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• W2023629636 abstract "Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of β-arrestin in the resensitization of the β2 adrenergic receptor (β2AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit β-arrestin to the plasma membrane and internalize in a β-arrestin- and clathrin-dependent manner. However, whereas β-arrestin dissociates from the β2AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of β-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of β-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of β-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which β-arrestins regulate the reestablishment of GPCR responsiveness. Resensitization of G protein-coupled receptors (GPCRs) following agonist-mediated desensitization is a necessary step for maintaining physiological responsiveness. However, the molecular mechanisms governing the nature of GPCR resensitization are poorly understood. Here, we examine the role of β-arrestin in the resensitization of the β2 adrenergic receptor (β2AR), known to recycle and resensitize rapidly, and the vasopressin V2 receptor (V2R), known to recycle and resensitize slowly. Upon agonist activation, both receptors recruit β-arrestin to the plasma membrane and internalize in a β-arrestin- and clathrin-dependent manner. However, whereas β-arrestin dissociates from the β2AR at the plasma membrane, it internalizes with the V2R into endosomes. The differential trafficking of β-arrestin and the ability of these two receptors to dephosphorylate, recycle, and resensitize is completely reversed when the carboxyl-terminal tails of these two receptors are switched. Moreover, the ability of β-arrestin to remain associated with desensitized GPCRs during clathrin-mediated endocytosis is mediated by a specific cluster of phosphorylated serine residues in the receptor carboxyl-terminal tail. These results demonstrate that the interaction of β-arrestin with a specific motif in the GPCR carboxyl-terminal tail dictates the rate of receptor dephosphorylation, recycling, and resensitization, and thus provide direct evidence for a novel mechanism by which β-arrestins regulate the reestablishment of GPCR responsiveness. G protein-coupled receptor (GPCR) 1The abbreviations used are:GPCRG protein-coupled receptorGRKG protein-coupled receptor kinaseβ2ARβ2-adrenergic receptorV2Rvasopressin V2 receptorAVParginine vasopressinβarr2-GFPβ-arrestin2-green fluorescent protein conjugateβarr1-GFPβ-arrestin1-green fluorescent protein conjugateD1ARdopamine D1A receptorETARendothelin type A receptorNTR1neurotensin receptor 1AT1ARangiotensin II type 1A receptorHAhemagglutininBSAbovine serum albuminPBSphosphate-buffered saline signaling plays a pivotal role in regulating various physiological functions including vision, olfaction, neurotransmission, cardiac output, and fluid and electrolyte balance. The magnitudes of these physiological responses are linked intimately to the delicate balance between GPCR signal generation and signal termination. The termination of GPCR signaling is tightly regulated by a family of intracellular proteins termed β-arrestins (1Lohse M.J. Benovic J.L. Codina J. Caron M.G. Lefkowitz R.J. Science. 1990; 248: 1547-1550Crossref PubMed Scopus (909) Google Scholar, 2Pippig S. Andexinger S. Daniel K. Puzicha M. Caron M.G. Lefkowitz R.J. Lohse M.J. J. Biol. Chem. 1993; 268: 3201-3208Abstract Full Text PDF PubMed 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, 4Ferguson S.S. Downey 3rd, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (846) Google Scholar, 5Zhang 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). β-Arrestins bind with high affinity to agonist-activated GPCRs that have been phosphorylated by G protein-coupled receptor kinases (GRKs). The interaction of β-arrestins with a phosphorylated receptor uncouples the receptor from heterotrimeric G proteins, producing a nonsignaling, desensitized receptor. For many GPCRs, like the β2-adrenergic receptor (β2AR), β-arrestins target the desensitized receptor to clathrin-coated pits for endocytosis (4Ferguson S.S. Downey 3rd, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (846) Google Scholar, 6Aramori I. Ferguson S.S. Bieniasz P.D. Zhang J. Cullen B. Cullen M.G. EMBO J. 1997; 16: 4606-4616Crossref PubMed Scopus (225) Google Scholar, 7Zhang J. Ferguson S.S. Barak L.S. Bodduluri S.R. Laporte S.A. Law P.Y. Caron M.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7157-7162Crossref PubMed Scopus (465) Google Scholar, 8McConalogue K. Corvera C.U. Gamp P.D. Grady E.F. Bunnett N.W. Mol. Biol. Cell. 1998; 9: 2305-2324Crossref PubMed Scopus (76) Google Scholar, 9Nakamura K. Krupnick J.G. Benovic J.L. Ascoli M. J. Biol. Chem. 1998; 273: 24346-24354Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 10Lazari M.d. F.M. Bertrand J.E. Nakamura K. Liu X. Krupnick J.G. Benovic J.L. Ascoli M. J. Biol. Chem. 1998; 273: 18316-18324Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In this process, β-arrestins function as docking proteins that link receptors to components of the endocytic machinery such as AP-2 and clathrin (11Laporte S.A. Oakley R.H. Zhang J. Holt J.A. Ferguson S.S. Caron M.G. Barak L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3712-3717Crossref PubMed Scopus (524) Google Scholar,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). Intracellular trafficking of GPCRs following β-arrestin-mediated desensitization is necessary for receptor resensitization (5Zhang 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, 13Yu S.S. Lefkowitz R.J. Hausdorff W.P. J. Biol. Chem. 1993; 268: 337-341Abstract Full Text PDF PubMed Google Scholar, 14Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar). Therefore, β-arrestins are involved not only in terminating receptor G protein coupling but also in initiating processes that regulate re-establishment of receptor responsiveness. G protein-coupled receptor G protein-coupled receptor kinase β2-adrenergic receptor vasopressin V2 receptor arginine vasopressin β-arrestin2-green fluorescent protein conjugate β-arrestin1-green fluorescent protein conjugate dopamine D1A receptor endothelin type A receptor neurotensin receptor 1 angiotensin II type 1A receptor hemagglutinin bovine serum albumin phosphate-buffered saline While it is evident that responsiveness to most GPCR-activating ligands can be regained, the biochemical and kinetic properties of the cellular processes mediating resensitization may differ considerably among receptors. Some internalized GPCRs recycle rapidly back to the plasma membrane fully resensitized, while others are retained inside the cell and recycle slowly or not at all (5Zhang 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, 14Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 15von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 16Fonseca M.I. Button D.C. Brown R.D. J. Biol. Chem. 1995; 270: 8902-8909Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Garland 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, 18Tarasova N.I. Stauber R.H. Choi J.K. Hudson E.A. Czerwinski G. Miller J.L. Pavlakis G.N. Michejda C.J. Wank S.A. J. Biol. Chem. 1997; 272: 14817-14824Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 19Innamorati G. Sadeghi H. Birnbaumer M. J. Biol. Chem. 1998; 273: 7155-7161Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 20Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar, 21Vogler O. Bogatkewitsch G.S. Wriske C. Krummenerl P. Jakobs K.H. van Koppen C.J. J. Biol. Chem. 1998; 273: 12155-12160Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 22Ishii I. Saito E. Izumi T. Ui M. Shimizu T. J. Biol. Chem. 1998; 273: 9878-9885Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The molecular mechanisms governing the rate at which receptors recycle and re-establish agonist responsiveness are poorly understood; however, the dephosphorylation of GRK-phosphorylated receptors in early endosomes appears to be a critical step in the resensitization pathway (14Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 17Garland 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, 23Sibley 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, 24Krueger 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, 25Shih M. Lin F. Scott J.D. Wang H. Malbon C.C. J. Biol. Chem. 1999; 274: 1588-1595Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). An event presumably necessary for the dephosphorylation of GPCRs is their dissociation from β-arrestin, as suggested by in vitroevidence that the binding of visual arrestin to GRK-phosphorylated rhodopsin prevents rhodopsin dephosphorylation (26Palczewski K. McDowell J.H. Jakes S. Ingebritsen T.S. Hargrave P.A. J. Biol. Chem. 1989; 264: 15770-15773Abstract Full Text PDF PubMed Google Scholar). A common assumption is that β-arrestins do not dissociate from desensitized receptors at the plasma membrane but traffic with them into early endosomes (27Ferguson S.S. Barak L.S. Zhang J. Caron M.G. Can. J. Physiol. Pharmacol. 1996; 74: 1095-1110Crossref PubMed Scopus (320) Google Scholar, 28Krupnick J.G. Benovic J.L. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 289-319Crossref PubMed Scopus (857) Google Scholar). However, recent observations have demonstrated that the fate of the GPCR-β-arrestin complex can differ among receptors (29Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). For some receptors, the receptor-β-arrestin complex dissociates at or near the plasma membrane shortly after the formation of clathrin-coated pits, and β-arrestin is excluded from receptor-containing endocytic vesicles. For other receptors, the receptor-β-arrestin complex remains intact and is internalized into endosomes. The ability of β-arrestin to remain associated with some receptors but not others suggests that β-arrestin may regulate the cellular trafficking and dephosphorylation of receptors and ultimately their kinetics of resensitization. In order to investigate the role of β-arrestin in the regulation of GPCR resensitization, two GPCRs, the β2AR and the vasopressin V2 receptor (V2R), which share many of the same signaling properties but differ markedly in their ability to recycle and resensitize (5Zhang 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, 14Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 20Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar), were chosen. We demonstrate using mutagenesis and chimeric receptors that the ability of β-arrestin to remain associated with desensitized GPCRs and internalize with them into endosomes dictates the properties of GPCR resensitization. These observations, therefore, provide direct evidence for an important mechanism by which β-arrestins can regulate the physiological responsiveness of GPCRs. Isoproterenol was purchased from Research Biochemicals Inc., and arginine vasopressin (AVP) was obtained from Sigma. The anti-HA 12CA5 mouse monoclonal antibody and the rhodamine-conjugated anti-HA 12CA5 mouse monoclonal antibody were purchased from Roche Molecular Biochemicals. [125I]Cyanopindolol, [3H]AVP, [3H]adenine, [14C]cAMP, [32P]ATP, [3H]ATP, [3H]cAMP, and [32P]orthophosphate were purchased from NEN Life Science Products. Construction of plasmids containing the hemagglutinin epitope (HA)-tagged β2AR, βarr2-GFP, βarr1-GFP, β-arrestin1, β-arrestin2, β-arrestin1 dominant negative mutant V53D, and dynaminI dominant negative mutant K44A have been described previously (4Ferguson S.S. Downey 3rd, W.E. Colapietro A.M. Barak L.S. Menard L. Caron M.G. Science. 1996; 271: 363-366Crossref PubMed Scopus (846) Google Scholar, 29Zhang J. Barak L.S. Anborgh P.H. Laporte S.A. Caron M.G. Ferguson S.S. J. Biol. Chem. 1999; 274: 10999-11006Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 30Barak 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, 31Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). The HA-tagged V2R cDNA was kindly provided by Dr. Jurgen Wess (National Institutes of Health, Bethesda, MD). All other constructs were generated by polymerase chain reaction following standard protocols and contain the HA epitope. The β2AR-V2R chimera contains the first 341 amino acids of the β2AR (Met-1 to Cys-341) fused to the last 29 amino acids of the V2R (Ala-343 to Ser-371). The V2R-β2AR chimera contains the first 342 amino acids of the V2R (Met-1 to Cys-342) fused to the last 72 amino acids of the β2AR (Leu-342 to Leu-413). The V2R-S362X truncation mutant was generated by replacing nucleotides CCG encoding Ser-362 of the V2R with nucleotides TAA encoding a stop codon. The V2R-SSSTSS/AAAAAA mutant was generated by replacing Ser-362, Ser-363, Ser-364, Thr-369, Ser-370, and Ser-371 of the V2R with alanine residues. The V2R-TSS/AAA mutant was generated by replacing Thr-369, Ser-370, and Ser-371 of the V2R with alanine residues. The V2R-SSS/AAA and β2AR-V2R-SSS/AAA mutants were generated by replacing Ser-362, Ser-363, and Ser-364 of the V2R with alanine residues. The β2AR413-V2R10 chimera contains the full-length β2AR (Met-1 to Leu-413) fused to the last 10 amino acids of the V2R (Ser-362 to Ser-371). The β2AR360-V2R10 chimera contains the first 360 amino acids of the β2AR (Met-1 to Thr-360) fused to the last 10 amino acids of the V2R (Ser-362 to Ser-371). Sequences of the DNA constructs were confirmed by DNA sequencing. HEK-293 and COS-7 cells, grown as described previously (32Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), were seeded at a density of 2 × 106 cells/100-mm dish and 5 × 105cells/100-mm dish, respectively. Transient transfections were performed using a modified calcium phosphate coprecipitation method as described previously (32Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Membrane binding assays were performed on transfected HEK-293 cells as described previously (33Hausdorff 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). Briefly, membrane proteins (2 μg) from cells expressing the β2AR and β2AR-V2R chimera were incubated in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) at room temperature in the presence of 15 pm[125I]cyanopindolol and increasing concentrations of isoproterenol (10 pm to 30 μm). Membrane proteins (10 μg) from cells expressing the V2R and V2R-β2AR chimera were incubated in PBS containing 2% BSA at room temperature with increasing concentrations of [3H]AVP (0.5 nm to 16.0 nm). Binding was terminated by rapid filtration and consecutive washes with ice-cold wash buffer (120 mm NaCl, 50 mmTris-HCl, pH = 7.2). Wild-type and chimeric receptor expression levels were measured on whole cells as described previously (32Ferguson S.S.G. Menard L. Barak L.S. Koch W.J. Colapietro A.M. Caron M.G. J. Biol. Chem. 1995; 270: 24782-24789Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Transfected HEK-293 cells expressing the V2R and V2R-β2AR chimera were incubated 2 h on ice in PBS containing 2% BSA with a saturating concentration of [3H]AVP, and bound radioactivity was extracted with 0.1 m NaOH. Nonspecific binding was determined under each respective condition in the presence of 10 μm propranolol or 10 μm unlabeled AVP. Receptor expression levels varied between 2000 and 4000 fmol/mg of whole cell protein for experiments with βarr2-GFP and between 500 and 1500 fmol/mg of whole cell protein for all other experiments. Receptor sequestration was assessed by flow cytometry as described previously (30Barak 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). Sequestration was defined as the fraction of total cell surface receptors that, after exposure to agonist, were removed from the plasma membrane and thus not accessible to antibodies from outside the cell. For recycling experiments, isoproterenol was removed by three rapid washes with serum-free medium at 37 °C and AVP was removed by successive washes with ice-cold PBS, acid (150 mm NaCl/5 mm acetic acid), PBS, and serum-free medium. βarr2-GFP trafficking was visualized in transfected HEK-293 cells on a heated (37 °C) microscope stage as described previously (31Barak L.S. Ferguson S.S. Zhang J. Caron M.G. J. Biol. Chem. 1997; 272: 27497-27500Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Images were collected sequentially using single line excitation (488 nm) with a Zeiss laser scanning confocal microscope (LSM-510). For experiments assessing βarr2-GFP trafficking after agonist removal, cells were washed as described above to remove agonist and returned to a 37 °C incubator for 60 min. Colocalization of βarr2-GFP with rhodamine-labeled receptors was performed on transfected cells pre-incubated in serum-free medium containing a rhodamine-conjugated anti-HA 12CA5 mouse monoclonal antibody (1:100) for 45 min at 37 °C. Cells were then washed three times with serum-free medium, treated with the appropriate agonist at 37 °C for 30 min, and imaged by confocal microscopy. βarr2-GFP and rhodamine-labeled receptor fluorescence were performed using dual excitation (488, 568 nm) and emission (515–540 nm, GFP; 590–610 nm, rhodamine) filter sets. Whole cell cyclase assays were performed on transfected HEK-293 cells using varying concentrations of isoproterenol (1 × 10−12m to 1 × 10−5m) or AVP (1 × 10−12m to 1 × 10−5m) as described previously (5Zhang 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). For membrane adenylyl cyclase assays, transfected HEK-293 cells were harvested by scraping in ice-cold lysis buffer (10 mm Tris-HCl, 5 mmEDTA, pH = 7.4) and membranes were prepared by disruption with a Polytron homogenizer for 20 s at 20,000 rpm followed by centrifugation at 40,000 × g. The cell membrane was resuspended in lysis buffer by Polytron homogenization for 15 s at 20,000 rpm, centrifuged, and resuspended in ice-cold assay buffer (75 mm Tris-HCl, 2 mm EDTA, 15 mmMgCl2, pH = 7.4) to a final concentration of 1–2 μg/μl membrane protein. Equivalent amounts of membrane protein in 20-μl aliquots were assayed for agonist-stimulated adenylyl cyclase activity in a final volume of 50 μl as described previously (5Zhang 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). Receptor phosphorylation was performed as described previously (5Zhang 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). In brief, transfected HEK-293 cells were labeled for 1 h at 37 °C with [32P]orthophosphate (100 μCi/ml) in phosphate-free medium. Cells were stimulated with agonist for 10 min at 37 °C and then washed three times on ice with ice-cold PBS. For resensitization experiments, cells were washed to remove agonist as described above and either maintained on ice or allowed to recover at 37 °C. All cells were scraped in radioimmune precipitation buffer (150 mmNaCl, 50 mm Tris, 5 mm EDTA, 10 mmNaF, 10 mm disodium pyrophosphate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) containing protease inhibitors and solubilized for 1 h at 4 °C. After centrifugation, supernatants were collected and assayed for protein concentration (Bio-Rad DC protein assay kit). HA-tagged receptors were immunoprecipitated at 4 °C using the anti-HA 12CA5 mouse monoclonal antibody. Equivalent amounts of receptor, as determined by receptor expression and the amount of solubilized protein in each sample, were subjected to SDS-polyacrylamide gel electrophoresis and processed for autoradiography. Receptor phosphorylation was quantitated using a Molecular Dynamics PhosphorImager and ImageQuant software. The mean and standard error of the mean are expressed for values obtained from the number of independent experiments indicated. Statistical significance was determined using a two-tailed t test. Binding and dose-response data were analyzed using GraphPad Prism software. The β2AR and V2R share many of the same signaling properties: both agonist-activated receptors couple to Gsα and stimulate adenylyl cyclase, desensitize in a GRK-dependent fashion, and internalize into early endosomes (5Zhang 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, 15von Zastrow M. Kobilka B.K. J. Biol. Chem. 1992; 267: 3530-3538Abstract Full Text PDF PubMed Google Scholar, 34Zhang 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, 35Birnbaumer M. Antaramian A. Themmen A.P. Gilbert S. J. Biol. Chem. 1992; 267: 11783-11788Abstract Full Text PDF PubMed Google Scholar, 36Pfeiffer R. Kirsch J. Fahrenholz F. Exp. Cell Res. 1998; 244: 327-339Crossref PubMed Scopus (35) Google Scholar, 37Innamorati G. Sadeghi H. Eberle A.N. Birnbaumer M. J. Biol. Chem. 1997; 272: 2486-2492Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, whereas the β2AR recycles rapidly back to the plasma membrane fully resensitized, the V2R is retained inside the cell (5Zhang 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, 14Pippig S. Andexinger S. Lohse M.J. Mol. Pharmacol. 1995; 47: 666-676PubMed Google Scholar, 20Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). β-Arrestin binding to many GPCRs is mediated by GRK-phosphorylated serine and threonine residues located in the receptor carboxyl-terminal tails. In addition, residues in the carboxyl terminus of the V2R have been shown to play a critical, but undefined, role in the intracellular retention of this sequestered receptor (20Innamorati G. Sadeghi H.M. Tran N.T. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2222-2226Crossref PubMed Scopus (111) Google Scholar). Therefore, to gain insight into the mechanisms regulating the ability of these two receptors to recycle and resensitize, chimeric receptors were constructed in which the carboxyl-terminal tails of the β2AR and V2R were exchanged, one for the other, after the putative sites of palmitoylation. The β2AR-V2R chimera contains the first 341 amino acids of the β2AR (Met-1 to Cys-341) fused to the last 29 amino acids of the V2R (Ala-343 to Ser-371). The V2R- β2AR chimera contains the first 342 amino acids of the V2R (Met-1 to Cys-342) fused to the last 72 amino acids of the β2AR (Leu-342 to Leu-413). The chimeric receptors were essentially indistinguishable from their wild-type counterparts with respect to their affinity for agonist, level of expression, and half-maximal effective concentration (EC50) for adenylyl cyclase activation (Table I).Table IBinding, expression, and adenylyl cyclase activation parameters for the wild-type and chimeric receptorsReceptorKdBmaxEC50nmpmol/mgnmβ2ARKh = 7.7 ± 0.6 Kl = 360 ± 301.7 ± 0.10.3 ± 0.01β2AR-V2RKh = 7.5 ± 1.0 Kl = 400 ± 301.4 ± 0.10.6 ± 0.15V2R2.4 ± 0.21.5 ± 0.20.3 ± 0.1V2R-β2AR2.5 ± 0.31.1 ± 0.10.4 ± 0.1The ligand binding properties, expression, and half-maximal effective concentration (EC50) for the activation of adenylyl cyclase were determined for the wild-type and chimeric receptors in transfected HEK-293 cells. Isoproterenol competition binding assays were performed on membranes to determine the equilibrium dissociation binding constants (Kd) for the β2AR and β2AR-V2R chimera. Kh andKl indicate the Kd values for the high and low affinity states, respectively. [3H]AVP saturation binding assays were performed on membranes to determine the affinity of AVP for the V2R and V2R-β2AR chimera. Saturation binding assays were performed on whole cells using [125I]cyanopindolol or [3H]AVP to determine the Bmax of the wild-type and chimeric receptors. The EC50for activation of adenylyl cyclase was determined on whole cells using varying concentrations of isoproterenol or AVP. All values are expressed as the mean ± S.E. (n = 3). Open table in a new tab The ligand binding properties, expression, and half-maximal effective concentration (EC50) for the activation of adenylyl cyclase were determined for the wild-type and chimeric receptors in transfected HEK-293 cells. Isoproterenol competition binding assays were performed on membranes to determine the equilibrium dissociation binding constants (Kd) for the β2AR and β2AR-V2R chimera. Kh andKl indicate the Kd values for the high and low affinity states, respectively. [3H]AVP saturation binding assays were performed on membranes to determine the affinity of AVP for the V2R and V2R-β2AR chimera. Saturation binding assays were performed on whole cells using [125I]cyanopindolol or [3H]AVP to determine the Bmax of the wild-type and chimeric receptors. The EC50for activation of adenylyl cyclase was determined on whole cells using varying concentrations of isoproterenol or AVP. All values are expressed as the mean ± S.E. (n = 3). Differences in the ability of the β2AR and V2R to recycle and resensitize might be due to differences in their sequestration pathways. The β2AR internalizes in a β-arrestin- and clathrin-dependent manner (34Zhang 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), but little is known about the sequestration pathway of the V2R other than it is blocked nonspecifically by sucrose (36Pfeiffer R. Kirsch J. Fahrenholz F. Exp. Cell Res. 1998; 244: 327-339Crossref PubMed Scopus (35) Google Scholar). Therefore, we evaluated the β-arrestin and clathrin dependence of the V2R sequestration pathway. HEK-293 cells were transfected with each receptor alone or each receptor together with the β-arrestin1 dominant negative mutant V53D (V53D), which blocks β2AR sequestration (34Zhang 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), or the dynaminI dominant negative mutant K44A (K44A), which blocks clathrin-mediated endocytosis (34Zhang 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). Agonist-induced sequestration of the β2AR, V2R, and their chimeras was blocked by overexpression of V53D or K44A (Fig.1 A). V53D was less effective inhibiting sequestration of the V2R (30 ± 4% reduction) compared with the β2AR (62 ± 1%" @default.
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