Feedback Regulation of β-Arrestin1 Function by Extracellular Signal-regulated Kinases
1999; Elsevier BV; Volume: 274; Issue: 23 Linguagem: Inglês
10.1074/jbc.274.23.15971
ISSN1083-351X
AutoresFang‐Tsyr Lin, William E. Miller, Louis M. Luttrell, Robert J. Lefkowitz,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe functions of β-arrestin1 to facilitate clathrin-mediated endocytosis of the β2-adrenergic receptor and to promote agonist-induced activation of extracellular signal-regulated kinases (ERK) are regulated by its phosphorylation/dephosphorylation at Ser-412. Cytoplasmic β-arrestin1 is almost stoichiometrically phosphorylated at Ser-412. Dephosphorylation of β-arrestin1 at the plasma membrane is required for targeting a signaling complex that includes the agonist-occupied receptors to the clathrin-coated pits. Here we demonstrate that β-arrestin1 phosphorylation and function are modulated by an ERK-dependent negative feedback mechanism. ERK1 and ERK2 phosphorylate β-arrestin1 at Ser-412 in vitro. Inhibition of ERK activity by a dominant-negative MEK1 mutant significantly attenuates β-arrestin1 phosphorylation, thereby increasing the concentration of dephosphorylated β-arrestin1. Under such conditions, β-arrestin1-mediated β2-adrenergic receptor internalization is enhanced as is its ability to bind clathrin. In contrast, if ERK-mediated phosphorylation is increased by transfection of a constitutively active MEK1 mutant, receptor internalization is inhibited. Our results suggest that dephosphorylated β-arrestin1 mediates endocytosis-dependent ERK activation. Following activation, ERKs phosphorylate β-arrestin1, thereby exerting an inhibitory feedback control of its function. The functions of β-arrestin1 to facilitate clathrin-mediated endocytosis of the β2-adrenergic receptor and to promote agonist-induced activation of extracellular signal-regulated kinases (ERK) are regulated by its phosphorylation/dephosphorylation at Ser-412. Cytoplasmic β-arrestin1 is almost stoichiometrically phosphorylated at Ser-412. Dephosphorylation of β-arrestin1 at the plasma membrane is required for targeting a signaling complex that includes the agonist-occupied receptors to the clathrin-coated pits. Here we demonstrate that β-arrestin1 phosphorylation and function are modulated by an ERK-dependent negative feedback mechanism. ERK1 and ERK2 phosphorylate β-arrestin1 at Ser-412 in vitro. Inhibition of ERK activity by a dominant-negative MEK1 mutant significantly attenuates β-arrestin1 phosphorylation, thereby increasing the concentration of dephosphorylated β-arrestin1. Under such conditions, β-arrestin1-mediated β2-adrenergic receptor internalization is enhanced as is its ability to bind clathrin. In contrast, if ERK-mediated phosphorylation is increased by transfection of a constitutively active MEK1 mutant, receptor internalization is inhibited. Our results suggest that dephosphorylated β-arrestin1 mediates endocytosis-dependent ERK activation. Following activation, ERKs phosphorylate β-arrestin1, thereby exerting an inhibitory feedback control of its function. The life cycle of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; ERK, extracellular signal-regulated kinase(s); PAGE, polyacrylamide gel electrophoresis. includes receptor activation, desensitization, sequestration, and either resensitization (recycling) or degradation (1Lefkowitz R.J. J. Biol. Chem. 1998; 273: 18677-18680Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar). β-Arrestins were initially discovered as molecules that bind to agonist-occupied receptors following receptor phosphorylation by G protein-coupled receptor kinases, thereby interdicting signal transduction to G proteins and causing receptor desensitization (2Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (919) Google Scholar, 3Attramadal 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). More recently, however, β-arrestins have been shown to be involved in the internalization and signaling of GPCRs (4Ferguson S.S.G. Downey III, W.E. Colapietro A.-M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (853) Google Scholar). For example, they serve as clathrin adaptors, which help to target agonist-occupied GPCRs to clathrin-coated pits for internalization (5Goodman 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 (1179) Google Scholar). This function is regulated by phosphorylation/dephosphorylation of β-arrestin1 at a carboxyl-terminal serine, Ser-412 (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). Cytosolic β-arrestin1 is constitutively phosphorylated by heretofore unidentified kinase(s) and is rapidly dephosphorylated when it is recruited to the plasma membrane in response to agonist stimulation. Dephosphorylation of β-arrestin1 at the plasma membrane is not required for receptor binding and receptor desensitization but is required for its clathrin binding and receptor internalization. The S412A mutant of β-arrestin1, which mimics the dephosphorylated form, has been shown to be more active than wild-type β-arrestin1 in promoting clathrin-mediated endocytosis of the β2-adrenergic receptor. In contrast, the S412D mutant, which simulates the phosphorylated form of β-arrestin1, acts as a dominant-negative inhibitor of receptor endocytosis (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). Moreover, in addition to regulating the internalization of classical GPCRs, such as the β2-adrenergic receptor, β-arrestin1 has been shown to bind to the tyrosine kinase insulin-like growth factor I receptor and mediate its endocytosis in an analogous fashion (7Lin F.-T. Daaka Y. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 31640-31643Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Recently, several studies have shown that clathrin-mediated internalization is required for mitogenic signaling by various GPCRs and tyrosine kinase growth factor receptors (7Lin F.-T. Daaka Y. Lefkowitz R.J. J. Biol. Chem. 1998; 273: 31640-31643Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Vieira A.V. Lamaze C. Schmid S.L. Nature. 1996; 274: 2086-2089Google Scholar, 9Riccio A. Pierchala B.A. Ciarallo C.L. Ginty D.D. Science. 1997; 277: 1097-1100Crossref PubMed Scopus (368) Google Scholar, 10Chow J.C. Condorelli G. Smith R.J. J. Biol. Chem. 1998; 273: 4672-4680Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 11Luttrell L.M. Daaka Y. Della Rocca G.J. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31648-31656Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 12Daaka 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 (465) Google Scholar, 15Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). Thus, inhibition of clathrin-mediated internalization reduces agonist-induced activation of ERK1 and 2. The Ras-dependent activation of ERKs by GPCRs also requires c-Src (13van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar, 14Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). Very recently, it has been shown that β-arrestins serve to recruit the activated c-Src to the agonist-occupied β2-adrenergic receptors as well as to target this signaling complex to the clathrin-coated pits for internalization and activation of the ERK cascade (15Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). Like clathrin targeting, the recruitment and activation of c-Src kinase is modulated by phosphorylation/dephosphorylation of β-arrestin1 (15Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). The S412D β-arrestin1 mutant, defective in both binding to Src and targeting the receptors to clathrin-coated pits, acts as a dominant-negative inhibitor of agonist-induced ERK activation. In contrast, the S412A β-arrestin1 mutant, which binds to Src as well as the wild-type β-arrestin1, is active in promoting agonist-induced ERK phosphorylation. ERK activity appears to be tightly regulated by an activation/inactivation cycle. GPCR-mediated activation of ERKs involves the sequential involvement of components of a Ras activation complex, including c-Src, Shc, Grb2, Gab1, and Sos1, followed by activation of Raf-1 kinase and MEK1 (13van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar, 14Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). It has been shown that the inactivation of this cascade is associated with the induction of mitogen-activated protein kinase phosphatase (MKP-1) by agonist stimulation (16Charles C.H. Sun H. lau L.F. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5292-5296Crossref PubMed Scopus (185) Google Scholar). Previous studies also suggest that it may involve the negative feedback phosphorylation of upstream activators, including Sos1, Raf-1 kinase, and MEK1, by the activated ERK (17Profiri E. McCormick F. J. Biol. Chem. 1996; 271: 5871-5877Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 18Foschi M. Chari S. Dunn M.J. Sorokin A. EMBO J. 1997; 16: 6439-6451Crossref PubMed Scopus (142) Google Scholar, 19Lee R. Cobb M.H. Blackshear P.J. J. Biol. Chem. 1992; 267: 1088-1092Abstract Full Text PDF PubMed Google Scholar, 20Ueki K. Matsuda S. Tobe K. Gotoh Y. Tamemoto H. Yachi M. Akanuma Y. Yazaki Y. Nishida E. Kadowaki T. J. Biol. Chem. 1994; 269: 15756-15761Abstract Full Text PDF PubMed Google Scholar, 21Brunet A. Pages G. Pouyssegur J. FEBS Lett. 1994; 346: 299-303Crossref PubMed Scopus (91) Google Scholar). Recently ERK has been reported to phosphorylate IRS-1 and reduce its function, thereby inhibiting further insulin signaling (22De Fea K. Roth R.A. J. Biol. Chem. 1997; 272: 31400-31406Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). These findings underscore the requirement for stringent control of cellular ERK activity by feedback regulatory mechanisms. Here we demonstrate a novel form of feedback regulation controlling GPCR-mediated activation of ERKs. Once stimulated, the ERKs phosphorylate β-arrestin1 at Ser-412, thereby reducing its endocytic functions and thus ultimately reducing ERK activation. A 1.26-kilobaseKpnI/HindIII fragment encoding (S412D)βarr1-His6 was removed from pBS/(S412D)βarr1-His6 (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar) and subcloned into pKK223–3 vector (Amersham Pharmacia Biotech). After transfection of pKK/βarr1-His6 or pKK/(S412D)βarr1-His6plasmid into E. coli, expression of the proteins was induced by isopropyl-1-thio-β-d-galactopyranoside. β-Arrestin1 was purified by nickel affinity chromatography followed by Heparin-Sepharose chromatography as described before (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). 20 pmol of wild-type or S412D β-arrestin1 were incubated with either 0.9 μg of GST-ERK1 (Upstate Biotechnologies Inc.), 0.15 μg of ERK2 (Upstate Biotechnologies Inc.), or 10 units of GSK-3 (New England Biolabs) in the presence of 20 mm Tris, pH 7.4, 2 mm EDTA, 10 mm MgCl2, 1 mm dithiothreitol, 100 μm ATP, and 1 μCi of [γ-32P]ATP at 30 °C for 30 min. The phosphoproteins were fractionated by SDS-PAGE. The gel was dried and developed by autoradiography. Purified phospho-β-arrestin1 (wild-type or S412D) was resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon, Millipore). The phospho-β-arrestin1 band was cut out, digested with trypsin in situ, and oxidized in performic acid (23Luo K. Hurley T.R. Sefton B.M. Methods Enzymol. 1991; 201: 149-152Crossref PubMed Scopus (131) Google Scholar). Lyophilized peptides were resolved by electrophoresis at pH 3.5 in the first dimension and ascending chromatography in the second dimension as described (24Huganir R.L. Miles K. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6968-6972Crossref PubMed Scopus (181) Google Scholar). Phosphopeptides were detected by autoradiography. The His-tagged β-arrestin1 expression vector was transfected alone or with the dominant-negative MEK1(K97A) plasmid (25Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar) into HEK 293 cells. Cells were labeled with [32P]orthophosphate for 1 h and then harvested for β-arrestin1 purification as described (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). The FLAG-tagged β-arrestin1 expression vector was transfected alone or with the MEK1(K97A) plasmid into HEK 293 cells. Two days after transfection, cells were harvested and lysed for co-immunoprecipitation as described (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). The FLAG-tagged β-Arrestin1 (15Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar) was immunoprecipitated with a polyclonal antibody directed against the FLAG epitope (Santa Cruz Inc.). After SDS-PAGE, the immunoblot was probed with a monoclonal antibody specific to clathrin heavy chain (Transduction Laboratories) and was visualized by enhanced chemiluminescence assay (ECL, Amersham Pharmacia Biotech). The expression levels of phospho-ERKs, total cellular ERKs, and MEK1 mutants (K97A and S218D/S222D) (25Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar, 26Huang W. Kessler D.S. Erikson R.L. Mol. Biol. Cell. 1995; 6: 237-245Crossref PubMed Scopus (110) Google Scholar) in whole cell extracts were determined by probing the immunoblots separately with the antibodies specific to phospho-ERK (Promega), cellular ERK2 (Transduction Laboratories), or MEK1 (Transduction Laboratories). HEK 293 cells were transiently transfected with the plasmid encoding FLAG-tagged β2-adrenergic receptors with or without the expression vectors of β-arrestin and a MEK1 mutant. Two days after transfection, cells were incubated with 10 μm (−)-isoproterenol in 0.1 mm ascorbic acid for 30 min before harvesting. The agonist-promoted sequestration of β2-adrenergic receptors was determined by immunofluorescence flow cytometry as described previously (27Barak L.S. Tiber M. Freedman N.J. Kwatra M.M. Lefkowitz R.J. Caron M.G. J. Biol. Chem. 1994; 269: 2790-2795Abstract Full Text PDF PubMed Google Scholar). Previously we have shown that cytosolic β-arrestin1 is highly phosphorylated and is dephosphorylated only when it is recruited to the plasma membrane in response to agonist stimulation (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). The major phosphorylation site is located at the carboxyl-terminal Ser-412, which accounts for 90% of β-arrestin1 phosphorylation. To identify the candidate kinase(s) that phosphorylate β-arrestin1 at Ser-412, we tested the ability of several kinases to phosphorylate β-arrestin1in vitro. Because Ser-412 is followed by a proline residue, a consensus phosphorylation sequence recognized by members of the mitogen-activated protein kinase family as well as by glycogen synthase kinase-3 (GSK-3), we speculated that these kinases might be potential candidates for mediating Ser-412 phosphorylation. Therefore, equal amounts of wild-type and S412D β-arrestin1 purified from E. coli were subjected to phosphorylation by ERK1, ERK2, or GSK-3in vitro. As shown in Fig. 1, wild-type β-arrestin1 was highly phosphorylated by either ERK1 or ERK2. The stoichiometry was ∼0.8 mol of Pi/mol of protein. Mutation of Ser-412 to Asp markedly reduced ERK-mediated β-arrestin1 phosphorylation. Both wild-type and S412D β-arrestin1 were equally but weakly phosphorylated by GSK-3, indicating that GSK-3 is not the kinase responsible for Ser-412 phosphorylation. Next, we compared the two-dimensional tryptic phosphopeptide map of cellular β-arrestin1 with those of wild-type and S412D β-arrestin1 phosphorylated by ERK2 in vitro. As shown in Fig.2 A, the two-dimensional phosphopeptide mapping of cellular β-arrestin1 purified from HEK 293 cells indicates that it contains three phosphopeptides: a1, a2, and b. The major phosphopeptides, a1 and a2, are partial digestion products (amino acids 401–418 and 398–418) containing Ser-412 (Fig.2 B) as confirmed by amino acid sequencing (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). The two-dimensional phosphopeptide map of β-arrestin1 phosphorylated by ERK2 in vitro (Fig. 2 C) was identical with the pattern derived from cellular phospho-β-arrestin1. This was further confirmed by the identical map derived from a mixture of equal amounts of cellular phospho-β-arrestin1 and ERK2-phosphorylated β-arrestin1 (data not shown). These two phosphopeptides, a1 and a2, were missing in the map of S412D β-arrestin1 phosphorylated by ERK2 (Fig.2 D). Taken together, our results indicate that ERK is capable of phosphorylating Ser-412 of β-arrestin1. Although β-arrestin1 could be phosphorylated by protein kinases A and C, GRK2, and casein kinase I and II in vitro, in no case did the two-dimensional tryptic phosphopeptide maps match those of β-arrestin1 purified from cells (data not shown). Moreover, mutation of Ser-412 to Asp did not reduce in vitro phosphorylation of β-arrestin1 by these kinases, indicating that these kinases are not responsible for Ser-412 phosphorylation. To investigate whether ERK1 and ERK2 mediate β-arrestin1 phosphorylation in cells, we employed a dominant-negative MEK1(K97A) inhibitor (25Seger R. Seger D. Reszka A.A. Munar E.S. Eldar-Finkelman H. Dobrowolska G. Jensen A.M. Campbell J.S. Fischer E.H. Krebs E.G. J. Biol. Chem. 1994; 269: 25699-25709Abstract Full Text PDF PubMed Google Scholar) to determine whether inhibition of ERK activity might affect β-arrestin1 phosphorylation. Overexpression of the MEK1(K97A) mutant in HEK 293 cells significantly reduced ERK phosphorylation (Fig. 3,lower panel). This was associated with ∼70% reduction of β-arrestin1 phosphorylation (Fig. 3, upper panel). Increasing the level of activated ERKs with a constitutively active S218D/S222D mutant of MEK1 (26Huang W. Kessler D.S. Erikson R.L. Mol. Biol. Cell. 1995; 6: 237-245Crossref PubMed Scopus (110) Google Scholar) did not significantly elevate β-arrestin1 phosphorylation (data not shown), consistent with the high stoichiometry of cellular phosphorylation of β-arrestin1 at Ser-412 (0.85 mol Pi/mol protein) (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). These results demonstrate that inhibition of ERK activation blocks β-arrestin1 phosphorylation in HEK 293 cells, thus further implicating ERKs as the kinases responsible for phosphorylating β-arrestin1 in cells. Previously we have shown that dephosphorylation of β-arrestin1 at the plasma membrane is required for clathrin binding and agonist-induced internalization of the β2-adrenergic receptor (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). Thus, it would be expected that increasing the level of dephosphorylated β-arrestin1 in cells by inhibiting ERK activation with the dominant-negative MEK1(K97A) mutant would augment its clathrin binding ability and function in receptor internalization. As shown in Fig.4 A, the dominant-negative MEK1(K97A) mutant significantly enhances the co-immunoprecipitation of wild-type β-arrestin1 with clathrin heavy chain. The S412A mutant of β-arrestin1, which mimics the dephosphorylated form of β-arrestin1, also robustly co-immunoprecipitated clathrin (Fig. 4 A). We did not observe any effect of the constitutively active S218D/S222D mutant of MEK1 on clathrin binding of β-arrestin1 (data not shown), presumably because cellular β-arrestin1 is already so highly phosphorylated that we could not detect its binding with clathrin. We further investigated the effect of dominant-negative K97A and constitutively active S218D/S222D mutants of MEK1 on β-arrestin1-mediated sequestration of the β2-adrenergic receptors. In the presence of the MEK1(K97A) mutant, receptor sequestration was increased in control HEK 293 cells. It was further promoted by overexpressing β-arrestin1 (Fig. 4 B), presumably because the level of active, dephosphorylated β-arrestin1 is highly increased by MEK1(K97A) mutant (as it is by S412A β-arrestin1 (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar)). In contrast, the constitutively active S218D/S222D mutant of MEK1 slightly reduced receptor sequestration in control cells. This reduction was even more dramatic in cells overexpressing β-arrestin1 where receptor sequestration was now predominantly mediated by transfected β-arrestin1 (in contrast to control cells where both endogenous β-arrestin1 and 2 participate). In such cells, levels of the phosphorylated β-arrestin1 are increased to such high levels by the constitutively active MEK1 mutant that phospho-β-arrestin1 now acts essentially as a dominant-negative inhibitor of receptor internalization (as does S412D β-arrestin1 (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar)). To determine whether the effect of MEK mutants was specifically due to altered β-arrestin1 function, we also tested their effects on receptor sequestration mediated by β-arrestin2. Interestingly, this enhancement was not significantly affected by either MEK1 mutant in cells overexpressing β-arrestin2 (Fig. 4 B). This result suggests that ERKs can modulate the function of β-arrestin1 but not β-arrestin2. Although β-arrestin2 is also a phosphoprotein in cells (data not shown), it has no site corresponding to Ser-412 of β-arrestin1. This suggests that ERKs are not the kinases that phosphorylate β-arrestin2 in cells. Fig.5 provides a model for the feedback regulation of β-arrestin1 function by ERK-mediated phosphorylation of Ser-412. Cytosolic β-arrestin1, which is predominately phosphorylated at Ser-412 (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar), is recruited to the plasma membrane upon agonist stimulation. Membrane-bound β-arrestin1 is dephosphorylated by as yet unknown phosphatases. Although dephosphorylation of β-arrestin1 is not required for its receptor binding, it is required for several of its other functions including Src recruitment (15Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F.-T. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar) and clathrin binding (6Lin F.-T. Krueger K.K. Kendall H.E. Daaka Y. Fredericks Z.L. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 31051-31057Crossref PubMed Scopus (207) Google Scholar). These events in turn are necessary for GPCR-mediated activation of the Ras-dependent ERK pathway (13van Biesen T. Luttrell L.M. Hawes B.E. Lefkowitz R.J. Endocr. Rev. 1996; 17: 698-714Crossref PubMed Scopus (390) Google Scholar). Once activated, the ERKs are able to phosphorylate β-arrestin1 at Ser-412, thereby reducing these functions and, in a feedback regulatory fashion, reducing further ERK signaling. The model shown in this paper was kindly provided by Dr. Stuart Maudsley. The expression vectors of MEK1(K97A) and MEK1(S218D/S222D) mutants were generous gifts from Dr. Edwin G. Krebs and Dr. Raymond. L. Erikson, respectively. We thank Drs. Yehia Daaka, Julie A. Pitcher, and Randy Hall for helpful discussions. We also thank Donna Addison and Mary Holben for excellent secretarial assistance.
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