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• W2075415414 abstract "Little is known about endoplasmic reticulum (ER) export signals, particularly those of members of the G-protein-coupled receptor family. We investigated the structural motifs involved in membrane export of the human pituitary vasopressin V1b/V3 receptor. A series of V3 receptors carrying deletions and point mutations were expressed in AtT20 corticotroph cells. We analyzed the export of these receptors by monitoring radioligand binding and by analysis of a V3 receptor tagged with both green fluorescent protein and Myc epitopes by a novel flow cytometry-based method. This novel method allowed us to quantify total and membrane-bound receptor expression. Receptors lacking the C terminus were not expressed at the cell surface, suggesting the presence of an export motif in this domain. The distal C terminus contains two di-acidic (DXE) ER export motifs; however, mutating both these motifs had no effect on the V3 receptor export. The proximal C terminus contains a di-leucine 345LL346 motif surrounded by the hydrophobic residues Phe341, Asn342, and Leu350. The mutation of one or more of these five residues abolished up to 100% of the receptor export. In addition, these mutants colocalized with calnexin, demonstrating that they were retained in the ER. Finally, this motif was sufficient to confer export properties on a CD8α glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)2LL(X)3L, in the C terminus of the V3 receptor. Little is known about endoplasmic reticulum (ER) export signals, particularly those of members of the G-protein-coupled receptor family. We investigated the structural motifs involved in membrane export of the human pituitary vasopressin V1b/V3 receptor. A series of V3 receptors carrying deletions and point mutations were expressed in AtT20 corticotroph cells. We analyzed the export of these receptors by monitoring radioligand binding and by analysis of a V3 receptor tagged with both green fluorescent protein and Myc epitopes by a novel flow cytometry-based method. This novel method allowed us to quantify total and membrane-bound receptor expression. Receptors lacking the C terminus were not expressed at the cell surface, suggesting the presence of an export motif in this domain. The distal C terminus contains two di-acidic (DXE) ER export motifs; however, mutating both these motifs had no effect on the V3 receptor export. The proximal C terminus contains a di-leucine 345LL346 motif surrounded by the hydrophobic residues Phe341, Asn342, and Leu350. The mutation of one or more of these five residues abolished up to 100% of the receptor export. In addition, these mutants colocalized with calnexin, demonstrating that they were retained in the ER. Finally, this motif was sufficient to confer export properties on a CD8α glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)2LL(X)3L, in the C terminus of the V3 receptor. G-protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G-protein-coupled receptor; ER, endoplasmic reticulum; AVP, [Arg8]vasopressin; EGFP, enhanced green fluorescent protein; WT, wild type; FACS, fluorescence-activated cell sorting; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PNGase F, glycopeptide N-glycosidase F; ANOVA, analysis of variance; GFP, green fluorescent protein; Endo H, endo-β-N-acetylglucosaminidase H; LSD test, least significant difference test. form the largest superfamily of the signal transduction cell surface proteins (1Dohlman H.G. Thorner J. Caron M.G. Lefkowitz R.J. Annu. Rev. Biochem. 1991; 60: 653-688Crossref PubMed Scopus (1136) Google Scholar). GPCRs are comprised of seven transmembrane domains, an extracellular N terminus, and a cytosolic C terminus. Numerous studies (2Tsao P. von Cao T. Zastrow M. Trends Pharmacol. Sci. 2001; 22: 91-96Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 3Claing A. Laporte S.A. Caron M.G. Lefkowitz R.J. Prog. Neurobiol. 2002; 66: 61-79Crossref PubMed Scopus (452) Google Scholar, 4Luttrell L.M. Lefkowitz R.J. J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar) have identified sequences in these receptors that are essential for ligand binding, G-protein coupling, desensitization, and internalization. In contrast, little is known about the sequences required for the transport of these proteins from the endoplasmic reticulum (ER) to the plasma membrane. Naturally occurring mutations in certain GPCRs result in impaired membrane export and lead to several diseases including retinitis pigmentosa (rhodopsin receptor) and nephrogenic diabetes insipidus (vasopressin V2 receptor) (5Stojanovic A. Hwa J. Recept. Channels. 2002; 8: 33-50Crossref PubMed Scopus (52) Google Scholar, 6Morello J.P. Bichet D.G. Annu. Rev. Physiol. 2001; 63: 607-630Crossref PubMed Scopus (262) Google Scholar). Increasing our understanding of the molecular determinants and mechanisms underlying GPCR export is essential for elucidating the pathophysiology of these diseases and for improving the treatments available. Translocation from the ER is a critical and rate-limiting checkpoint of functional membrane-bound protein production and brings in two steps (7Petaja-Repo U.E. Hogue M. Laperriere A. Walker P. Bouvier M. J. Biol. Chem. 2000; 275: 13727-13736Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). First, quality control takes place within the lumen of the ER and involves ubiquitous ER resident chaperone proteins. These proteins ensure the correct folding, stability, and maturation of the membrane-bound proteins by means of hydrophobic interactions, formation of disulfide bridges, and addition of oligosaccharide moieties. A second set of chaperones, specific for a protein subset or family, then verifies the folding of the membrane cargo proteins before ER export. This system ensures that only native proteins reach their final destination. If the folding and maturation process fails, the protein is not transported to the plasma membrane and is retained in internal compartments. These proteins then undergo ER-associated degradation, a pathway linked to the proteasome (8Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1676) Google Scholar, 9Sitia R. Braakman I. Nature. 2003; 426: 891-894Crossref PubMed Scopus (575) Google Scholar, 10Trombetta E.S. Parodi A.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 649-676Crossref PubMed Scopus (364) Google Scholar). Little is known about the folding and maturation processes involved in GPCR expression and in particular about the chaperones interacting with the cytosolic domains of these receptors. Several conserved C-terminal motifs seem to be involved in GPCR folding such as the di-leucine motif E(X)3LL of the vasopressin V2 receptor and the di-leucine motif F(x)6I/LL of the α2B-adrenergic and angiotensin AT1 receptors (11Krause G. Hermosilla R. Oksche A. Rutz C. Rosenthal W. Schulein R. Mol. Pharmacol. 2000; 57: 232-242PubMed Google Scholar, 12Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). When the folding step is achieved, the protein leaves the ER. Evidence is mounting that the ER export of at least some membrane proteins is a selective process, involving the recruitment and concentration of cargo proteins in prebudding complexes. The formation of these complexes may involve interactions with the coat protein complex (COPII), COPII-associated proteins, or a cargo receptor. These interactions may result in either the masking of an ER retention signal (KDEL, KKXX, or RXR sequences) or the unmasking of an ER export signal (10Trombetta E.S. Parodi A.J. Annu. Rev. Cell Dev. Biol. 2003; 19: 649-676Crossref PubMed Scopus (364) Google Scholar, 13Barlowe C. Trends Cell Biol. 2003; 13: 295-300Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Two of these export signals have been extensively characterized: the C-terminal di-acidic DXE motif of the vesicular stomatitis virus glycoprotein, and the di-phenylalanine FF motif of endoplasmic reticulum-Golgi intermediate compartment-53 and members of the p24 protein family (14Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (398) Google Scholar, 15Kappeler F. Klopfenstein D.R. Foguet M. Paccaud J.P. Hauri H.P. J. Biol. Chem. 1997; 272: 31801-31808Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 16Fiedler K. Veit M. Stamnes M.A. Rothman J.E. Science. 1996; 273: 1396-1399Crossref PubMed Scopus (275) Google Scholar). None of these motifs have been found in the C terminus of GPCRs, but a recent report (17Bermak J.C. Li M. Bullock C. Zhou Q.Y. Nat. Cell Biol. 2001; 3: 492-498Crossref PubMed Scopus (235) Google Scholar) indicates that the proximal C terminus of the dopamine D1 receptor contains a F(X)3F(X)3F motif that is required for the proper export of this receptor from the ER. This motif confers export properties on a truncated CD8α glycoprotein that is normally retained and binds to the ER membrane-associated chaperone protein DRiP78 (17Bermak J.C. Li M. Bullock C. Zhou Q.Y. Nat. Cell Biol. 2001; 3: 492-498Crossref PubMed Scopus (235) Google Scholar). These findings highlight the role played by the C-terminal domain of GPCRs in mediating the folding and export of these receptors. This domain varies in length but is generally composed of a highly conserved hydrophobic proximal region, forming an amphipathic α-helix and a randomly coiled segment (18Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Trong LeI. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (5038) Google Scholar). Deletion of this C-terminal domain results in the retention of many GPCRs in the ER (12Duvernay M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 19Oksche A. Dehe M. Schulein R. Wiesner B. Rosenthal W. FEBS Lett. 1998; 424: 57-62Crossref PubMed Scopus (64) Google Scholar, 20Pankevych H. Korkhov V. Freissmuth M. Nanoff C. J. Biol. Chem. 2003; 278: 30283-30293Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), but the precise role of this region is not known, and the mechanism mediating the transport of GPCRs from the ER to the plasma membrane remains to be defined. In this study, we investigated intracellular trafficking and cell surface expression of the human pituitary vasopressin V1b/V3 receptor (V3). This receptor is involved in adrenocorticotropic hormone secretion and the stress response (21Tanoue A. Ito S. Honda K. Oshikawa S. Kitagawa Y. Koshimizu T.A. Mori T. Tsujimoto G. J. Clin. Investig. 2004; 113: 302-309Crossref PubMed Scopus (208) Google Scholar). The C-terminal region of this receptor contains two distal di-acidic motifs (DXE) and a proximal di-leucine motif surrounded by hydrophobic amino acids. We identified a novel ER export signal in the hydrophobic region of the proximal C terminus of the V3 receptor. This signal consists of a 341FN(X)2LL(X)3L350 motif. Its disruption impaired ER export and transport of the receptor to the cell surface. Moreover, this motif was sufficient to confer transport properties on a CD8α glycoprotein-V3 receptor chimera. Materials—Culture reagents were obtained from Sigma, BD Biosciences (Nu-serum), HyClone (Fetalclone III), or Invitrogen. Enzymes for molecular cloning were purchased from New England Biolabs. Oligonucleotides were synthesized by Invitrogen. Radioactive vasopressin, [3H]AVP (60–70 Ci/mmol), was obtained from PerkinElmer Life Sciences, and [Arg8]vasopressin (AVP) was obtained from Bachem. TOPRO-3 iodide was purchased from Molecular Probes. Endo-β-N-acetylglucosaminidase H (Endo H) (EC 3.2.1.96) and glycopeptide N-glycosidase F (PNGase F) (EC 3.5.1.52) were obtained from New England Biolabs. The following antibodies were used: mouse anti-c-Myc (9E10) (Santa Cruz Biotechnology); mouse anti-adaptin-γ, mouse anti-CD8α, and phycoerythrin-conjugated anti-mouse IgG (BD Biosciences); rabbit anti-calnexin and peroxidase-conjugated anti-rabbit IgG (Sigma); mouse anti-GFP (Roche Applied Science); rabbit anti-GFP (Clontech); mouse anti-CD8α-phycoerythrin-conjugated (Beckman Instruments); Alexa Fluor 594-conjugated anti-mouse IgG, Alexa Fluor 594-conjugated anti-rabbit IgG, and Alexa Fluor 488-conjugated anti-rabbit IgG (Molecular Probes). DNA Constructs and Site-directed Mutagenesis—Constructs were subcloned into the BamHI-XbaI sites of the ps-EGFP-6myc vector (22Miserey-Lenkei S. Parnot C. Bardin S. Corvol P. Clauser E. J. Biol. Chem. 2002; 277: 5891-5901Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This vector was constructed using the pEGFP-C3 vector (Clontech) and contained the signal peptide of the insulin receptor (ps) upstream of the EGFP sequence and six copies of the Myc epitope downstream of the EGFP sequence followed by the vector polylinker. An EGFP-deleted version of this vector (ps-6myc) was generated by deletion of the EGFP sequence and in-frame insertion of a linker. An initial expression vector coding for an N-terminal double-tagged wild type human V3 receptor was generated in the ps-EGFP-6myc vector (ps-EGFP-6myc-V3-WT) as follows: PCR was used to introduce BamHI (5′) and PmlI-XbaI (3′) restriction sites flanking the N-terminal and transmembrane sequences of the V3 receptor (residues 1–344), and to introduce EcoRV (5′) and XbaI (3′) restriction sites flanking the C terminus (residues 345–424) of the receptor. Both fragments were then ligated into the BamHI-XbaI sites in the polylinker of the ps-EGFP-6myc vector. Several C-terminally deleted constructs were generated as follows: the ps-EGFP-6myc-V3-ΔCter construct, lacking 80 C-terminal amino acid residues, was generated by PCR amplification of the N-terminal and transmembrane sequences of the V3 receptor, and insertion of this fragment into the BamHI-XbaI sites of the ps-EGFP-6myc vector. The ps-EGFP-6myc-V3-Δ345–403 (lacking 58 C-terminal amino acids), Δ355–424 (lacking 69 C-terminal amino acids), Δ359–424 (lacking 65 C-terminal amino acids), and Δ371–424 (lacking 53 C-terminal amino acids) constructs were produced by inserting various linkers into the PmlI-XbaI sites of the ps-EGFP-6myc-V3-ΔCter vector. The ps-EGFP-6myc-V3-Δ387–424 construct was obtained by inserting a linker into the NaeI-XbaI sites of the ps-EGFP-6myc-V3-Δ371–424 vector. The ps-EGFP-6myc-V3-Δ409–424 construct was generated by PCR amplification of the C-terminal fragment (residues 345–408) and subsequent subcloning of this fragment into the PmlI-XbaI sites of the ps-EGFP-6myc-V3-ΔCter vector. The ps-EGFP-6myc-V3-Δ344–364 construct was obtained by NaeI-XbaI digestion of ps-EGFP-6myc-V3-WT and insertion of the resulting fragment into the PmlI-XbaI sites of the ps-EGFP-6myc-V3-ΔCter. Point mutations of the proximal C-terminal 341FNSHLLPRPL350 and the two distal C-terminal DXE, 409DLELADGE416, motifs were generated from ps-EGFP-6myc-V3-WT vector using the QuickChange site-directed mutagenesis kit (Stratagene). Untagged or Myc-tagged versions of two of these mutants (F341T/N342T/L345T/L346T/L350T and D409A/E411A/D414A/D416A) and of the wild type (WT) receptor were subcloned into the BamHI-XbaI sites of the pcDNA3 vector (Invitrogen) and the ps-6myc vector. The chimeras, CD8-V3-WT and CD8-V3-MUT, were generated by in-frame fusion of the CD8α glycoprotein extracellular and transmembrane domains (residues 1–206) from the pCN plasmid (23Erdtmann L. Janvier K. Raposo G. Craig H.M. Benaroch P. Berlioz-Torrent C. Guatelli J.C. Benarous R. Benichou S. Traffic. 2000; 1: 871-883Crossref PubMed Scopus (42) Google Scholar) to the C terminus of the WT (residues 340–424) or mutated (341TT(X)2TT(X)3-T350) V3 receptors, respectively. Constructs were verified by restriction enzyme digestion and by DNA sequencing. Cell Culture and Transfection—Mouse corticotroph AtT20 cells (ATCC, CRL-1795) were cultured (37 °C, 5% CO2) in Dulbecco's modified Eagle's medium/F-12 supplemented with 7.5% Fetalclone III, 7.5% Nu-serum, and 0.5 mm glutamine. Cells were transiently transfected using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. All analyses were performed 24–48 h after transfection. A stable cell line was established for the ps-EGFP-6myc-V3-WT transfectant. Stable transfectants were generated using LipofectAMINE reagent followed by G418 (Invitrogen) selection. Clones were purified by fluorescence-activated cell sorting (FACS). FACS—Transient transfectants were grown to confluence in 6-well dishes, washed twice with PBS supplemented with 0.1% BSA, and dissociated by treatment with cell dissociation buffer (Invitrogen). Cells were incubated with monoclonal anti-c-Myc antibodies diluted (1:200) in PBS supplemented with 1% BSA for 1 h at 4 °C on a rotating wheel. Cells were then washed and incubated with phycoerythrin-conjugated anti-mouse secondary antibodies (diluted 1:50) for 1 h at 4 °C on a rotating wheel. For CD8-V3-WT and CD8-V3-MUT chimera labeling, cells were incubated with anti-CD8α phycoerythrin-conjugated antibodies (diluted 1:25) for 2 h at 4 °C on a rotating wheel. Unbound antibodies were removed by three rounds of washing (with PBS) followed by centrifugation (100 × g, 5 min, 4 °C). TOPRO-3 iodide (2 μg/ml) was added to detect living cells. Transfectants were then analyzed by FACS using a BD Biosciences FACScan flow cytometer. Cells positive for EGFP fluorescence were analyzed for phycoerythrin-derived fluorescence. Autofluorescence was determined by measuring nontransfected AtT20 cells treated with phycoerythrin-coupled secondary antibodies. The ratio of phycoerythrin fluorescence (surface expression) to EGFP fluorescence (total expression) was calculated from measurements of 2500 cells as follows: ((meanPE - meanauto)/meanEGFP, n = 2500), where auto is the autofluorescence and PE is the phycoerythrin fluorescence. Radioligand Binding Assay—Transiently transfected cells were grown to confluence in 24-well dishes. Cells were then washed with PBS supplemented with 5 mm MgCl2, 0.2% BSA, and 1 mg/ml bacitracin, pH 7.4, and incubated with [3H]AVP in the same buffer for2hat4 °C. After washing twice with cold PBS, cells were suspended in a solution containing 0.1% SDS and 0.1 n NaOH and transferred to liquid scintillation vials for counting. Nonspecific binding was measured after incubation in the presence of 2 μm AVP. Kinetic constants (Kd) were derived from saturation experiments as described previously (24Ventura M.A. Rene P. de Keyzer Y. Bertagna X. Clauser E. J. Mol. Endocrinol. 1999; 22: 251-260Crossref PubMed Scopus (31) Google Scholar). Crude membrane extracts from transiently transfected AtT20 cells were prepared, and incubations with [3H]AVP were carried out as described previously (25Derick S. Cheng L.L. Voirol M.J. Stoev S. Giacomini M. Wo N.C. Szeto H.H. Mimoun BenM. Andres M. Gaillard R.C. Guillon G. Manning M. Endocrinology. 2002; 143: 4655-4664Crossref PubMed Scopus (61) Google Scholar). Radioligand binding and flow cytometry assays were both performed on the same day, and binding data were normalized for each EGFP-tagged construct using the total expression level of receptor protein, determined by measuring EGFP fluorescence. Immunofluorescence Confocal Microscopy—For selective labeling of cell surface receptors, transiently or stably transfected cells were grown to confluence in Lab-Teck (Nunc) chambered cover glasses, washed with PBS, and fixed with 4% paraformaldehyde for 20 min. Cells were then washed (PBS) and incubated with anti-c-Myc primary antibodies (diluted 1:200) for 1 h at room temperature. After washing with PBS, cells were then incubated with Alexa Fluor 594-conjugated anti-mouse secondary antibodies (diluted 1:1000) for 1 h. To determine whether the receptors colocalized with internal compartment markers, cells were fixed and permeabilized at room temperature with 0.5% Triton X-100 and incubated with antibodies against calnexin (an ER marker) (diluted 1:1000) or against adaptin-γ (a Golgi apparatus marker) (diluted 1:600) for 1 h at room temperature. Alexa Fluor 594-conjugated anti-rabbit secondary antibodies or Alexa Fluor 594-conjugated anti-mouse secondary antibodies (diluted 1:1000) were used to detect anti-calnexin and anti-adaptin γ, respectively. For detection of the CD8-V3-WT and CD8-V3-MUT chimeras, permeabilized and nonpermeabilized cells were incubated with mouse anti-CD8α antibodies (diluted 1:50) for 1 h and then with Alexa Fluor 594-conjugated anti-mouse secondary antibodies (diluted 1:1000) for 1 h. Fluorescence was detected using a Leica TCS SP2 AOBS confocal microscope. Immunoprecipitation, Deglycosylation, and Western Blot Analysis— Cells were transfected in Petri dishes. Forty eight hours later, the transient transfectants were washed twice with ice-cold PBS, scraped, and incubated in 1 ml of lysis buffer, 50 mm Tris, pH 7.4, 150 mm NaCl, 10 mm EDTA, 1% Triton X-100, complete anti-protease mixture (Roche Applied Science), for 2 h at 4 °C on a rotating wheel and then centrifuged at 10,000 × g for 10 min. For immunoprecipitation, the total cell lysates were incubated with 50 μl of protein A-Sepharose beads and 3–5 μg of a mouse anti-GFP monoclonal antibody overnight with gentle rotation. The beads were then washed and boiled in SDS sample buffer. For deglycosylation, the beads were washed and boiled for 10 min with denaturing buffer and then incubated at 37 °C for 16 h with either PNGase F or Endo H, according to the instructions of the manufacturers. The reaction was stopped by adding SDS sample buffer. For Western blotting, proteins were subjected to 10% SDS-PAGE and then transferred onto nitrocellulose membranes (Schleicher & Schuell). Nonspecific binding was blocked by incubation of the membranes with 5% milk. Membranes were then incubated with an anti-GFP polyclonal antibody (diluted 1:1000) overnight at 4 °C, washed, and incubated for 1 h with peroxidase-conjugated anti-rabbit IgG (diluted 1:50,000). The blotted proteins were revealed using the ECL kit (Pierce). Statistical Analysis—Values are expressed as means (±S.D.). We used ANOVA to analyze differences between groups. When significant differences were detected by ANOVA, a posteriori comparisons between means were conducted using the Fisher least significant difference (LSD) test (α = 0.05). Calculations were carried out using the StatView program. Quantification of Receptor Export by an Original Flow Cytometry-based Method and Ligand Binding Assays—We tagged the N terminus of the wild type (WT) and mutant V3 receptors with both EGFP and Myc epitopes to prevent modification of the C-terminal domain conformation, a process suspected to be involved in receptor export to the cell surface. This double labeling also allowed us to quantify and to compare receptor trafficking by flow cytometry. Total chimeric receptor levels were measured by EGFP fluorescence, and surface receptor levels were measured indirectly by immunofluorescence labeling of the extracellular Myc epitopes (Fig. 1A). We calculated the ratio of immunolabel fluorescence to EGFP to compare the membrane export of the different receptor constructs. Moreover, an excellent correlation was observed between FACS and binding data (Figs. 2A, 3, 4, B and C, 5A, and 6, A and B). Our ligand binding assays showed that the affinities of the ps-EGFP-6myc-V3-WT and V3-WT receptors for [3H]AVP were similar (Kd = 3.7 ± 0.63 and 3.4 ± 0.25, respectively) (Fig. 1, B and C). Our analysis of [3H]AVP binding also indicated that the level of plasma membrane expression was lower for the ps-EGFP-6myc-V3-WT receptor than for the V3-WT receptor.Fig. 1Expression of the double-tagged V3 receptor. A, schematic representation of the double-tagged V3 receptor. An insulin receptor signal sequence (ps) followed by EGFP and 6 Myc epitopes were added to the N terminus of the V3 receptor to generate the ps-EGFP-6myc-V3 construct. B, [3H]AVP binding in AtT20 cells transiently expressing the nontagged V3-WT receptor (open circles) and the doubletagged ps-EGFP-6myc-V3-WT receptor (black squares). Saturation binding experiments were performed using increasing concentrations of [3H]AVP in the presence or absence of 2 μm cold AVP. C, Scatchard plot of the data from B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Effect of a series of C-terminal deletions on V3 receptor export. ps-EGFP-6myc was fused to the N termini of the following C-terminal deletion mutants: ΔCter, Δ345–403, Δ344–364, Δ355–424, Δ359–424, Δ371–424, Δ387–424, and Δ409–424. AtT20 transfectants expressing these constructs were analyzed by flow cytometry (gray columns). The ability of these mutant receptors to bind AVP was investigated using [3H]AVP (open columns). Results are compared with those obtained for cells expressing the ps-EGFP-6myc-V3-WT receptor as described in the legend to Fig. 2A. ANOVA, p < 0.0001. LSD test, mutants are listed according to their mean expression level (in order of increasing magnitude): Δ345–403 ΔCter Δ344–364 Δ355–424 Δ359–424 Δ371–424 Δ387–424 Δ409–424 WT. No significant differences were found for underlined subsets.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4The di-acidic motifs 409DXE411 and 414DXE416 are not required for ER export of the V3 receptor. A, the C-terminal residues of the human, mouse, and rat vasopressin V3 receptors. The boxes contain the distal DXE motifs. B, ps-EGFP-6myc was fused to the N termini of the following alanine substitution mutants: 409AXA411, 414AXA416, and 409AXA411-414AXA416. AtT20 cells expressing these constructs were analyzed by flow cytometry (gray columns). The ability of these mutant receptors to bind AVP was investigated using [3H]AVP (open columns). Results are compared with those obtained for cells expressing the ps-EGFP-6myc-V3-WT receptor as described in the legend to Fig. 2A. ANOVA revealed no significant differences. C, untagged (WT and 4A) and Myc-tagged versions (6myc-WT and 6myc-4A) of the WT and the 409AXA411-414AXA416 double mutant (called 4A) were expressed in AtT20 cells and analyzed by flow cytometry (gray columns) and/or by [3H]AVP binding (open columns). Results are expressed as the percentage of the respective versions of the V3-WT receptor detected. Values are means (± S.D.) of three different experiments. ANOVA revealed no significant differences.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5The di-leucine motif L345L346 is necessary for ER export of the V3 receptor. A, ps-EGFP-6myc was fused to the N termini of threonine substitution (L345T, L346T, and L345T/L346T) and deletion (ΔLeu345 or ΔLeu346 and ΔLeu345ΔLeu346) mutants. AtT20 cells expressing these constructs were analyzed by flow cytometry (gray columns). The ability of these mutant receptors to bind AVP was investigated using [3H]AVP (open columns). Results are compared with those obtained for cells expressing the ps-EGFP-6myc-V3-WT receptor as described in the legend to Fig. 2A. ANOVA, p < 0,0001. LSD test, mutants are listed according to their mean expression level (in order of increasing magnitude): ΔLeu345ΔLeu346 L345T/L346T ΔLeu345 or 346 L346/T L345/T WT. No significant differences were found for underlined subsets. B, cell surface staining of nonpermeabilized AtT20 cells transiently expressing the ps-EGFP-6myc-V3-L345T/L346T receptor. Cells were labeled using anti-c-Myc antibodies. C, ps-EGFP-6myc-V3-L345T/L346T receptor colocalization with calnexin (ER-specific marker) and adaptin-γ (Golgi apparatus-specific marker).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Hydrophobic residues are implicated in the V3 receptor export. A, ps-EGFP-6myc was fused to the N termini of the following threonine substitution mutants: F341T, N342T, F341T/L345T, L346T/L350T, N342T/L346T/L350T, F341T/N342T/L345T/L346T/L350T. AtT20 cells expressing these constructs were analyzed by flow cytometry (gray columns). The ability of these mutant receptors to bind AVP was investigated using [3H]AVP (open columns). Results are compared with those obtained for cells expressing the ps-EGFP-6myc-V3-WT receptor as described in the legend to Fig. 2A. ANOVA, p < 0,0001. LSD test, mutants are listed according to their mean expression level (in order of increasing magnitude): F341T/N342T/L345T/L346T/L350T F341T/L345T N342T/L346T/L350T F341T L346T/L350T N342T WT. No significant differences were found for underlined subsets. B, untagged (WT and 5T) and Myc-tagged versions (6myc-WT and 6myc-5T) of the WT and the F341T/N342T/L345T/L346T/L350T mutant (called 5T) were expressed in AtT20 cells and analyzed by flow cytometry (gray columns) and/or by [3H]AVP binding (open columns). Results are expressed as the percentage of the respective versions of the V3-WT receptor detected. Values are means (±S.D.) of three different experiments. *** indicates p < 0.0001 (ANOVA). C, cell surface staining of AtT20 cells transiently expressing the ps-EGFP-6myc-V3-F341T/N342T/L345T/L346T/L350T receptor. Nonpermeabilized cells were labeled usin" @default.
• W2075415414 created "2016-06-24" @default.
• W2075415414 creator A5000374695 @default.
• W2075415414 creator A5016276886 @default.
• W2075415414 creator A5061088960 @default.
• W2075415414 creator A5090025410 @default.
• W2075415414 date "2005-01-01" @default.
• W2075415414 modified "2023-10-18" @default.
• W2075415414 title "A Novel C-terminal Motif Is Necessary for the Export of the Vasopressin V1b/V3 Receptor to the Plasma Membrane" @default.
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