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A Central Role for ॆ-Arrestins and Clathrin-coated Vesicle-mediated Endocytosis in ॆ2-Adrenergic Receptor Resensitization

1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês

10.1074/jbc.272.43.27005

ISSN

1083-351X

Autores

Jie Zhang, Larry S. Barak, Katharine E. Winkler, Marc G. Caron, Stephen S. G. Ferguson,

Tópico(s)

Neuropeptides and Animal Physiology

Resumo

G protein-coupled receptor (GPCR) sequestration to endosomes is proposed to be the mechanism by which G protein-coupled receptor kinase (GRK)-phosphorylated receptors are dephosphorylated and resensitized. The identification of ॆ-arrestins as GPCR trafficking molecules suggested that ॆ-arrestins might represent critical determinants for GPCR resensitization. Therefore, we tested whether ॆ2-adrenergic receptor (ॆ2AR) resensitization was dependent upon ॆ-arrestins and an intact clathrin-coated vesicle endocytic pathway. The overexpression of either the ॆ-arrestin 1-V53D dominant negative inhibitor of ॆ2AR sequestration or dynamin I-K44A to block clathrin-coated vesicle-mediated endocytosis impaired both ॆ2AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration-impaired ॆ2AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or ॆ-arrestin 1. Moreover, ॆ2ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, ॆ2AR resensitization was restored in these cells following the overexpression of ॆ-arrestin 2. These findings demonstrate, using both loss and gain of function paradigms, that ॆ2AR dephosphorylation and resensitization are dependent upon an intact sequestration pathway. These studies also indicate that ॆ-arrestins play an integral role in regulating not only the desensitization and intracellular trafficking of GPCRs but their ability to resensitize. ॆ-Arrestin expression levels appear to underlie cell type-specific differences in the regulation of GPCR resensitization. G protein-coupled receptor (GPCR) sequestration to endosomes is proposed to be the mechanism by which G protein-coupled receptor kinase (GRK)-phosphorylated receptors are dephosphorylated and resensitized. The identification of ॆ-arrestins as GPCR trafficking molecules suggested that ॆ-arrestins might represent critical determinants for GPCR resensitization. Therefore, we tested whether ॆ2-adrenergic receptor (ॆ2AR) resensitization was dependent upon ॆ-arrestins and an intact clathrin-coated vesicle endocytic pathway. The overexpression of either the ॆ-arrestin 1-V53D dominant negative inhibitor of ॆ2AR sequestration or dynamin I-K44A to block clathrin-coated vesicle-mediated endocytosis impaired both ॆ2AR dephosphorylation and resensitization. In contrast, resensitization of a sequestration-impaired ॆ2AR mutant (Y326A) was reestablished following the overexpression of either GRK2 or ॆ-arrestin 1. Moreover, ॆ2ARs did not resensitize in COS-7 cells as the consequence of impaired sequestration and dephosphorylation. However, ॆ2AR resensitization was restored in these cells following the overexpression of ॆ-arrestin 2. These findings demonstrate, using both loss and gain of function paradigms, that ॆ2AR dephosphorylation and resensitization are dependent upon an intact sequestration pathway. These studies also indicate that ॆ-arrestins play an integral role in regulating not only the desensitization and intracellular trafficking of GPCRs but their ability to resensitize. ॆ-Arrestin expression levels appear to underlie cell type-specific differences in the regulation of GPCR resensitization. Reestablishment of the responsiveness of G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; ॆ2AR, ॆ2-adrenergic receptor; CCV, clathrin-coated vesicle; GRK, G protein-coupled receptor kinase; HEK 293 cells, human embryonic kidney cells; PKA, cAMP-dependent protein kinase; Iso, isoproterenol. after agonist-mediated desensitization is a multifaceted event involving both cellular and biochemical processes (1Ferguson S.S.G. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (320) Google Scholar, 2Ferguson S.S.G. Zhang J. Barak L.S. Caron M.G. Biochem. Soc. Trans. 1996; 24: 953-959Crossref PubMed Scopus (37) Google Scholar, 3Zhang J. Ferguson S.S.G. Barak L.S. Jaber M. Giros B. Lefkowitz R.J. Caron M.G. Receptors Channels. 1997; (in press)PubMed Google Scholar). Sequestration, the cellular event leading to GPCR endocytosis and recycling, is initiated by the agonist-promoted mobilization of cell surface receptors to an intracellular vesicular compartment, probably endosomes (4Moore R.H. Sadovnikoff N. Hoffenberg S. Liu S. Woodford P. Angelides K. Trial J.A. Carsrud N.D.V. Dickey B.F. Knoll B.J. J. Cell Sci. 1995; 108: 2983-2991Crossref PubMed Google Scholar, 5von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 6Barak L.S. Ferguson S.S.G. Zhang J. Martenson C. Meyer T. Caron M.G. Mol. Pharmacol. 1997; 51: 177-184Crossref PubMed Scopus (201) Google Scholar, 7Garland A.M. Grady E.F. Lovett M. Vigna S.R. Frucht M.M. Krause J.E. Bunnett N.W. Mol. Pharmacol. 1996; 49: 438-446PubMed Google Scholar). Receptors internalized via clathrin-coated vesicles (CCVs) and/or caveolae are thought to be resensitized in endosomes following their dephosphorylation by a membrane-associated phosphatase that exhibits specificity for G protein-coupled receptor kinase (GRK)-phosphorylated GPCRs residing in an acidified endosomal environment (8Sibley D.R. Strasser R.H. Benovic J.L. Daniel K. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9408-9412Crossref PubMed Scopus (176) Google Scholar, 9Krueger K.M. Daaka Y. Pitcher J.A. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 5-8Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 10Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). Subsequently, these dephosphorylated receptors must be mobilized back to the cell surface to reestablish normal receptor signaling (7Garland A.M. Grady E.F. Lovett M. Vigna S.R. Frucht M.M. Krause J.E. Bunnett N.W. Mol. Pharmacol. 1996; 49: 438-446PubMed Google Scholar, 10Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). However, the molecular mechanisms contributing to each of these events and their relative contributions to the resensitization of GPCR responsiveness are only now becoming recognized. A role for receptor phosphorylation in the sequestration process was suggested by the observation that overexpression of G protein-coupled receptor kinase 2 (GRK2) in cells expressing the m2 muscarinic acetylcholine receptor could augment agonist-mediated internalization (11Tsuga H. Kameyama K. Haga T. Kurose H. Nagao T. J. Biol. Chem. 1994; 269: 32522-32527Abstract Full Text PDF PubMed Google Scholar). Additionally using a sequestration-defective ॆ2-adrenergic receptor (ॆ2AR) mutant (Y326A), GRK-mediated phosphorylation, and ॆ-arrestin binding, the same molecular intermediates required for receptor desensitization were demonstrated to initiate ॆ2AR endocytosis (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Ferguson 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 (846) Google Scholar). These studies also demonstrated that GRK phosphorylation was not absolutely required for ॆ2AR sequestration (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Ferguson 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 (846) Google Scholar) but served to increase the affinity of the receptor for ॆ-arrestins, which functioned as ॆ2AR trafficking molecules (13Ferguson 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 (846) Google Scholar). A role of ॆ-arrestin in wild-type ॆ2AR sequestration was established using ॆ-arrestin mutants, which specifically impaired wild-type receptor sequestration without altering the ability of the receptor to become phosphorylated or desensitized (13Ferguson 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 (846) Google Scholar). Recently, we showed that GPCRs can utilize several distinct endocytic pathways (14Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). However, in the case of the ॆ2AR, a growing body of evidence supports the idea that the endocytic route employed by this receptor is mainly the same CCV-mediated pathway utilized by constitutively recycling transferrin and low density lipoprotein receptors (4Moore R.H. Sadovnikoff N. Hoffenberg S. Liu S. Woodford P. Angelides K. Trial J.A. Carsrud N.D.V. Dickey B.F. Knoll B.J. J. Cell Sci. 1995; 108: 2983-2991Crossref PubMed Google Scholar, 5von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 14Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 15Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1338) Google Scholar, 16Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar). An important step in CCV endocytosis is the GTP hydrolysis-dependent pinching off of the vesicles by a large GTPase, dynamin (15Mellman I. Annu. Rev. Cell Dev. Biol. 1996; 12: 575-625Crossref PubMed Scopus (1338) Google Scholar, 17Takel K. McPherson P.S. Schmid S.L. De Camilli P. Nature. 1995; 374: 186-190Crossref PubMed Scopus (656) Google Scholar, 18Hinshaw J.E. Schmid S.L. Nature. 1995; 374: 190-192Crossref PubMed Scopus (659) Google Scholar, 19Shpetner H.S. Herskovits J.S. Vallee R.B. J. Biol. Chem. 1996; 271: 13-16Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 20Wang L.-H. Südhof T.C. Anderson R.G.W. J. Biol. Chem. 1995; 270: 10079-10083Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Dynamin mutants defective in GTP binding, such as dynamin I-K44A, specifically block clathrin-mediated endocytosis (21Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1040) Google Scholar, 22Herskovits J.S. Burgess C.C. Obar R.A. Vallee R.B. J. Cell Biol. 1993; 122: 565-578Crossref PubMed Scopus (398) Google Scholar, 23Vieira A.V. Lamaze C. Schmid S.L. Science. 1996; 274: 2086-2089Crossref PubMed Scopus (828) Google Scholar). We have used this dynamin mutant to demonstrate that agonist-promoted internalization of the ॆ2AR occurs via dynamin-dependent CCV-mediated endocytosis (14Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Moreover, we demonstrated that ॆ-arrestins serve as adaptor proteins specifically targeting receptors to CCVs (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Ferguson 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 (846) Google Scholar, 14Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Biochemical studies have now shown a direct interaction between ॆ-arrestins and clathrin triskelions (24Goodman 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, 25Krupnick 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, 26Goodman 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). Several recent reports suggest that GRK-mediated phosphorylation followed by ॆ-arrestin binding may represent a common mechanism required for the sequestration of many other GPCRs (11Tsuga H. Kameyama K. Haga T. Kurose H. Nagao T. J. Biol. Chem. 1994; 269: 32522-32527Abstract Full Text PDF PubMed Google Scholar, 12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Ferguson 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 (846) Google Scholar, 14Zhang J. Ferguson S.S.G. Barak L.S. Menard L. Caron M.G. J. Biol. Chem. 1996; 271: 18302-18305Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar,27Pals-Rylaarsdam R. Hosey M.M. J. Biol. Chem. 1997; 272: 14152-14158Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 28Itokawa M. Toru M. Ito K. Tsuga H. Kameyama K. Haga T. Arinami T. Hamaguchi H. Mol. Pharmacol. 1996; 49: 560-566PubMed Google Scholar, 29.Schlador, M. L., Nathanson, N. M., Abstracts Society for Neuroscience, 26th Annual Meeting, November 16–21, 1996, Washington, D. C., 1996, 1257, Society for Neuroscience, Washington, D. C.Google Scholar, 30Aramori I. Ferguson S.S.G. Bieniasz P.D. Zhang J. Cullen B.R. Caron M.G. EMBO J. 1997; 15: 4606-4616Crossref Scopus (225) Google Scholar). The identification of ॆ-arrestins as GPCR-specific endocytic trafficking proteins provides the first opportunity to directly assess, using GPCR-interacting proteins, the hypothesis that sequestration and more importantly normal ॆ-arrestin function (as a GPCR-specific adaptor protein) is absolutely required for normal ॆ2AR dephosphorylation and resensitization. In addition, we examine, using dynamin I-K44A as an inhibitor of CCV-mediated endocytosis, whether ॆ2AR resensitization is dependent upon an intact CCV endocytic pathway. Moreover, we establish that ॆ2AR resensitization in different cell types is regulated by differences in the endogenous expression levels of ॆ-arrestin proteins. Human embryonic kidney cells (HEK 293) and COS-7 cells were provided by the American Type Culture Collection. Tissue culture media and fetal bovine serum were obtained from Life Technologies, Inc. Isoproterenol (Iso) was purchased from Research Biochemicals Inc. Anti-hemagglutinin 12CA5 monoclonal antibody was from Boehringer Mannheim, anti-FLAG M2 antibody was from Eastman Kodak Co., and fluorescein-secondary antibody was from Sigma. Dithiobis(succinimidylproprionate) (Lomant's reagent, DSP) was obtained from Pierce. [125I]Pindolol, [3H]adenine, [32P]ATP, [3H]ATP, [14C]cAMP, and [32P]orthophosphate were purchased from NEN Life Science Products. HEK 293 and COS-7 cells were grown in Eagle's minimal essential medium with Earle's salt and Dulbecco's modified Eagle's medium, respectively, supplemented with heat-inactivated fetal bovine serum (107, v/v) and gentamicin (100 ॖg/ml). The cells were transiently transfected using a modified calcium phosphate method (31Cullen B.R. Methods Enzymol. 1987; 152: 684-704Crossref PubMed Scopus (662) Google Scholar) as described previously (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Ferguson 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 (846) Google Scholar). Receptor expression was measured using saturating concentrations of [125I]pindolol (∼1 nm) at 30 °C for 30 min (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Bound ligand was separated on glass fiber filters (Whatman, GF/C) by vacuum filtration and counted in a γ-counter. Protein concentrations were determined using a Bio-Rad assay kit with bovine serum albumin as the standard. Receptor sequestration was assessed by flow cytometry as described previously (32Barak L.S. Tiberi 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). In brief, sequestration was defined as the fraction of total cell surface receptors that, after exposure to agonist, are removed from the plasma membrane and thus are not accessible to antibodies from outside the cell. The cells were exposed to 10 ॖm isoproterenol before antibody staining. HEK 293 cells were transfected with FLAG epitope-tagged ॆ2AR with or without either ॆ-arrestin 1 or ॆ-arrestin 1-V53D. Cells were incubated for 10 min with 10 ॖm Iso at room temperature and then incubated an additional 30 min in the presence of a thio-cleavable chemical cross-linker (Lomant's reagent) at a final concentration of 2.5 mm in 107 Me2SO. Cells were solubilized in radioimmune precipitation buffer with protease inhibitors (150 mm NaCl, 50 mm Tris-HCl, 5 mm EDTA, 10 mm NaF, 10 mm Na2-pyrophosphate, 17 Nonidet P-40, 0.57 deoxycholate, 0.17 SDS, 0.1 mmphenylmethylsulfonyl fluoride, 10 ॖg/ml leupeptin, 5 ॖg/ml aprotinin, 1 ॖg/ml pepstatin A, pH 7.4) for 1 h and FLAG epitope-tagged ॆ2ARs immunoprecipitated with monoclonal anti-FLAG M2 antibody as described previously (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Samples were subjected to SDS-polyacrylamide gel electrophoresis followed by electroblotting with a Millipore Milliblot semidry electroblotting system onto nitrocellulose membranes. The membranes were blocked in phosphate-buffered saline with 37 bovine serum albumin, probed with an anti-ॆ-arrestin 1/2 rabbit polyclonal antibody (33Attramadal 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), and exposed using the ECL Western blotting analysis system (Amersham Corp.), as described previously (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Ferguson 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 (846) Google Scholar). Receptor phosphorylation was performed as described previously (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 13Ferguson 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 (846) Google Scholar). In brief, the intracellular ATP pool was 32P-labeled by incubating transfected cells seeded in six-well dishes with [32P]orthophosphate (100 ॖCi/ml) in phosphate- and serum-free media at 37 °C for 45 min. Cells were then stimulated with or without 10 ॖm Iso in 100 ॖm ascorbate for 10 min at 37 °C and then washed three times on ice with phosphate-buffered saline. Resensitized cells were allowed to recover at 37 °C for 20 min in phosphate-buffered saline. The cells were solubilized in radioimmune precipitation buffer with protease inhibitors and 12CA5 epitope-tagged ॆ2AR immunoprecipitated with 12CA5 antibody as described previously (12Ferguson S.S.G. Menard L. Barak L.S. Colapietro A.-M. Koch W.J. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In each experiment, equivalent amounts of receptor protein, as determined by receptor expression and the amount of solubilized protein in each lysate, were then subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. The extent of receptor phosphorylation was quantitated using a Molecular Dynamics PhosphorImager system and ImageQuant software. Membranes from cells, treated as described in the legends to Figs. 2, 5, 7, and 9, were prepared by disruption with a Polytron homogenizer for 20 s at 20,000 rpm followed by centrifugation at 40,000 × g in lysis buffer (10 mm Tris-HCl, 5 mm EDTA, pH 7.4). The cell membrane pellet was resuspended in lysis buffer by Polytron, recentrifuged, and resuspended in cold assay buffer (75 mm Tris-HCl, 2 mm EDTA, 15 mmMgCl2, pH 7.4) to a final concentration of 1–2 ॖg/ॖl membrane protein. 20-ॖl aliquots of membrane preparations were assayed for agonist-stimulated adenylyl cyclase activity in a final volume of 50 ॖl as described previously (34Walseth T.F. Johnson R.A. Biochim. Biophys. Acta. 1979; 562: 11-31Crossref PubMed Scopus (426) Google Scholar, 35Hausdorff W.P. Hnatowich M. O'Dowd B.F. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1990; 265: 1388-1393Abstract Full Text PDF PubMed Google Scholar).Figure 5The effect of dynamin I-K44A on the desensitization and resensitization of ॆ2AR-stimulated adenylyl cyclase activity in HEK 293 cells. HEK 293 cells were transiently transfected to overexpress ॆ2ARs in the absence (803 ± 109 fmol/mg protein) (A) or presence (1206 ± 156 fmol/mg protein) (B) of 3 ॖg of cotransfected dynamin I-K44A in pCB1. Cells were preincubated for 10 min in serum-free media at 37 °C in the absence (naive (•)) or presence (desensitized (○); resensitized (▾)) of a desensitizing stimulus, 2 ॖm isoproterenol, washed three times, and either allowed to resensitize for 20 min at 37 °C (▾) or kept on ice (○, •). Cell membranes were prepared, and adenylyl cyclase activity was determined under basal conditions as well as in the presence of increasing concentrations of isoproterenol (10−10 to 10−4m) or 10 ॖm forskolin as described under 舠Experimental Procedures.舡 Adenylyl cyclase activity was normalized according to forskolin-stimulated cyclase response and was expressed as the percentage of the maximal response of naive cells (•). The EC50 values for ॆ2AR-mediated adenylyl cyclase activation were 54 ± 10 and 39 ± 7 nm, respectively, in the absence and presence of overexpressed dynamin I-K44A. The data represent the mean ± S.E. of four different experiments done in triplicate. C, cells transfected with ॆ2ARs in the absence (•) or presence (○) of dynamin I-K44A were desensitized with 2 ॖm isoproterenol for different periods of time (0–45 min) at 37 °C in serum-free media and washed. Cell membranes were prepared, and then adenylyl cyclase activity was determined in the presence of 10 ॖmisoproterenol or 10 ॖm forskolin. Adenylyl cyclase activity was normalized according to forskolin-stimulated cyclase response, and desensitization was expressed as the percentage of adenylyl cyclase activity in cells that had not been preincubated with agonist (i.e. t = 0). The data represent the mean ± S.E. of three different experiments done in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Effect of GRK2, ॆ-arrestin 1, and ॆ-arrestin 1-V53D on the desensitization and resensitization of ॆ2AR-Y326A mutant-stimulated adenylyl cyclase activity. HEK 293 cells were transiently transfected to overexpress the ॆ2AR-Y326A mutant in the absence (2220 ± 870 fmol/mg protein) (A) or the presence of cotransfected GRK2 (2450 ± 390 fmol/mg protein) (B), ॆ-arrestin 1 cDNA (2040 ± 100 fmol/mg protein) (C), ॆ-arrestin 1-V53D cDNA (1910 ± 120 fmol/mg protein) (D), GRK2 and ॆ-arrestin 1 cDNA (1470 ± 220 fmol/mg protein) (E) or GRK2 and ॆ-arrestin 1-V53D (1490 ± 340 fmol/mg protein) (F). Cells were treated as described in the legend to Fig. 2, and adenylyl cyclase activity in response to Iso (10−9.7 to 10−4.5m) was determined. •, naive cells; ○, desensitized cells; ▴, resensitized cells. Adenylyl cyclase activity is expressed as the percentage of the maximal response of control cells (•) to 30 ॖm Iso. The V max and EC50 values for ॆ2AR-Y326A mutant-mediated adenylyl cyclase activation were 62 ± 6 pmol/min/mg protein and 72 ± 9 nm, respectively, and were not affected by the co-expression of GRK and ॆ-arrestin regulatory proteins. Forskolin-stimulated adenylyl cyclase activity was 533 ± 40 pmol/min/mg protein. The data represent the mean ± S.E. of three different experiments done in duplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Desensitization and resensitization of ॆ2AR-stimulated adenylyl cyclase activity in COS-7 cells in the absence and presence of overexpressing ॆ-arrestin 2.COS-7 cells were transiently transfected to overexpress ॆ2ARs in the absence (1444 ± 277 fmol/mg protein) (A) and presence (2182 ± 350 fmol/mg protein) (B) of cotransfected ॆ-arrestin 2. Cells were treated as described in the legend of Fig. 5, and adenylyl cyclase activity in response to isoproterenol (10−10 to 10−4) was determined. •, naive cells; ○, desensitized cells; ▾, resensitized cells. Adenylyl cyclase activity was expressed as the percentage of the maximal response of naive cells (•). The EC50 values for ॆ2AR-mediated adenylyl cyclase activation were 62 ± 10 and 38 ± 5 nm, respectively, in the absence and presence of overexpressed ॆ-arrestin 2. The data represent the mean ± S.E. of three different experiments done in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cells were grown in 12-well Falcon dishes at a density of 400,000 cells/well and were labeled overnight with 1 ॖCi/ml/well of [3H]adenine in culture medium. The cells were then washed with fresh medium without serum and treated with varying concentrations of isoproterenol in serum-free medium containing 10 mm HEPES, 1 mmisobutylmethylxanthine, and 100 ॖm ascorbate at pH 7.4 for 10 min at 37 °C. The medium was aspirated, and 1 ml of ice-cold stop solution (2.57 (v/v) perchloric acid, 0.1 mm cAMP, and 2 ॖCi of [14C]cAMP per 500 ml) was added to each well followed by incubation on ice for 20–30 min. The cell lysate was added to tubes containing 100 ॖl of 4.2 m KOH, and the cAMP accumulated in the cells was quantitated chromatographically by the method of Salomon (36Salomon Y. Methods Enzymol. 1991; 195: 22-28Crossref PubMed Scopus (130) Google Scholar). Statistical significance was determined by analysis of variance when appropriate and multiple comparisons between groups were made using a two-tailed t test for independent samples. Dose response data were analyzed using GraphPad Prism. We have reported that overexpression of ॆ-arrestin 1-V53D, a dominant negative inhibitor of ॆ2AR sequestration, increased the extent of agonist-induced ॆ2AR phosphorylation, suggesting that ॆ-arrestin might play a role in ॆ2AR dephosphorylation and resensitization (13Ferguson 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 (846) Google Scholar). To begin to test this question, the effect of overexpressing either wild-type ॆ-arrestin or ॆ-arrestin 1-V53D on ॆ2AR dephosphorylation was examined in HEK 293 cells. In the absence of overexpressed ॆ-arrestins, agonist stimulation with 10 ॖm isoproterenol for 10 min increased the extent of ॆ2AR phosphorylation 3.1 ± 0.3-fold over basal level (Fig. 1 A). When cells were allowed to recover for 20 min following the removal of agonist, ॆ2AR dephosphorylation (61 ± 67) was observed (Fig. 1, A and B). Overexpression of ॆ-arrestin 1 led to a reduction in agonist-induced ॆ2AR phosphorylation (75 ± 47 of control ॆ2AR phosphorylation) without significantly affecting the ability of the receptor to be dephosphorylated (54 ± 37 of matched control) (Fig. 1, A and B). In contrast, ॆ-arrestin 1-V53D not only increased the extent of agonist-induced ॆ2AR phosphorylation (137 ± 147 of control ॆ2AR phosphorylation) but significantly impaired the ability of the receptor to be dephosphorylated (28 ± 127 of matched control) (Fig. 1, A and B). While complete inhibition of ॆ2AR dephosphorylation was not observed in the presence of ॆ-arrestin 1-V53D, the absolute value of ॆ2AR phosphorylation remained 95 ± 97 of control ॆ2AR phosphorylation. Taken together, these data indicate that normal ॆ-arrestin function contributes to the regulation of ॆ2AR dephosphorylation. To determine whether normal ॆ-arrestin function and receptor dephosphorylation were absolutely required for ॆ2AR resensitization, the ability of the ॆ2AR to both desensitize and resensitize in the presence of either wild-type or V53D mutant ॆ-arrestin was tested in HEK 293 cells. When expressed alone, the ॆ2AR desensitized in response to a desensitizing stimulus (10 ॖm isoproterenol for 15 min), which was measured as a 2-fold rightward shift in the half-maximal effective concentration (EC50) and a 43 ± 37 reduction in the maximal velocity (V max) for agonist-stimulated ॆ2AR-responsive adenylyl cyclase activity (Fig. 2 A). When cells were allowed to recover for 30 min in agonist-free media, following the desensitizing stimulus, complete resensitization of ॆ2AR-responsive adenylyl cyclase activity was observed (Fig. 2 A). While the overexpression of either wild-type or V53

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