Termination of Protease-activated Receptor-1 Signaling by β-Arrestins Is Independent of Receptor Phosphorylation (2004)

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and β-arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with β-arrestin 1 (βarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas β-arrestin 2 (βarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by βarr1 compared with βarr2. In contrast, both β-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated βarr1 in an agonist-dependent manner, whereas βarr2 association was virtually undetectable. Remarkably, βarr1 also interacted with phosphorylation defective PAR1 mutant, whereas βarr2 did not. Moreover, constitutively active β-arrestin mutants, βarr1 R169E and βarr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, β-arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine2A receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, β-arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling. Protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is the prototypic member of a family of protease-activated receptors. PAR1 is irreversibly proteolytically activated; thus, the magnitude and duration of thrombin cellular responses are determined primarily by mechanisms responsible for termination of receptor signaling. Both phosphorylation and β-arrestins contribute to rapid desensitization of PAR1 signaling. However, the relative contribution of each of these pathways to the termination of PAR1 signaling is not known. Co-expression of PAR1 with β-arrestin 1 (βarr1) in COS-7 cells resulted in a marked inhibition of PAR1 signaling, whereas β-arrestin 2 (βarr2) was essentially inactive. Strikingly, signaling by a PAR1 cytoplasmic tail mutant defective in agonist-induced phosphorylation was also attenuated more effectively by βarr1 compared with βarr2. In contrast, both β-arrestin isoforms were equally effective at desensitizing the substance P receptor, a classic reversibly activated GPCR. PAR1 coimmunoprecipitated βarr1 in an agonist-dependent manner, whereas βarr2 association was virtually undetectable. Remarkably, βarr1 also interacted with phosphorylation defective PAR1 mutant, whereas βarr2 did not. Moreover, constitutively active β-arrestin mutants, βarr1 R169E and βarr2 R170E, that bind to activated receptor independent of phosphorylation failed to enhance either wild type or mutant PAR1 desensitization compared with normal versions of these proteins. In contrast, β-arrestin mutants displayed enhanced activity at desensitizing the serotonin 5-hydroxytryptamine2A receptor. Taken together, these results suggest that, in addition to PAR1 cytoplasmic tail phosphorylation itself, β-arrestin binding independent of phosphorylation promotes desensitization of PAR1 signaling. These findings reveal a new level of complexity in the regulation of protease-activated GPCR signaling. Thrombin, a coagulant protease, is generated at sites of vascular injury and produces a variety of cellular effects critical for hemostasis, thrombosis, and inflammatory and proliferative responses triggered by vascular damage (1Coughlin S.R. Trends Cardiovasc. Med. 1994; 4: 77-83Crossref PubMed Scopus (97) Google Scholar, 2O'Brien P.J. Molino M. Kahn M. Brass L.F. Oncogene. 2001; 20: 1570-1581Crossref PubMed Scopus (199) Google Scholar). Thrombin activates cells through at least three proteolytically activated G protein-coupled receptors: PAR1, 1 -3, and -4 (3Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2106) Google Scholar). The prototype of this family, PAR1, is activated by an unusual irreversible proteolytic mechanism in which thrombin binds to and cleaves the amino-terminal exodomain of the receptor. This cleavage generates a new amino terminus that functions as a tethered ligand by binding intramolecularly to the body of the receptor to cause transmembrane signaling (4Vu T.-K.H. Hung D.T. Wheaton V.I. Coughlin S.R. Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2651) Google Scholar, 5Vu T.-K.H. Wheaton V.I. Hung D.T. Coughlin S.R. Nature. 1991; 353: 674-677Crossref PubMed Scopus (467) Google Scholar, 6Chen J. Ishii M. Wang L. Ishii K. Coughlin S.R. J. Biol. Chem. 1994; 269: 16041-16045Abstract Full Text PDF PubMed Google Scholar). The synthetic peptide SFLLRN, which represents the newly formed amino terminus of the receptor, can activate PAR1 independent of thrombin and receptor cleavage. PAR1 is irreversibly activated; thus, the mechanisms that contribute to the termination of signaling are critical determinants of the magnitude and kinetics of the thrombin response in cells. Given the irreversible nature of PAR1 activation, we hypothesize that signal termination events are probably unique, since all other GPCRs are reversibly activated. The molecular events responsible for GPCR desensitization and resensitization have been extensively studied using the β2-adrenergic receptor (7Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 8Kohout T.A. Lefkowitz R.J. Mol. Pharmacol. 2003; 63: 9-18Crossref PubMed Scopus (352) Google Scholar). In the classic paradigm, GPCRs are initially desensitized by rapid phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs) and other kinases. Receptor phosphorylation enhances the affinity of interaction with arrestins, and arrestin binding prevents receptor-G protein interaction, thereby uncoupling the receptor from signaling. Arrestins also interact with components of the endocytic machinery to facilitate recruitment of GPCRs to clathrin-coated pits and internalization from the plasma membrane (9Goodman 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: 447-450Crossref PubMed Scopus (1156) Google Scholar, 10Ferguson S.S. Downey W.r. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (840) Google Scholar). Once internalized into endosomes, GPCRs dissociate from their ligands, become dephosphorylated, and then return to the cell surface in a state capable of responding to ligand. Thus, for most classic, reversibly activated GPCRs, signaling is terminated at the plasma membrane, and receptor trafficking is linked to resensitization of signaling. Phosphorylation of activated PAR1 also appears to be important for rapid uncoupling from G protein signaling. Overexpression of either GRK3 or GRK5 enhances PAR1 phosphorylation and K. J. Ishii M. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Benovic J.L. 2000; PubMed Scopus Google Scholar). PAR1 mutant in which all of the and in the cytoplasmic tail are to is extensively by GRK3 in cell K. J. Ishii M. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Wang L. M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, S.R. Coughlin S.R. 38: PubMed Scopus Google Scholar). In we that are also critical for the termination of PAR1 signaling. of is in both arrestin arrestin 2 and arrestin β-arrestin 1 and β-arrestin whereas PAR1 internalization T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, in both and phosphorylation of activated PAR1 is for internalization through clathrin-coated Moreover, classic GPCRs, proteolytically activated PAR1 is internalized and to an critical for termination of receptor signaling J. S.R. Coughlin S.R. 1998; PubMed Scopus Google Scholar, J. Coughlin S.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, PAR1 a new of GPCRs that a and for that independent of β-arrestins and receptor trafficking is linked to termination of signaling. The of in signal regulation of a GPCR as PAR1 that not these for internalization through clathrin-coated pits not been Moreover, the relative contribution of phosphorylation β-arrestins to the termination of PAR1 signaling to be In the we COS-7 cells to the of phosphorylation and β-arrestins in uncoupling PAR1 from G protein signaling. findings suggest that β-arrestins are to bind and activated PAR1 independent of Thus, these reveal a regulation of PAR1 signaling that both PAR1 phosphorylation and binding of and was from peptide was as the and by P peptide was from was from and were from was T.A. Lefkowitz R.J. 2001; Google and by J. Lefkowitz was generated as D.T. Wheaton V.I. Ishii K. Coughlin S.R. J. PubMed Scopus Google Scholar). and were from and PAR1 and phosphorylation mutant were K. J. Ishii M. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The PAR1 mutants in which and were to were generated using the were by wild type substance P receptor an amino-terminal was generated as J. Coughlin S.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and β-arrestins were from J. Lefkowitz protein β-arrestins were from Caron βarr1 R169E and βarr2 were by Gurevich and have been J. Gurevich V.V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The 5-hydroxytryptamine2A serotonin receptor was by L. The wild type and constitutively active mutant were by of COS-7 cells were from the and in with and cells were at in and were then with a of of either PAR1, PAR1 mutants, or receptor and either mutants, or using to the at 2 in were and with a of 2 of either PAR1 or PAR1 mutants and either or using cells at of were and then with 2 in 1 for were with 1 and were then in the or of either substance or in for at was and were with were with and were by as T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was then to in this were using and was determined using The of PAR1 desensitization was determined by the in thrombin response The were to the of formed in cells for each COS-7 cells at in were either or with or substance P for at were with for at and then with for 1 at in 1 and were then and with for 1 at were and with for at was and the was determined at using a and were as J. Coughlin S.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google from cells at of with PAR1 and of were determined using protein and of were by and and were with PAR1 and β-arrestin COS-7 cells were at 2 in a with PAR1 and β-arrestin and then with for at were in and and of were for with were by and with or were with to and were with by and by a COS-7 cells were and with for 1 at and to at were and then for as T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were using an with an with a The was using of PAR1 of Receptor to and through the of Nature. PubMed Scopus Google Scholar). Thus, we to the of phosphorylation and β-arrestins in PAR1 desensitization by of in COS-7 cells. COS-7 cells are to of β-arrestins L. J. Lefkowitz R.J. Caron M.G. Barak L.S. Mol. Pharmacol. Scopus Google Scholar). initially compared the signaling of PAR1 wild type and a mutant that all phosphorylation sites and is to desensitization in cell COS-7 K. J. Ishii M. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, S.R. Coughlin S.R. 38: PubMed Scopus Google Scholar). The for thrombin at wild type and mutant PAR1 were determined by cells with and of thrombin for at The of was then The effective of thrombin to a response was for both PAR1 wild type and mutant in these However, activated PAR1 mutant an enhanced signaling response compared with wild type receptor These findings suggest that each activated PAR1 mutant to signaling was Both phosphorylation and β-arrestins contribute to PAR1 desensitization K. J. Ishii M. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the relative contribution of each of these pathways to termination of PAR1 signaling to be initially compared the of agonist-induced in COS-7 cells with PAR1 and either βarr1 or βarr2 to that β-arrestins are capable of PAR1 signaling in these cells. were in the or of a of thrombin for at and were then The of was in all of a marked in was in cells PAR1 a in in cells PAR1 and βarr2 that βarr2 not a in PAR1 uncoupling from G protein signaling. In contrast, signaling was in cells PAR1 and an in was of that βarr1 is more effective βarr2 at PAR1 signaling. the contribution of phosphorylation β-arrestin binding to PAR1 we signaling by the PAR1 mutant in cells either βarr1 or βarr2. In COS-7 cells the PAR1 mutant, thrombin an in a response that with of wild type receptor in these cells of βarr2 failed to signaling by PAR1 mutant to that with wild type receptor. In contrast, βarr1 a marked inhibition of PAR1 signaling that PAR1 uncoupling from G protein signaling is independent of the of activated PAR1 to was by either βarr1 or βarr2. PAR1 wild type or mutant was with either βarr1 or and the of receptor to was The for thrombin at wild type and mutant PAR1 with either or was in The for of by thrombin were in each The of thrombin for of by PAR1 wild type and mutant with either or was also these findings that the of activated PAR1 wild type and mutant to G signaling response is not by of β-arrestins relative and of thrombin at PAR1 wild type and in a new desensitization COS-7 cells PAR1 wild type or mutant with either or were to a of thrombin for at The of PAR1 signaling activity of thrombin was then determined by the addition of and of the of In the of was not in these cells not In cells PAR1 wild type and either βarr1 or the of desensitization were not These findings suggest that the of PAR1 wild type desensitization is independent of β-arrestin PAR1 mutant also a of desensitization in cells with either βarr2 or In contrast, in cells the PAR1 mutant desensitization to more these findings suggest that βarr1 enhances the of PAR1 desensitization independent of receptor other GPCRs are by β-arrestins in COS-7 we the effects of β-arrestins signaling by the substance P receptor also as the receptor. In COS-7 cells a in was of In to responses with PAR1, signaling is in cells either βarr1 or βarr2 that both βarr1 and βarr2 are equally effective at uncoupling activated from G protein signaling in these cells. These findings are with that activated is desensitized a and of β-arrestins to the receptor in other cell L.S. K. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. J.H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In these results that of βarr2 is to GPCR signaling in COS-7 cells. also the of to signaling by to that of β-arrestins not signaling by this G protein in COS-7 cells. In cells wild type the of in was with that in cells with wild type or the constitutively active mutant of a in of either βarr1 or βarr2 failed to the signaling response that βarr1 βarr2 signaling by in COS-7 cells. in cells wild type and mutant PAR1 and of the β-arrestin isoforms to the that the effects of β-arrestins PAR1 signaling are to in the of β-arrestin COS-7 cells with either PAR1 wild type or mutant and of βarr1 or βarr2 were in the or of for at The of was then or cell were and β-arrestin was by In the of β-arrestin an and in was in PAR1 wild and cells of and In cells wild type PAR1 and of activated PAR1 signaling was by whereas βarr1 a inhibition of signaling In PAR1 was more effectively by βarr1 compared with βarr2 to the results with wild type receptor. However, both β-arrestin isoforms were at that the PAR1 mutant is more wild type receptor to in cells of βarr1 and the βarr1 appears more effective βarr2 at activated PAR1 signaling in the of receptor to PAR1 in COS-7 the effects of β-arrestins PAR1 signaling from in receptor we agonist-induced receptor COS-7 cells PAR1 and either βarr1 or βarr2 were in the or of of for at thrombin the amino terminus of PAR1 the the peptide was the of PAR1 the cell surface was by cell surface In an of receptor from the cell surface with PAR1 internalization in other cell T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Coughlin S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). of PAR1 internalization was by in cells either βarr1 or βarr2 The of β-arrestins to enhance PAR1 internalization that receptor trafficking independent of β-arrestins in COS-7 cells. These are with internalization of activated PAR1 in in β-arrestin T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the effects of β-arrestins agonist-induced internalization of PAR1 with internalization of activated PAR1 T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Coughlin S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google to PAR1 internalization whereas wild type PAR1 is internalized Moreover, βarr1 βarr2 enhance agonist-induced PAR1 mutant internalization that the regulation of PAR1 signaling by the isoforms of β-arrestins is not to effects receptor also determined internalization is by β-arrestins in COS-7 cells. In to wild type and mutant PAR1, both βarr1 and βarr2 enhance agonist-induced internalization of with a internalization of F. PubMed Scopus Google Scholar). these results suggest that the regulation of PAR1 signaling by the isoforms of β-arrestin is not to in their to receptor are with a of β-arrestins to enhance internalization of COS-7 cells were with PAR1 wild type or mutant with either βarr1 or and internalization of PAR1 was by In the of both wild type and mutant PAR1 are to the cell surface and However, a of receptor was in an in both wild and mutant with of these receptors as J. Coughlin S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In cells wild type PAR1, to for at internalization of receptor into endocytic of agonist-induced PAR1 internalization was in both and cells In contrast, failed to PAR1 mutant in cells βarr1 and βarr2 These findings for an internalization of PAR1 in COS-7 cells. with PAR1 of Receptor determined activated PAR1 and β-arrestins by COS-7 cells with and either βarr1 or βarr2 were with or for at were and PAR1 was with and the of β-arrestins was by In cells PAR1 was and a of βarr1 coimmunoprecipitated with the receptor, that receptor with βarr1 In contrast, from cells a more in βarr1 with activated PAR1, whereas βarr2 was at most with PAR1 Strikingly, a of βarr1 with PAR1 mutant in both and cells this from activity with this mutant with a of interaction wild type PAR1 and a association βarr2 and PAR1 mutant was in cells a of receptor was The in the of βarr1 βarr2 in COS-7 cell is to the affinity of for βarr1 protein T.A. Lefkowitz R.J. 2001; Google Scholar). This affinity is not responsible for the of association PAR1 and since results were in cells PAR1 and the of β-arrestins in was using not these findings suggest that enhances binding of βarr1 to wild type PAR1, and the PAR1 mutant binds βarr1 in the of receptor wild type and constitutively active mutants are equally effective at desensitizing PAR1 signaling. and COS-7 cells with PAR1 wild type or mutant and either or βarr1 were with and in the or of for at and were then The in are the of with and are of The of PAR1 wild type and mutant surface with or βarr1 R169E were or and or The in PAR1 wild type or mutant signaling with compared with either βarr1 or βarr1 R169E was was determined using an and COS-7 cells with PAR1 wild type or mutant and either βarr2 or βarr2 were with and as The of PAR1 wild type and mutant with βarr2 or βarr2 were or and or The in are the of with and are of to PAR1 β-arrestins are capable of binding to activated PAR1 independent of we the β-arrestin mutants, βarr1 R169E and βarr2 R170E, that bind with affinity to receptors independent of phosphorylation J. Gurevich V.V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Gurevich J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the of wild type and mutant β-arrestins to signaling by wild type PAR1 in COS-7 cells. with cells was by and in cells either βarr1 wild type or βarr1 R169E mutant, Thus, both wild type and mutant R169E βarr1 are equally effective at signaling by wild type with a of βarr2 at desensitizing PAR1, βarr2 wild type βarr2 mutant signaling by activated PAR1 these results that desensitization of activated PAR1 is equally to wild type and mutant mutant β-arrestins are capable of binding to activated receptors independent of these findings suggest that phosphorylation of activated PAR1 is not for β-arrestin we the of wild type and mutant β-arrestins to PAR1 mutant signaling. In cells PAR1 mutant a in signaling was compared with cells of wild type receptor These findings suggest that the PAR1 mutant is at the least of either βarr1 or βarr1 R169E a in both and agonist-induced signaling by PAR1 mutant These findings suggest that βarr1 is to activated PAR1 from signaling independent of βarr2 and βarr2 mutant also both and agonist-induced signaling by PAR1 mutant were effective βarr1 and compared with wild type βarr1 R169E and βarr2 to signaling of either PAR1 wild type or of three in the of PAR1 contribute to β-arrestin binding and desensitization of PAR1 signaling. these are important for termination of PAR1 signaling, the of both PAR1 wild type and mutant were to COS-7 cells PAR1 wild type or mutant and either βarr1 or βarr1 R169E mutant were to for and was The of the failed to the of either βarr1 or βarr1 R169E mutant to PAR1 signaling of the three in the of PAR1 mutant also failed to desensitization of signaling by either βarr1 or βarr1 R169E Both βarr2 wild type and βarr2 mutant also failed to signaling by PAR1 wild type or mutant in which the was not these findings the that β-arrestin binding to PAR1 the βarr1 R169E and βarr2 mutants enhanced activity at desensitizing GPCRs in COS-7 cells as in other cell J. Gurevich J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Gurevich V.V. Mol. Pharmacol. 2003; 63: PubMed Scopus Google we their effects desensitization of the serotonin receptor. In COS-7 cells receptor in the of the addition of a in of was in cells receptor and either wild type βarr1 or βarr2 an was by both βarr1 and both βarr1 R169E and βarr2 mutants were more effective wild type β-arrestins and virtually inhibition in activated receptor signaling compared with cells β-arrestin The of β-arrestins to receptor signaling is not to in of receptor at the cell surface These findings are with that mutant βarr1 R169E and βarr2 are to bind to activated GPCRs with affinity and signaling responses more effectively wild type PAR1 is proteolytically irreversibly and mechanisms that PAR1 signaling the magnitude and duration of thrombin cellular In this we that β-arrestins bind to activated PAR1 independent of phosphorylation and termination of receptor signaling. Moreover, βarr1 is more effective βarr2 at uncoupling activated PAR1 from signaling, that β-arrestins can PAR1 signaling independent of receptor with these activated PAR1 with whereas PAR1 interaction with βarr2 was virtually undetectable. contrast, both βarr1 and βarr2 were equally effective at desensitizing the classic reversibly activated these findings suggest that PAR1 signaling is by independent mechanisms receptor phosphorylation and the binding of β-arrestins independent of The β-arrestin isoforms to have functions in desensitization of most classic GPCRs T.A. Lefkowitz R.J. 2001; Google Scholar). However, their to GPCR internalization that these are not that βarr1 is more effective βarr2 at thrombin signaling responses 1 and that β-arrestins PAR1 signaling in the of receptor These results are with in which desensitization of PAR1 signaling is in that βarr1 βarr2 T.A. Lefkowitz R.J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Moreover, we that βarr1 βarr2 enhances PAR1 internalization in COS-7 cells and that receptor trafficking is not responsible for effects of β-arrestins PAR1 signaling. The molecular for the of the isoforms of β-arrestin to GPCR signaling is not known. is that the β-arrestin isoforms have determinants for binding to is also that of either βarr1 or βarr2 their to or Phosphorylation and the endocytic functions of Lefkowitz R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T.A. Lefkowitz R.J. Science. 2001; PubMed Scopus Google these the of β-arrestins to PAR1 signaling is not known. have that interact with the cytoplasmic of GPCRs J. Gurevich Benovic J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Krupnick J.G. Benovic J.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in suggest that the of classic GPCRs are also in β-arrestin interaction J. Barak L.S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the β-arrestin binding promotes GPCR is that the binding of β-arrestins to a GPCR functions desensitization The of PAR1 is the of phosphorylation and is in desensitization K. J. Ishii M. Lefkowitz R.J. Coughlin S.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, Wang L. M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). However, is that PAR1 phosphorylation is responsible for β-arrestin interaction, since β-arrestins bind to PAR1 mutant and desensitization 1 and Moreover, we also that βarr1 binds to an activated PAR1 mutant the not that the is not for β-arrestin is to suggest that other in the of PAR1 are sites of a of three in the cytoplasmic of PAR1 contribute to β-arrestin However, in both PAR1 wild type and mutant in which the were to we in the of β-arrestins to signaling responses these findings the that phosphorylation and β-arrestin binding both contribute to PAR1 activated GPCRs phosphorylation for β-arrestin binding and receptor In contrast, β-arrestins bind to activated PAR1 independent of phosphorylation to uncoupling from G protein signaling. The mutant arrestins, βarr1 R169E and βarr2 R170E, which bind with affinity to activated GPCRs independent of phosphorylation J. Gurevich V.V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Gurevich J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google are equally effective at desensitization of both PAR1 wild type and These findings that PAR1 phosphorylation is not critical for β-arrestin Moreover, agonist-induced enhanced association of β-arrestins with activated PAR1 the that β-arrestins the active of the receptor. Thus, of PAR1 or critical the cytoplasmic of the receptor that phosphorylation and thereby promotes binding of with these wild type and mutant are equally effective at desensitizing the receptor Gurevich V.V. L. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This receptor is desensitized in a phosphorylation independent and a to the for β-arrestin binding and In we the contribution of phosphorylation β-arrestin binding to the termination of PAR1 signaling in COS-7 cells. In these we that β-arrestins can bind to activated PAR1 independent of phosphorylation and termination of receptor signaling. also that βarr1 is more effective βarr2 at desensitizing both PAR1 and These findings suggest that the isoforms of β-arrestins can GPCR desensitization independent of receptor PAR1 to as as and and PAR1 to G protein is not known. Thus, desensitization of PAR1 signaling is by independent mechanisms phosphorylation and binding of β-arrestins independent of The mechanisms by which β-arrestins bind to and activated PAR1 to be These findings new into signaling by irreversibly proteolytically activated GPCRs is and of for and