonstrate a role for β-arrestins in regulation of PAR1 signaling. Moreover, in cells that lack only βarr1, the rate of PAR1 desensitization was markedly impaired compared with βarr2 lacking cells and wild-type controls. These results strongly suggest that the βarr1 isoform functions as the predominant regulator of PAR1 desensitization. These findings contrast with our recent report in which both β-arrestin isoforms were found to be equally effective in regulating desensitization of β2-AR and AT1A-R using these same knockout cells (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Thus, these studies provide the first example in which the β-arrestin isoforms can differentially regulate GPCR desensitization.In addition to regulating GPCR desensitization, β-arrestins can facilitate GPCR internalization. Arrestins bind phosphorylated GPCRs and interact with clathrin and the adaptor protein complex-2 (AP-2) to promote receptor internalization (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). PAR1 requires phosphorylation for internalization through a dynamin- and clathrin-dependent pathway (13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar, 16Trejo J. Altschuler Y. Fu H.-W. Mostov K.E. Coughlin S.R. J. Biol. Chem. 2000; 275: 31255-31265Google Scholar), raising the possibility that arrestin might function in this process. In contrast, we found that activated PAR1 is internalized through a dynamin- and clathrin-dependent pathway even when both β-arrestin isoforms are absent. Constitutive internalization of PAR1 was also intact in β-arrestin knockout cells. Interestingly, PAR1 internalization required phosphorylation even in the absence of β-arrestins. Together, these findings are consistent with a distinct phosphorylation-dependent but arrestin-independent pathway for PAR1 internalization. By contrast, the β2-AR failed to internalize in these same β-arrestin knockout cells. The β2-AR is known to bind arrestin and internalize through a classic dynamin- and clathrin-dependent pathway (39Ferguson S.S. Downey W.r. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Google Scholar). Thus, the failure of β2-AR to internalize in β-arrestin knockout cells that show robust PAR1 internalization suggests that these receptors have distinct requirements for internalization through clathrin-coated pits. Together, these studies provide strong evidence for a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of arrestins.The relative contributions of receptor uncoupling via phosphorylation and arrestin binding versus receptor internalization to the rapid termination of PAR1 signaling remain poorly understood. In this study we demonstrate that, in the absence of β-arrestins, rapid desensitization of PAR1 signaling is markedly impaired while internalization remains intact. Thus, internalization is not required for rapid desensitization of PAR1 signaling. In many cases, arrestins can mediate both desensitization and internalization of GPCRs (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar); our observations, however, are consistent with a distinct arrestin-independent mechanism for PAR1 internalization. This is also consistent with a recent report in which termination of PAR1 signaling and internalization were separated by mutation of phosphorylation sites within PAR1's cytoplasmic tail (14Hammes S.R. Shapiro M.J. Coughlin S.R. Biochemistry. 1999; 38: 2486-2493Google Scholar). Similar mutants have been reported for the m2 muscarinic acetylcholine receptor (40Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Google Scholar). In cells lacking arrestins PAR1 signaling was eventually slowed; this may be due to internalization and lysosomal sorting of activated PAR1. Indeed, a mutant PAR1 that internalized and recycled back to the cell surface signaled persistently following activation by thrombin (19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). These studies strongly suggest that internalization and lysosomal sorting are critical for termination of PAR1 signaling (18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). Thus, β-arrestin is required for rapid desensitization of PAR1 signaling, whereas internalization and lysosomal sorting appear to contribute to termination of PAR1 signaling observed at later times.Internalization of GPCRs through clathrin-coated pits is a multistep process involving numerous proteins. β-Arrestins bind to clathrin and the adaptor protein complex-2 (AP-2) and thereby link phosphorylated GPCRs to the endocytic machinery (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). It is becoming increasingly clear that the individual β-arrestin isoforms can differentially regulate GPCR internalization. The β2-AR preferentially utilizes βarr2 for sequestration through clathrin-coated pits, whereas both β-arrestin isoforms were equally effective for internalization of the AT1A-R (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Interestingly, upon recruitment to activated receptors, βarr1 undergoes dephosphorylation at a carboxyl-terminal serine residue (41Lin F.-T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Google Scholar). This is thought to be critical for interaction with clathrin but not for uncoupling the receptor from signaling. In this study we demonstrate that βarr1 is required for rapid PAR1 desensitization but not for internalization through clathrin-coated pits. Thus, it is possible that upon PAR1 activation βarr1 is recruited to the receptor but fails to undergo dephosphorylation and is, therefore, unable to promote receptor interaction with clathrin. This possibility remains to be tested. In addition to arrestins, AP-2 can function as an adaptor to recruit receptor tyrosine kinases and other membrane proteins to clathrin-coated pits (42Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Google Scholar); whether AP-2 functions in PAR1 internalization is not known.In summary, mouse embryonic fibroblasts derived from β-arrestin knockouts provided an opportunity to assess arrestin function in PAR1 signaling and trafficking. These studies show that βarr1 functions as the predominant regulator of PAR1 desensitization and strongly suggest that the individual β-arrestin isoforms can differentially regulate GPCR desensitization. Moreover, these studies reveal a novel arrestin-independent mechanism for PAR1 internalization from the plasma membrane. The challenge now is to elucidate the mechanisms by which activated PAR1 is recruited to clathrin-coated pits and internalized from the plasma membrane; an event critical for termination of receptor signaling (18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). Protease-activated receptor-1 (PAR11), a G protein-coupled receptor (GPCR) for thrombin, is the prototype member of a family of protease-activated receptors. PAR1 couples to Gq, Gi, and G12/13 to elicit a variety of signaling events important for hemostasis, thrombosis, and embryonic development (1Coughlin S.R. Nature. 2000; 407: 258-264Google Scholar, 2Griffin C.T. Srinivasan Y. Zheng Y.-W. Huang W. Coughlin S.R. Science. 2001; 293: 1666-1670Google Scholar). PAR1 is activated by thrombin through an unusual proteolytic mechanism. Thrombin, a serine protease, binds to and cleaves the extracellular amino terminus of PAR1 (3Vu T.-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Google Scholar). The newly formed amino terminus of PAR1 then functions as a tethered peptide ligand by interacting with the receptor to trigger signaling (3Vu T.-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Google Scholar, 4Vu T.-K.H. Wheaton V.I. Hung D.T. Coughlin S.R. Nature. 1991; 353: 674-677Google Scholar, 5Chen J. Ishii M. Wang L. Ishii K. Coughlin S.R. J. Biol. Chem. 1994; 269: 16041-16045Google Scholar). Despite PAR1's irreversible proteolytic mechanism of activation and the generation of a tethered ligand that cannot diffuse away, signaling by the receptor is rapidly terminated at the plasma membrane. The molecular mechanisms responsible for termination of PAR1 signaling are not clearly understood. The molecular mechanisms responsible for GPCR desensitization and resensitization have been extensively studied for the β2-adrenergic receptor (β2-AR) (6Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Google Scholar, 7Pitcher J.A. Freedman N.J. Lefkowitz R.J. Annu. Rev. Biochem. 1998; 67: 653-692Google Scholar). In this model GPCRs are initially desensitized by rapid phosphorylation of the activated form of the receptor by G protein-coupled kinases (GRKs). Phosphorylated receptor then binds arrestin, which impedes interaction with G proteins. Arrestin also facilitates GPCR internalization by interacting with clathrin and the adaptor protein complex-2 (AP-2), components of the endocytic machinery (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). Once internalized into endosomes, receptor dissociates from the ligand, becomes dephosphorylated, and is then recycled back to the plasma membrane ready for activation again. Phosphorylation of activated PAR1 initiates rapid desensitization and internalization from the plasma membrane. Overexpression of GRK3 and GRK5 enhances PAR1 phosphorylation and markedly inhibits PAR1 signaling (10Ishii K. Chen J. Ishii M. Koch W.J. Freedman N.J. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: 1125-1130Google Scholar, 11Tiruppathi C. Yan W. Sandoval R. Naqvi T. Pronin A.N. Benovic J.L. Malik A.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7440-7445Google Scholar). Activation of PAR1 cytoplasmic tail mutants that were not phosphorylated signaled more robustly than wild-type PAR1 (12Nanevicz T. Wang L. Chen M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: 702-706Google Scholar, 13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar). Moreover, these same mutants also failed to exhibit agonist-triggered internalization. Interestingly, a cytoplasmic tail mutant of PAR1 with limited alanine substitution for serines between residues 391 and 406 is phosphorylated but defective in its ability to uncouple from signaling (14Hammes S.R. Shapiro M.J. Coughlin S.R. Biochemistry. 1999; 38: 2486-2493Google Scholar). This same receptor mutant is internalized following activation like wild-type PAR1. Together, these findings suggest that phosphorylation of PAR1's cytoplasmic tail is important for both rapid desensitization and internalization, however, distinct mechanisms may regulate these processes. Internalization and lysosomal sorting of activated PAR1 is also important for termination of receptor signaling. Activated PAR1 is internalized through a dynamin- and clathrin-dependent pathway, like many recycling receptors (15Hoxie J.A. Ahuja M. Belmonte E. Pizarro S. Parton R. Brass L.F. J. Biol. Chem. 1993; 268: 13756-13763Google Scholar, 16Trejo J. Altschuler Y. Fu H.-W. Mostov K.E. Coughlin S.R. J. Biol. Chem. 2000; 275: 31255-31265Google Scholar). Once internalized, PAR1 is sorted away from recycling receptors and targeted to lysosomes for degradation; an event critical for termination of receptor signaling (17Hein L. Ishii K. Coughlin S.R. Kobilka B.K. J. Biol. Chem. 1994; 269: 27719-27726Google Scholar, 18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). In transfected fibroblasts, a mutant PAR1 able to internalize and recycle to the cell surface signaled persistently after activation by thrombin (19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). This prolonged signaling is apparently due to recycling and continued signaling by receptors that return to the plasma membrane with their tethered ligands intact. Thus, phosphorylation of activated PAR1 promotes rapid desensitization at the plasma membrane while internalization and lysosomal sorting prevents the receptor from recycling and continuing to signal on the cell surface. The β-arrestin isoforms 1 and 2 are widely expressed and bind to phosphorylated GPCRs to mediate desensitization and internalization (20Krupnick J.G. Benovic J.L. Annu. Rev. Pharm. Toxicol. 1998; 38: 289-319Google Scholar). Phosphorylation of PAR1 is important for desensitization and internalization, however, the role of arrestins in signaling and trafficking of PAR1 is not known. In this study, we investigate the function of arrestin in PAR1 signaling and trafficking through the use of mouse embryonic fibroblasts (MEFs) derived from β-arrestin (βarr) knockouts (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Our findings strongly suggest that βarr1 functions as the predominant regulator of PAR1 desensitization. Moreover, these studies reveal a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of β-arrestins. DISCUSSIONPAR1's proteolytic mechanism of activation is clearly distinct from that of most GPCRs and raises questions regarding the molecular mechanisms responsible for termination of PAR1 signaling. Phosphorylation of activated PAR1 is important for both rapid termination of receptor signaling and internalization from the plasma membrane (12Nanevicz T. Wang L. Chen M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: 702-706Google Scholar, 13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar). Many phosphorylated GPCRs bind arrestins, which uncouple the receptor from signaling and facilitate receptor internalization (20Krupnick J.G. Benovic J.L. Annu. Rev. Pharm. Toxicol. 1998; 38: 289-319Google Scholar). The role of arrestins in the regulation of PAR1 signaling and trafficking has not previously been determined. Prior studies have used heterologous overexpression of wild-type and dominant-negative forms of arrestin to assess function; however, such studies are often complicated by the expression of endogenous protein. Our recent generation of mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockouts offered an opportunity to assess function in cells in which the expression of the individual arrestin isoforms was eliminated genetically by gene knockout (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). We therefore assessed signaling and trafficking of PAR1 in cells that lack either one or both isoforms of β-arrestin.PAR1 is endogenously expressed in MEFs, and thrombin signaling is completely abolished in MEFs derived from PAR1 knockout mice, thus PAR1 is the predominant mediator of thrombin signaling in these cells (38Connolly A.J. Ishihara H. Kahn M.L. Farese R.V. Coughlin S.R. Nature. 1996; 381: 516-519Google Scholar). In cells lacking both β-arrestin isoforms (βarr1,2−/−) endogenous PAR1 signaling was significantly more robust than wild-type controls. Consistent with these results, in transfected MEFs expressing similar amounts of PAR1, the rate of PAR1 desensitization was significantly slowed, resulting in a greater accumulation of inositol phosphates in β-arrestin knockouts compared with wild-type control cells. Thus, in the absence of β-arrestins, desensitization of PAR1 signaling is significantly impaired. To our knowledge these findings are the first to demonstrate a role for β-arrestins in regulation of PAR1 signaling. Moreover, in cells that lack only βarr1, the rate of PAR1 desensitization was markedly impaired compared with βarr2 lacking cells and wild-type controls. These results strongly suggest that the βarr1 isoform functions as the predominant regulator of PAR1 desensitization. These findings contrast with our recent report in which both β-arrestin isoforms were found to be equally effective in regulating desensitization of β2-AR and AT1A-R using these same knockout cells (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Thus, these studies provide the first example in which the β-arrestin isoforms can differentially regulate GPCR desensitization.In addition to regulating GPCR desensitization, β-arrestins can facilitate GPCR internalization. Arrestins bind phosphorylated GPCRs and interact with clathrin and the adaptor protein complex-2 (AP-2) to promote receptor internalization (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). PAR1 requires phosphorylation for internalization through a dynamin- and clathrin-dependent pathway (13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar, 16Trejo J. Altschuler Y. Fu H.-W. Mostov K.E. Coughlin S.R. J. Biol. Chem. 2000; 275: 31255-31265Google Scholar), raising the possibility that arrestin might function in this process. In contrast, we found that activated PAR1 is internalized through a dynamin- and clathrin-dependent pathway even when both β-arrestin isoforms are absent. Constitutive internalization of PAR1 was also intact in β-arrestin knockout cells. Interestingly, PAR1 internalization required phosphorylation even in the absence of β-arrestins. Together, these findings are consistent with a distinct phosphorylation-dependent but arrestin-independent pathway for PAR1 internalization. By contrast, the β2-AR failed to internalize in these same β-arrestin knockout cells. The β2-AR is known to bind arrestin and internalize through a classic dynamin- and clathrin-dependent pathway (39Ferguson S.S. Downey W.r. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Google Scholar). Thus, the failure of β2-AR to internalize in β-arrestin knockout cells that show robust PAR1 internalization suggests that these receptors have distinct requirements for internalization through clathrin-coated pits. Together, these studies provide strong evidence for a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of arrestins.The relative contributions of receptor uncoupling via phosphorylation and arrestin binding versus receptor internalization to the rapid termination of PAR1 signaling remain poorly understood. In this study we demonstrate that, in the absence of β-arrestins, rapid desensitization of PAR1 signaling is markedly impaired while internalization remains intact. Thus, internalization is not required for rapid desensitization of PAR1 signaling. In many cases, arrestins can mediate both desensitization and internalization of GPCRs (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar); our observations, however, are consistent with a distinct arrestin-independent mechanism for PAR1 internalization. This is also consistent with a recent report in which termination of PAR1 signaling and internalization were separated by mutation of phosphorylation sites within PAR1's cytoplasmic tail (14Hammes S.R. Shapiro M.J. Coughlin S.R. Biochemistry. 1999; 38: 2486-2493Google Scholar). Similar mutants have been reported for the m2 muscarinic acetylcholine receptor (40Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Google Scholar). In cells lacking arrestins PAR1 signaling was eventually slowed; this may be due to internalization and lysosomal sorting of activated PAR1. Indeed, a mutant PAR1 that internalized and recycled back to the cell surface signaled persistently following activation by thrombin (19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). These studies strongly suggest that internalization and lysosomal sorting are critical for termination of PAR1 signaling (18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). Thus, β-arrestin is required for rapid desensitization of PAR1 signaling, whereas internalization and lysosomal sorting appear to contribute to termination of PAR1 signaling observed at later times.Internalization of GPCRs through clathrin-coated pits is a multistep process involving numerous proteins. β-Arrestins bind to clathrin and the adaptor protein complex-2 (AP-2) and thereby link phosphorylated GPCRs to the endocytic machinery (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). It is becoming increasingly clear that the individual β-arrestin isoforms can differentially regulate GPCR internalization. The β2-AR preferentially utilizes βarr2 for sequestration through clathrin-coated pits, whereas both β-arrestin isoforms were equally effective for internalization of the AT1A-R (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Interestingly, upon recruitment to activated receptors, βarr1 undergoes dephosphorylation at a carboxyl-terminal serine residue (41Lin F.-T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Google Scholar). This is thought to be critical for interaction with clathrin but not for uncoupling the receptor from signaling. In this study we demonstrate that βarr1 is required for rapid PAR1 desensitization but not for internalization through clathrin-coated pits. Thus, it is possible that upon PAR1 activation βarr1 is recruited to the receptor but fails to undergo dephosphorylation and is, therefore, unable to promote receptor interaction with clathrin. This possibility remains to be tested. In addition to arrestins, AP-2 can function as an adaptor to recruit receptor tyrosine kinases and other membrane proteins to clathrin-coated pits (42Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Google Scholar); whether AP-2 functions in PAR1 internalization is not known.In summary, mouse embryonic fibroblasts derived from β-arrestin knockouts provided an opportunity to assess arrestin function in PAR1 signaling and trafficking. These studies show that βarr1 functions as the predominant regulator of PAR1 desensitization and strongly suggest that the individual β-arrestin isoforms can differentially regulate GPCR desensitization. Moreover, these studies reveal a novel arrestin-independent mechanism for PAR1 internalization from the plasma membrane. The challenge now is to elucidate the mechanisms by which activated PAR1 is recruited to clathrin-coated pits and internalized from the plasma membrane; an event critical for termination of receptor signaling (18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). PAR1's proteolytic mechanism of activation is clearly distinct from that of most GPCRs and raises questions regarding the molecular mechanisms responsible for termination of PAR1 signaling. Phosphorylation of activated PAR1 is important for both rapid termination of receptor signaling and internalization from the plasma membrane (12Nanevicz T. Wang L. Chen M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: 702-706Google Scholar, 13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar). Many phosphorylated GPCRs bind arrestins, which uncouple the receptor from signaling and facilitate receptor internalization (20Krupnick J.G. Benovic J.L. Annu. Rev. Pharm. Toxicol. 1998; 38: 289-319Google Scholar). The role of arrestins in the regulation of PAR1 signaling and trafficking has not previously been determined. Prior studies have used heterologous overexpression of wild-type and dominant-negative forms of arrestin to assess function; however, such studies are often complicated by the expression of endogenous protein. Our recent generation of mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockouts offered an opportunity to assess function in cells in which the expression of the individual arrestin isoforms was eliminated genetically by gene knockout (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). We therefore assessed signaling and trafficking of PAR1 in cells that lack either one or both isoforms of β-arrestin. PAR1 is endogenously expressed in MEFs, and thrombin signaling is completely abolished in MEFs derived from PAR1 knockout mice, thus PAR1 is the predominant mediator of thrombin signaling in these cells (38Connolly A.J. Ishihara H. Kahn M.L. Farese R.V. Coughlin S.R. Nature. 1996; 381: 516-519Google Scholar). In cells lacking both β-arrestin isoforms (βarr1,2−/−) endogenous PAR1 signaling was significantly more robust than wild-type controls. Consistent with these results, in transfected MEFs expressing similar amounts of PAR1, the rate of PAR1 desensitization was significantly slowed, resulting in a greater accumulation of inositol phosphates in β-arrestin knockouts compared with wild-type control cells. Thus, in the absence of β-arrestins, desensitization of PAR1 signaling is significantly impaired. To our knowledge these findings are the first to demonstrate a role for β-arrestins in regulation of PAR1 signaling. Moreover, in cells that lack only βarr1, the rate of PAR1 desensitization was markedly impaired compared with βarr2 lacking cells and wild-type controls. These results strongly suggest that the βarr1 isoform functions as the predominant regulator of PAR1 desensitization. These findings contrast with our recent report in which both β-arrestin isoforms were found to be equally effective in regulating desensitization of β2-AR and AT1A-R using these same knockout cells (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Thus, these studies provide the first example in which the β-arrestin isoforms can differentially regulate GPCR desensitization. In addition to regulating GPCR desensitization, β-arrestins can facilitate GPCR internalization. Arrestins bind phosphorylated GPCRs and interact with clathrin and the adaptor protein complex-2 (AP-2) to promote receptor internalization (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). PAR1 requires phosphorylation for internalization through a dynamin- and clathrin-dependent pathway (13Shapiro M.J. Trejo J. Zeng D. Coughlin S.R. J. Biol. Chem. 1996; 271: 32874-32880Google Scholar, 16Trejo J. Altschuler Y. Fu H.-W. Mostov K.E. Coughlin S.R. J. Biol. Chem. 2000; 275: 31255-31265Google Scholar), raising the possibility that arrestin might function in this process. In contrast, we found that activated PAR1 is internalized through a dynamin- and clathrin-dependent pathway even when both β-arrestin isoforms are absent. Constitutive internalization of PAR1 was also intact in β-arrestin knockout cells. Interestingly, PAR1 internalization required phosphorylation even in the absence of β-arrestins. Together, these findings are consistent with a distinct phosphorylation-dependent but arrestin-independent pathway for PAR1 internalization. By contrast, the β2-AR failed to internalize in these same β-arrestin knockout cells. The β2-AR is known to bind arrestin and internalize through a classic dynamin- and clathrin-dependent pathway (39Ferguson S.S. Downey W.r. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Google Scholar). Thus, the failure of β2-AR to internalize in β-arrestin knockout cells that show robust PAR1 internalization suggests that these receptors have distinct requirements for internalization through clathrin-coated pits. Together, these studies provide strong evidence for a novel mechanism by which GPCRs can internalize through a dynamin- and clathrin-dependent pathway that is independent of arrestins. The relative contributions of receptor uncoupling via phosphorylation and arrestin binding versus receptor internalization to the rapid termination of PAR1 signaling remain poorly understood. In this study we demonstrate that, in the absence of β-arrestins, rapid desensitization of PAR1 signaling is markedly impaired while internalization remains intact. Thus, internalization is not required for rapid desensitization of PAR1 signaling. In many cases, arrestins can mediate both desensitization and internalization of GPCRs (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar); our observations, however, are consistent with a distinct arrestin-independent mechanism for PAR1 internalization. This is also consistent with a recent report in which termination of PAR1 signaling and internalization were separated by mutation of phosphorylation sites within PAR1's cytoplasmic tail (14Hammes S.R. Shapiro M.J. Coughlin S.R. Biochemistry. 1999; 38: 2486-2493Google Scholar). Similar mutants have been reported for the m2 muscarinic acetylcholine receptor (40Pals-Rylaarsdam R. Gurevich V.V. Lee K.B. Ptasienski J.A. Benovic J.L. Hosey M.M. J. Biol. Chem. 1997; 272: 23682-23689Google Scholar). In cells lacking arrestins PAR1 signaling was eventually slowed; this may be due to internalization and lysosomal sorting of activated PAR1. Indeed, a mutant PAR1 that internalized and recycled back to the cell surface signaled persistently following activation by thrombin (19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). These studies strongly suggest that internalization and lysosomal sorting are critical for termination of PAR1 signaling (18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). Thus, β-arrestin is required for rapid desensitization of PAR1 signaling, whereas internalization and lysosomal sorting appear to contribute to termination of PAR1 signaling observed at later times. Internalization of GPCRs through clathrin-coated pits is a multistep process involving numerous proteins. β-Arrestins bind to clathrin and the adaptor protein complex-2 (AP-2) and thereby link phosphorylated GPCRs to the endocytic machinery (8Goodman Jr., O.B. Krupnick J.G. Santini F. Gurevich V.V. Penn R.B. Gagnon A.W. Keen J.H. Benovic J.L. Nature. 1996; 383: 447-450Google Scholar, 9Laporte S. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S.G. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Google Scholar). It is becoming increasingly clear that the individual β-arrestin isoforms can differentially regulate GPCR internalization. The β2-AR preferentially utilizes βarr2 for sequestration through clathrin-coated pits, whereas both β-arrestin isoforms were equally effective for internalization of the AT1A-R (21Kohout T.A. Lin F.-T. Perry S.J. Conner D.A. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1601-1606Google Scholar). Interestingly, upon recruitment to activated receptors, βarr1 undergoes dephosphorylation at a carboxyl-terminal serine residue (41Lin F.-T. Krueger K.M. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Google Scholar). This is thought to be critical for interaction with clathrin but not for uncoupling the receptor from signaling. In this study we demonstrate that βarr1 is required for rapid PAR1 desensitization but not for internalization through clathrin-coated pits. Thus, it is possible that upon PAR1 activation βarr1 is recruited to the receptor but fails to undergo dephosphorylation and is, therefore, unable to promote receptor interaction with clathrin. This possibility remains to be tested. In addition to arrestins, AP-2 can function as an adaptor to recruit receptor tyrosine kinases and other membrane proteins to clathrin-coated pits (42Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Google Scholar); whether AP-2 functions in PAR1 internalization is not known. In summary, mouse embryonic fibroblasts derived from β-arrestin knockouts provided an opportunity to assess arrestin function in PAR1 signaling and trafficking. These studies show that βarr1 functions as the predominant regulator of PAR1 desensitization and strongly suggest that the individual β-arrestin isoforms can differentially regulate GPCR desensitization. Moreover, these studies reveal a novel arrestin-independent mechanism for PAR1 internalization from the plasma membrane. The challenge now is to elucidate the mechanisms by which activated PAR1 is recruited to clathrin-coated pits and internalized from the plasma membrane; an event critical for termination of receptor signaling (18Trejo J. Coughlin S.R. J. Biol. Chem. 1999; 274: 2216-2224Google Scholar, 19Trejo J. Hammes S.R. Coughlin S.R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13698-13702Google Scholar). We thank Dr. David P. Siderovski for helpful suggestions and critical review of this manuscript and Dr. Shaun R. Coughlin for generously providing reagents." @default.
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• W2073037108 title "β-Arrestins Regulate Protease-activated Receptor-1 Desensitization but Not Internalization or Down-regulation" @default.
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