Characterization of Dominant Negative Arrestins That Inhibit β2-Adrenergic Receptor Internalization by Distinct Mechanisms

Arrestins have been shown to act as adaptor proteins that mediate the interaction of G protein-coupled receptors with the endocytic machinery. In this study, the role of arrestin-3 in receptor internalization was investigated by constructing different arrestin-3 minigenes that could potentially act as dominant negative inhibitors of arrestin function. Expression of arrestin-3 proteins containing amino acids 1–320 or 201–409 resulted in the inhibition of β2-adrenergic receptor internalization in HEK-293 cells by approximately 40%. Both of these arrestins were diffusely localized within the cytoplasm of transfected cells, were unable to mediate redistribution of receptors to clathrin-coated pits, and did not localize to coated pits in either the presence or absence of receptor and agonist. Arrestin-3(1–320), but not arrestin-3(201–409), bound to light-activated phosphorylated rhodopsin with an affinity comparable with that of wild-type arrestin-3. In contrast, expression of arrestin-3 proteins composed of only the clathrin binding domain, arrestin-3(284–409), and arrestin-3(290–409) resulted in the constitutive localization of these arrestins to coated pits. Arrestin-3(284–409) and arrestin-3(290–409) acted as dominant negative inhibitors of wild-type arrestin function, inhibiting receptor internalization by 70 and 30%, respectively. Carboxyl-terminal deletions of arrestin-3 retained the ability to promote internalization until residues amino-terminal to amino acid 350 were deleted, suggesting that residues in this region also compose part of the clathrin binding domain in addition to the major binding site between residues 371–379. These studies characterize at least two distinct mechanisms, competition for either receptor or clathrin binding, by which dominant negative arrestins inhibit receptor internalization and further define residues within arrestin-3 that constitute the clathrin binding domain. Arrestins have been shown to act as adaptor proteins that mediate the interaction of G protein-coupled receptors with the endocytic machinery. In this study, the role of arrestin-3 in receptor internalization was investigated by constructing different arrestin-3 minigenes that could potentially act as dominant negative inhibitors of arrestin function. Expression of arrestin-3 proteins containing amino acids 1–320 or 201–409 resulted in the inhibition of β2-adrenergic receptor internalization in HEK-293 cells by approximately 40%. Both of these arrestins were diffusely localized within the cytoplasm of transfected cells, were unable to mediate redistribution of receptors to clathrin-coated pits, and did not localize to coated pits in either the presence or absence of receptor and agonist. Arrestin-3(1–320), but not arrestin-3(201–409), bound to light-activated phosphorylated rhodopsin with an affinity comparable with that of wild-type arrestin-3. In contrast, expression of arrestin-3 proteins composed of only the clathrin binding domain, arrestin-3(284–409), and arrestin-3(290–409) resulted in the constitutive localization of these arrestins to coated pits. Arrestin-3(284–409) and arrestin-3(290–409) acted as dominant negative inhibitors of wild-type arrestin function, inhibiting receptor internalization by 70 and 30%, respectively. Carboxyl-terminal deletions of arrestin-3 retained the ability to promote internalization until residues amino-terminal to amino acid 350 were deleted, suggesting that residues in this region also compose part of the clathrin binding domain in addition to the major binding site between residues 371–379. These studies characterize at least two distinct mechanisms, competition for either receptor or clathrin binding, by which dominant negative arrestins inhibit receptor internalization and further define residues within arrestin-3 that constitute the clathrin binding domain. G protein-coupled receptor β2-adrenergic receptor enzyme-linked immunosorbent assay polymerase chain reaction fluorescein isothiocyanate. The processes of desensitization, internalization, down-regulation, and resensitization of G protein-coupled receptors (GPCRs)1 have been shown to be promoted in part by arrestins (reviewed in Refs. 1Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar, 2Carman C.V. Benovic J.L. Curr. Opin. Neurobiol. 1998; 8: 335-344Crossref PubMed Scopus (238) Google Scholar, 3Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar). Arrestins promote these processes by interacting with the agonist-occupied form of GPCRs. Recruitment of arrestins to receptors is promoted by phosphorylation of the receptor by G protein-coupled receptor kinases (4Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 5Gurevich V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. Chem. 1993; 268: 16879-16882Abstract Full Text PDF PubMed Google Scholar, 6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), although binding of arrestins to receptor in the absence of receptor phosphorylation has also been observed (7Ferguson S.S.G. Downey W.E.I. Colapietro A-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (846) Google Scholar, 8Wu G. Krupnick J.G. Benovic J.L. Lanier S.M. J. Biol. Chem. 1997; 272: 17836-17842Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The binding of arrestin appears to sterically inhibit interaction of the receptor with G-proteins, thus attenuating receptor-mediated signaling (9Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar, 10Krupnick J.G. Gurevich V.V. Benovic J.L. J. Biol. Chem. 1997; 272: 18125-18131Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 11Lohse M.J. Andexinger S. Pitcher J. Trukawinski S. Codina J. Faure J-P. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 8558-8564Abstract Full Text PDF PubMed Google Scholar). Arrestins mediate the internalization of receptors from the cell surface by interacting with clathrin, the major component of clathrin-coated pits, thus facilitating receptor endocytosis through the clathrin-coated vesicle pathway (12Goodman 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 (1172) Google Scholar, 13Goodman Jr., O.B. Krupnick J.G. Gurevich V.V. Benovic J.L. Keen J.H. J. Biol. Chem. 1997; 272: 15017-15022Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 14Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Arrestins have recently been shown to be involved in both receptor down-regulation (15Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), the overall decrease in receptor number after prolonged agonist exposure, as well as resensitization (16Zhang J. Barak L.S. Winkler K.E. Caron M.G. Ferguson S.S.G. J. Biol. Chem. 1997; 272: 27005-27014Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar), the dephosphorylation of the receptor and its recycling back to the cell surface after agonist removal. Recently, a role for arrestin-mediated internalization of GPCRs in the activation of the p42/p44 mitogen-activated protein kinase pathway has been proposed (17Daaka Y. Luttrell L.M. Ahn S. Della Rocca G.J. Ferguson S.S.G. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 685-688Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). Extensive study of visual arrestin, which is involved in quenching phototransduction via its specific interaction with metarhodopsin II, has served as a basis for the model of nonvisual arrestin function. The four mammalian arrestins, visual arrestin (arrestin-1), β-arrestin (arrestin-2), β-arrestin-2 (arrestin-3), and cone arrestin (arrestin-4), 2Although a variety of names have been used for the various mammalian arrestins, we propose that the following nomenclature be used based on the order of discovery of the various arrestins: arrestin-1 (visual arrestin, S-antigen, 48 kDa protein); arrestin-2 (β-arrestin, β-arrestin-1); arrestin-3 (β-arrestin-2, arrestin-3, thy-X arrestin); arrestin-4 (C-arrestin, X-arrestin). share approximately 45% identity and 70% similarity to one another (18Sterne-Marr R. Benovic J.L. Vitam. Horm. 1995; 51: 193-234Crossref PubMed Scopus (113) Google Scholar). Initial binding of arrestin-1 to phosphorylated metarhodopsin II occurs through the activation- and phosphorylation-recognition domains. These domains are located within the amino-terminal half of arrestins and include a conserved arginine residue (Arg-175 in arrestin-1) within the phosphorylation recognition domain (6Gurevich V.V. Benovic J.L. J. Biol. Chem. 1995; 270: 6010-6016Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 19Gurevich V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (123) Google Scholar). This residue, upon interaction with receptor, is thought to act as a molecular switch that promotes a conformational change in arrestin. This conformational change is proposed to disrupt the intramolecular interaction of the basic amino terminus and acidic carboxyl terminus (20Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, V.V. Kim C.M. Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which binding of a of arrestin to V.V. Richardson R.M. Kim C.M. Hosey M.M. Benovic J.L. J. Biol. 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The ability to arrestin-mediated internalization of GPCRs is a potentially by which to both receptor and the of of negative of arrestin-2 have been that to by with arrestins for binding to in arrestin-2 has been shown to affinity for clathrin and affinity for receptor (7Ferguson S.S.G. Downey W.E.I. Colapietro A-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (846) Google Scholar, J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of a the arrestin-2 clathrin binding domain has been shown to inhibit internalization of both β2-adrenergic receptors and receptors in a dominant negative J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, Krupnick J.G. Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). dominant negative of that binding H. J. Biol. PubMed Scopus Google has also in the role of endocytosis in various receptor-mediated but not be specific for the clathrin-coated vesicle pathway J. Biol. 1998; PubMed Scopus Google Scholar). In this study, we have further the role of arrestin-3 in receptor by a of arrestin-3 the of these on internalization in both and HEK-293 These studies resulted in the of dominant negative arrestin-3 proteins that inhibit arrestin at either the of receptor or we residues that the clathrin binding domain. The of arrestin-3 that with arrestin at in the endocytic pathway in the role of arrestins in the of GPCRs by different endocytic and role in wild-type arrestin-3 and were J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Gagnon A.W. Penn R.B. Benovic J.L. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The arrestin-3 minigenes and were as was to include a site and a by the amino terminus of arrestin-3. The was composed of a an site and a by the for the amino acids of the These were used in a reaction with wild-type as for for for for by a at The were with and to with the the arrestin-3 and the was used with the that the after the and the either a or The were with and or and with and were by and HEK-293 cells were in with and at in a of cells were to were transfected for in either or and or to the binding to receptor internalization, cells were after and at internalization by cells from one were of a that been coated with and of was by either binding or by binding was as (15Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), with the that cells were with were in the presence of or with a of of and in in containing for at in the presence or absence of were by a cell The assay was as by C. L. J. F. Mol. Pharmacol. 1997; 51: PubMed Scopus Google Scholar). cells were as and were with with or for with The and the were used at a of binding was an were at in a by arrestin cells were in and on to amino-terminal arrestin were with a a conserved in the amino terminus of arrestins, Curr. 1992; PubMed Scopus Google Scholar). expression of were with a a protein containing residues of arrestin-3 was the in by by or cells in were transfected as with and of the arrestin-3 or of the arrestin-3 cells were and to on in cell surface cells were with for at in with were with for with with for and with fluorescein arrestins, cells were and as after agonist The used to arrestins were either or the were a the clathrin adaptor proteins were with either or or was with a were and by on a a the of transfected but of cells, were for were and with were and in the to the arrestin-3 minigenes were to a reaction containing of amino acid of and of in a of The were for at and was by of reaction and by for in in and in was in was and the were in a were phosphorylated with rhodopsin kinase to a of and with as (20Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar). receptor binding, arrestins were for at with rhodopsin in a of in and were on and arrestins were by as V.V. J. Kim C.M. R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). Extensive of with the of its has distinct domains involved in arrestin (20Gurevich V.V. Benovic J.L. J. Biol. Chem. 1993; 268: 11628-11638Abstract Full Text PDF PubMed Google Scholar, V.V. J. Kim C.M. R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. G. Nature. 1998; PubMed Scopus Google Scholar). is the of the nonvisual arrestins, arrestin-2 and arrestin-3. have the clathrin binding domain of arrestin-3 J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google and have shown that expression of the domain of arrestin-2 wild-type arrestin J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). that expression of the arrestin-3 clathrin binding domain, which has been shown to have a affinity for clathrin (12Goodman 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 (1172) Google Scholar), act as a of arrestin function. we to expression of the receptor binding domain of arrestin-3 also inhibit receptor these we a of arrestin-3 minigenes containing either receptor or clathrin binding domains. These shown in were on the basis of of arrestin-3 domains to in arrestin-1 V.V. Benovic J.L. Mol. Pharmacol. 1997; 51: 161-169Crossref PubMed Scopus (123) Google Scholar). we the ability of of the to be in cells to which were for further shown in of the arrestin-3 amino-terminal minigenes were in with wild-type arrestin-3, was and of as a of The minigenes were also for which was only after of the not on to its and these the and arrestin-3 were not in the of arrestin-3 protein by its ability to promote the internalization of the in has been shown that internalization of the in cells is but be promoted to by of nonvisual arrestins (15Gagnon A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Ferguson S.S.G. J. Lefkowitz Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: PubMed Scopus Google Scholar). different were used to receptor internalization, an to the surface of the and a binding assay to the binding of the which to cell surface The of the assay are in A. the amino-terminal which deletions of the carboxyl and were unable to promote internalization In contrast, the and arrestin-3 proteins promoted internalization to an comparable with that of wild-type arrestin-3. or of the major clathrin binding domain between residues of arrestin-3 did not clathrin binding or of internalization (12Goodman 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 (1172) Google Scholar, 14Krupnick J.G. Goodman Jr., O.B. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: 15011-15016Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), this that clathrin binding that between amino acids the acidic carboxyl which has been proposed to mediate an intramolecular interaction with the basic amino terminus to arrestin-3 in an V.V. Kim C.M. Benovic J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of this interaction in arrestin-3 in a arrestin. appears to promote affinity agonist binding to the and receptor with wild-type arrestin-3 V.V. R. Benovic J.L. Hosey J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). shown in promoted receptor internalization as as wild-type arrestin-3, but receptor internalization upon the presence of agonist. In the absence of the number of surface receptors in cells with was to that in cells with wild-type arrestin-3 not of the amino terminus of arrestin-3, in which both receptor binding and phosphorylation recognition domains were deleted, resulted in the proteins arrestin-3(201–409), arrestin-3(284–409), and These arrestins were unable to promote internalization of the that internalization was by the and to the of different of receptor we also internalization by binding of the shown in we that were to in the In this the was at approximately were the was at not In both promoted receptor internalization, the and arrestin-3 proteins did did not in in cells with wild-type arrestin-3 or arrestin-3 minigenes in the absence of agonist not These that the ability of arrestins to receptors be in two different a of receptor expression the ability of and arrestin-3 proteins to mediate the redistribution of the in we cells with and the different arrestin-3 by cells with the agonist In this only the of receptors on the cell surface be agonist exposure, cells were and the was with shown in redistribution of the promoted by arrestins was observed in cells in the absence of arrestin-3 redistribution of the to clathrin-coated pits and was observed in cells that wild-type arrestin-3. of promoted a of receptor was redistribution of the upon of the or arrestin-3 These the of arrestin by binding and and that the ability of a arrestin-3 protein to promote internalization well with the redistribution of to clathrin-coated pits and In cells the receptor and arrestin-2 is only with clathrin-coated pits after agonist (12Goodman 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 (1172) Google Scholar, J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, of the clathrin binding domain of was shown to a and with clathrin in the absence of receptor and agonist J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the localization of the arrestin-3 minigenes in the absence of receptor and cells were transfected with the various arrestin-3 and was arrestin-3 or a major component of cell surface clathrin-coated pits. arrestin-3, and to be diffusely localized within the cytoplasm In contrast, the and arrestin-3 proteins to be and the of of cells for with These that expression of the arrestin-3 clathrin binding domain in constitutive localization to clathrin-coated pits. the presence of the domain between residues appears to be to of the clathrin binding domain with clathrin-coated pits. is that in coated pits, but that is to be The arrestin-3 minigenes that were unable to promote internalization of the in cells, as by binding, and were as negative inhibitors of receptor The in HEK-293 with cells of of arrestins (7Ferguson S.S.G. Downey W.E.I. Colapietro A-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-365Crossref PubMed Scopus (846) Google Scholar, A.W. Kallal L. Benovic J.L. J. Biol. Chem. 1998; 273: 6976-6981Abstract Full Text Full Text PDF PubMed Scopus (202) Google J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, L. Ferguson S.S.G. J. Lefkowitz Caron M.G. Barak L.S. Mol. Pharmacol. 1997; 51: PubMed Scopus Google Scholar). we have shown that the of arrestins in HEK-293 cells be in a dominant negative by the clathrin binding domain of arrestin-2 J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). the various arrestin-3 were with HEK-293 cells, and internalization of the was by expression approximately which the of receptor internalization, expression resulted in internalization not shown in and internalization by approximately 40%. Arrestin-3(284–409) internalization by arrestin-3(290–409) internalization of the by approximately and were also receptor internalization, in were observed in cells with wild-type arrestin-3 or arrestin-3 minigenes in the absence of agonist not of the expression of the various arrestin-3 proteins in these cells that the and arrestin-3 proteins were at expression of arrestin-3(290–409) was which the of inhibition by this with the of a dominant negative arrestin appears to be to its of studies that receptor internalization by with arrestins for receptor binding and arrestin-3(290–409) act by with arrestins for binding to the of of is the presence of the clathrin binding domain, did not localize to coated pits of its localization and the presence of a domain to receptor binding V.V. J. Kim C.M. R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), was that this act by with receptor to these we receptor binding of in arrestin-3 The and of the in arrestins to be to in cells binding was light-activated of interaction with the be binding of nonvisual arrestins to rhodopsin appears to to to either or receptors V.V. J. Kim C.M. R. Hosey M.M. Benovic J.L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). arrestin-3 and bound to rhodopsin In contrast, the and arrestin-3 proteins did not to This that is to inhibit receptor internalization by for receptor binding and that not in this In we have and a of arrestin-3 minigenes to the role of arrestin-3 in receptor that expression of an arrestin-3 protein composed of the major receptor activation and phosphorylation recognition domains inhibit wild-type arrestin by for receptor binding expression of arrestin-3 composed of the clathrin binding domain act in a dominant negative by with arrestins for binding to arrestin-3(284–409), the internalization of by which is inhibition that by expression of J.G. Santini F. Gagnon A.W. Keen J.H. Benovic J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google and not we have two distinct in the endocytic pathway at which dominant negative arrestins inhibit receptor internalization by although this protein did not to receptor and was not with clathrin-coated pits the presence of the clathrin binding domain. is that the presence of the domain between amino acids and the constitutive of this protein with clathrin-coated pits. a of this protein be with coated pits in that are to be Recently, has been that arrestins are by agonist of at least different GPCRs L.S. Ferguson S.S.G. J. Caron M.G. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). dominant negative arrestins are to be in the role of arrestins in the and signaling of a number of GPCRs. The of dominant negative arrestin-2 proteins in studies the role of arrestins in and signaling has been (7Ferguson S.S.G. Downey W.E.I. Colapietro A-M. Barak L.S. Menard L. Caron M.G. 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