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Originally published In Press as doi:10.1074/jbc.M410655200 on November 4, 2004

J. Biol. Chem., Vol. 280, Issue 3, 2300-2308, January 21, 2005
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A Novel C-terminal Motif Is Necessary for the Export of the Vasopressin V1b/V3 Receptor to the Plasma Membrane*

Jessica Robert{ddagger}, Eric Clauser{ddagger}, Patrice Xavier Petit§, and Maria Angeles Ventura{ddagger}

From the Départements {ddagger}d'Endocrinologie and §Génétique Développement et Pathologies Moléculaires, Institut Cochin, INSERM U567, CNRS UMR8104, Université René Descartes, Paris 75014, France

Received for publication, September 16, 2004 , and in revised form, October 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 CD8{alpha} 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
G-protein-coupled receptors (GPCRs)1 form the largest superfamily of the signal transduction cell surface proteins (1). GPCRs are comprised of seven transmembrane domains, an extracellular N terminus, and a cytosolic C terminus. Numerous studies (2–4) 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) (5, 6). 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 (7). 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 (8–10). 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 {alpha}2B-adrenergic and angiotensin AT1 receptors (11, 12).

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 (10, 13). 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 (14–16). None of these motifs have been found in the C terminus of GPCRs, but a recent report (17) 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 CD8{alpha} glycoprotein that is normally retained and binds to the ER membrane-associated chaperone protein DRiP78 (17). 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 {alpha}-helix and a randomly coiled segment (18). Deletion of this C-terminal domain results in the retention of many GPCRs in the ER (12, 19, 20), 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 (21). 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 CD8{alpha} glycoprotein-V3 receptor chimera.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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-{beta}-N-acetylglucosaminidase H (Endo H) (EC 3.2.1.96 [EC] ) and glycopeptide N-glycosidase F (PNGase F) (EC 3.5.1.52 [EC] ) were obtained from New England Biolabs. The following antibodies were used: mouse anti-c-Myc (9E10) (Santa Cruz Biotechnology); mouse anti-adaptin-{gamma}, mouse anti-CD8{alpha}, 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-CD8{alpha}-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 (22). 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-{Delta}Cter 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-{Delta}345–403 (lacking 58 C-terminal amino acids), {Delta}355–424 (lacking 69 C-terminal amino acids), {Delta}359–424 (lacking 65 C-terminal amino acids), and {Delta}371–424 (lacking 53 C-terminal amino acids) constructs were produced by inserting various linkers into the PmlI-XbaI sites of the ps-EGFP-6myc-V3-{Delta}Cter vector. The ps-EGFP-6myc-V3-{Delta}387–424 construct was obtained by inserting a linker into the NaeI-XbaI sites of the ps-EGFP-6myc-V3-{Delta}371–424 vector. The ps-EGFP-6myc-V3-{Delta}409–424 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-{Delta}Cter vector. The ps-EGFP-6myc-V3-{Delta}344–364 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-{Delta}Cter.

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 CD8{alpha} glycoprotein extracellular and transmembrane domains (residues 1–206) from the pCN plasmid (23) 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-CD8{alpha} 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 x 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 (24). Crude membrane extracts from transiently transfected AtT20 cells were prepared, and incubations with [3H]AVP were carried out as described previously (25). 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-{gamma} (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 {gamma}, respectively. For detection of the CD8-V3-WT and CD8-V3-MUT chimeras, permeabilized and nonpermeabilized cells were incubated with mouse anti-CD8{alpha} 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 x 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 ({alpha} = 0.05). Calculations were carried out using the StatView program.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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.



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  		FIG. 1.
Expression 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.

 



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  		FIG. 2.
The C terminus of the V3 receptor is required for cell surface expression and maturation of the receptor. A, flow cytometry analysis (gray columns) and [3H]AVP binding (open columns) in AtT20 cells transiently expressing ps-EGFP-6myc-V3-WT or ps-EGFP-6myc-V3-{Delta}Cter (mutant lacking the C terminus). For FACS analysis, total receptor levels were evaluated by measuring EGFP fluorescence, and surface receptor levels were evaluated indirectly by measuring immunofluorescence of the extracellular Myc epitopes. The ratio of both fluorescences and the binding results, normalized according to the EGFP fluorescence, were expressed as the percentage of ps-EGFP-6myc-V3-WT detected. Values are the means (±S.D.) of at least three different experiments. *** indicates p < 0.0001 (ANOVA). B, EGFP fluorescence (left panels) and cell surface staining using anti-c-Myc antibodies and Alexa Fluor 594-conjugated secondary antibodies (right panels) of nonpermeabilized AtT20 cells stably expressing ps-EGFP-6myc-V3-WT (upper panels) or transiently expressing ps-EGFP-6myc-V3-{Delta}Cter (lower panels). C, ps-EGFP-6myc-V3-WT and ps-EGFP-6myc-V3-{Delta}Cter colocalization with the ER-specific marker, calnexin, and the Golgi apparatus-specific marker, adaptin {gamma}. D, SDS-PAGE immunoblot analysis of transiently transfected AtT20 cells expressing ps-EGFP-6myc-V3-WT and ps-EGFP-6myc-V3-{Delta}Cter. After immunoprecipitation using monoclonal anti-GFP antibodies, the samples were either left untreated (-) or treated with Endo H (H) or PNGase F (F). Nontransfected AtT20 cells were used as a control.

 



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  		FIG. 3.
Effect 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: {Delta}Cter, {Delta}345–403, {Delta}344–364, {Delta}355–424, {Delta}359–424, {Delta}371–424, {Delta}387–424, and {Delta}409–424. 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): {Delta}345–403 {Delta}Cter {Delta}344–364 {Delta}355–424 {Delta}359–424 {Delta}371–424 {Delta}387–424 {Delta}409–424 WT. No significant differences were found for underlined subsets.

 



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  		FIG. 4.
The 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.

 



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  		FIG. 5.
The 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 ({Delta}Leu345 or {Delta}Leu346 and {Delta}Leu345{Delta}Leu346) 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): {Delta}Leu345{Delta}Leu346 L345T/L346T {Delta}Leu345 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-{gamma} (Golgi apparatus-specific marker).

 



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  		FIG. 6.
Hydrophobic 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 using anti-c-Myc antibodies. D, staining of non permeabilized (NP) and permeabilized (P) AtT20 cells transiently expressing ps-6myc-V3-F341T/N342T/L345T/L346T/L350T (6myc-5T) and ps-6myc-V3-WT (6myc-WT) constructs. Cells were labeled using anti-c-Myc antibodies. E, ps-6myc-V3-F341T/N342T/L345T/L346T/L350T (6myc-5T) colocalization with the ER-specific marker, calnexin. F, SDS-PAGE immunoblot analysis of transiently transfected AtT20 cells expressing ps-EGFP-6myc-V3-F341T/N342T/L345T/L346T/L350T. After immunoprecipitation with monoclonal anti-GFP antibodies, the samples were either left untreated (-) or treated with Endo H (H) or PNGase F (F). Nontransfected AtT20 cells were used as a control.

 
Transport of the V3 Receptor Requires a C-terminal Motif— The deletion of 80 amino acids from the C terminus of the V3 receptor ({Delta}Cter) prevented cell surface expression of the receptor and ligand binding (Fig. 2, A and B). The intracellular fluorescence pattern of the {Delta}Cter mutant overlapped with that of the ER-specific marker calnexin. There was no overlap between the {Delta}Cter mutant and the Golgi apparatus-specific marker, adaptin-{gamma} (Fig. 2C). In contrast, we found that the full-length receptor localized to the plasma membrane (Fig. 2, A and B) and the internal compartments, particularly the Golgi apparatus (Fig. 2C). We confirmed these results by Western blot analysis of Endo H- and PNGase F-treated cells (Fig. 2D). Extracts from the wild type transfectants contained one 100-kDa polypeptide corresponding to the complex-glycosylated mature receptor protein, which was resistant to Endo H treatment, and one 90-kDa immature receptor polypeptide, which was degraded by this enzyme to give a band at 85 kDa. The profile of extracts from PNGase F-treated wild type transfectants contained only a faint 85-kDa band. In contrast, the profile of the {Delta}Cter mutant transfectants contained one 75-kDa band. This apparent molecular weight is consistent with the deletion of 80 amino acids from the wild type protein. Treatment with Endo H and PNGase F gave rise to a 65-kDa band, indicating that this mutant protein was only core-glycosylated and failed to traffic along the Golgi apparatus.

To locate the region of the C terminus responsible for ER export, we generated a series of deletion mutants (Fig. 3). We found that the entire C-terminal region was required for ER export and that the extent of the loss of cell surface expression was directly proportional to the extent of the deletion. Receptor export was 50% lower in transfectants expressing the receptor lacking the last 15 residues ({Delta}409–424) than in wild type transfectants. The receptor was sequestered in both the ER and the Golgi apparatus in this mutant (data not shown). This region (409–424) contains two di-acidic motifs (DXE) (14).

The Di-acidic Motifs Are Not Required for ER Export of the V3 Receptor—The di-acidic residues are conserved in human, mouse, and rat vasopressin V3 receptors (Fig. 4A). We investigated the role played by these residues in receptor export by mutating 409DXE411 and 414DXE416, either individually or in combination, to alanine (409AXA411 and 414AXA416). Alanine scanning revealed no differences in the plasma membrane expression and ligand binding between the wild type and the mutated receptors (Fig. 4B). Untagged and Myc-tagged double DXE mutants were also normally expressed at the cell surface (Fig. 4C). These results suggest that neither 409DXE411 nor 414DXE416 is required for ER export.

The Di-leucine Motif Is Required for ER Export of the V3 Receptor—The deletion of the proximal region at the end of the seventh transmembrane domain ({Delta}345–403 and {Delta}344–364) prevented receptor expression at the plasma membrane and led to its retention in the ER (Fig. 3 and data not shown). This region contains a putative di-leucine export motif (26). We investigated whether this di-leucine motif 345LL346 was involved in V3 receptor trafficking by analyzing transfectants expressing receptors in which threonine residues had been substituted for leucine. Export was 43, 66, and 84% lower for the single (L345T and L346T) and multiple (L345T/L346T) mutants, respectively, than for the wild type (Fig. 5A). The mutations had a similar effect on ligand binding (Fig. 5A), whereas the affinity of the mutant receptors for [3H]AVP was unaffected (Kd for wild type = 3.4 nM ± 0.25, Kd for L345T = 2.6 nM ± 1.58, Kd for L346T = 5.5 nM ± 1.19, and Kd for L345T/L346T = 4.9 nM ± 1.91). These mutant receptors mainly localized to the ER and Golgi apparatus; however, small amounts were detected at the plasma membrane (Fig. 5, B and C). Similar results were observed in cells transfected with receptors carrying single ({Delta}Leu345 or {Delta}Leu 346) and double ({Delta}Leu345{Delta}Leu346) deletions (Fig. 5A) (Kd = 5.7 nM ± 2.44 and 4.85 nM ± 2.45, respectively). In contrast, export was 57% lower than wild type for mutants in which the leucine residues of the di-leucine motif had been substituted with valine (L345V/L346V) (data not shown). This suggests that the structure of the amino acid is more important than its noncharged nature for ER export of the receptor. These results highlight the importance of the di-leucine motif in correct export of the V3 receptor.

A Novel Motif Required for ER Export of the V3 Receptor— The proximal C-terminal sequence surrounding the di-leucine motif of the V3 receptor (341FNSHLLPRPL350) contains hydrophobic amino acids. This sequence may correspond to the {Phi}(X)3{Phi}(X)3{Phi} export motif identified in the dopamine D1 receptor. We investigated the role played by these hydrophobic residues in the V3 receptor export by using site-directed mutagenesis to generate receptor mutants in which these residues were mutated to threonine. Cell surface expression and ligand binding were 95% lower than wild type for the single mutant F341T (Fig. 6A). This mutation also led to ER accumulation of the receptor, and no label was detected in the Golgi apparatus or plasma membrane (data not shown). No [3H]AVP binding was detected in crude membrane extracts (data not shown), suggesting that the F341T mutation resulted in a folding defect. Similar results were observed for the double mutant F341T/L345T (Fig. 6A). In contrast, cell surface expression and ligand binding were 81 and 87% lower than wild type for the single N342T and double L346T/L350T mutants, respectively (Fig. 6A). As shown in Fig. 5A, the effect of single mutation L346T on the cell surface expression was substantially lower (66%) than the double mutation L346T/L350T (87%), indicating that Leu350 plays an important role in V3 receptor export. Cell surface expression of the V3 receptor was 95% lower than wild type for the N342T/L346T/L350T triple mutant (Fig. 6A). In addition, we found that export of the receptor was 44% lower than wild type for mutants in which Leu345, Leu346, and Leu350 had been mutated to phenylalanine (L345F/L346F/L350F), suggesting that the structure of these amino acids is more important than their noncharged nature for correct export of the receptor (data not shown). These studies show that the following residues are required for V3 receptor transport from the ER to the cell surface: Phe341, Asn342, Leu345, Leu346, and Leu350. These residues, either individually or in combination, are crucial for the biogenesis of the V3 receptor and thus constitute a new export motif, FN(X)2LL(X)3L.

We then investigated the effect of mutating the whole motif on receptor export; the residues in the motif were substituted to threonine (F341N342(X)2L345L346(X)3L350 to T341T342(X)2T345T346(X)3T350). As expected, this construct was not detected at the plasma membrane (Fig. 6, A and C). Untagged and Myc-tagged versions of this mutant were not expressed at the plasma membrane either, indicating that the absence of export is not related to the presence of the N-terminal EGFP tag (Fig. 6, B and D). Its colocalization with calnexin showed that it was retained within the ER (Fig. 6E). These results were confirmed by Western blot analysis (Fig. 6F). The mutated protein has an apparent molecular mass of 90 kDa, the immature core-glycosylated molecular mass. Its apparent molecular mass shifted to 85 kDa after Endo H and PNGase F treatment, indicating that the transport of this protein was impaired and that it was unable to traffic along the Golgi apparatus. Taken together, our results show that the 341FN(X)2LL(X)3L350 motif is necessary for ER export of the V3 receptor.

The V3 Receptor C terminus Confers Transport Properties on the CD8{alpha} Glycoprotein—If the proximal C-terminal 341FN(X)2LL(X)3L350 motif functions as an ER exit signal, then these residues should confer export properties on a protein that is normally retained in the ER. We investigated this by generating chimeras in which the cytoplasmic domain of the CD8{alpha} glycoprotein was replaced by the C terminus of the V3 receptor (Fig. 7A). CD8{alpha} was unable to exit the ER in the absence of its cytoplasmic tail (27). Flow cytometry and immunocytochemical analysis revealed that a chimera containing the extracellular and transmembrane domains of CD8{alpha} and the cytoplasmic C-terminal domain of the V3 receptor (CD8-V3-WT) was efficiently exported to the cell surface (Fig. 7, B and C). Export of this chimera was blocked when the residues of the FN(X)2LL-(X)3L motif were mutated to threonine, TT(X)2TT(X)3T (CD8-V3-MUT) (Fig. 7, B and C). This mutant protein was sequestered in the ER (Fig. 7D). These results show that the cytoplasmic FN(X)2LL(X)3L motif is sufficient to direct ER export.



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  		FIG. 7.
The V3 receptor C terminus confers transport properties on the CD8{alpha} glycoprotein. A and B, the N-terminal and transmembrane domains of the CD8{alpha} glycoprotein were fused to the WT V3 receptor C terminus (CD8-V3-WT) and to the C terminus of the F341T/N342T/L345T/L346T/L350T mutant (CD8-V3-MUT). AtT20 cells expressing these constructs were analyzed by flow cytometry. Surface protein levels were measured indirectly by immunofluorescence staining of the extracellular N terminus of CD8{alpha}. Values are means (±S.D.) of three different experiments. *** indicates p < 0.0001 (ANOVA). C, staining of non permeabilized (NP) and permeabilized (P) AtT20 cells transiently expressing CD8-V3-WT and CD8-V3-MUT. Cells were labeled using anti-CD8{alpha} antibodies. D, CD8-V3-MUT colocalization with the ER-specific marker, calnexin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Three vasopressin receptors, encoded by different genes, have been identified in the mammalian genome: the vascular V1a, the renal V2, and the pituitary V3 receptors (28–30). The vasopressin V3 receptor is expressed mainly in pituitary corticotroph cells, where it associates with the Gq protein and activates adrenocorticotropic hormone secretion (21, 24, 30). The sequences required for desensitization, internalization, G-protein coupling, and dimerization are well documented for the V1a and V2 receptors (31–34). Several mutations of the V2 receptor have been studied extensively, because they are responsible for nephrogenic diabetes insipidus. These mutations result in ER retention of the V2 receptor (11, 26, 35). In contrast, only limited data are available on the V3 receptor (36).

In this study, we show that the C-terminal domain of the V3 receptor is important for protein transport to the plasma membrane; deletion of this region prevented receptor export from the ER. This result strongly suggests that a trafficking signal, located in the C-terminal domain of the V3 receptor, is necessary for its export from the ER. This finding is consistent with those obtained from studies of the export of other GPCRs, including the rhodopsin (37), vasopressin V2 (38), luteinizing hormone (39), adenosine A1 (20), dopamine D1 (17), and {alpha}2B-adrenergic and angiotensin AT1 (12) receptors. Moreover, the full-length C-terminal sequence is required for total ER export of the receptor since the progressive deletion of this domain leads to progressively lower levels of ER sorting. Cell surface expression of the vasopressin V2 receptor is also influenced by reductions in the length of the protein (19); however, reductions in protein length do not alter the expression of the vasopressin V1a receptor (33). Export motifs found in other membrane proteins are present in the C terminus of the V3 receptor: two distal di-acidic motifs (DXE) and a proximal di-leucine motif surrounded by hydrophobic amino acids. We investigated systematically the role of these motifs in ER export by analyzing mutants generated by site-directed mutagenesis.

The C-terminal domain of the V3 receptor is one of the longest (84 amino acids) in the vasopressin receptor family and the only one to possess di-acidic motifs (DXE). These motifs are involved in ER export of several transmembrane proteins, such as vesicular stomatitis virus glycoprotein, LAP, CD3{gamma}, CD3{delta}, and E-cadherin (14). However, our data show that the DXE motifs are not necessary for V3 receptor ER export. Similarly, the C terminus of prenylin contains a di-acidic motif that does not influence the ER export of this multipass transmembrane protein (40). It is unclear why this DXE sequence is an export signal for some proteins and not for others.

The proximal region of the C terminus contains a di-leucine motif. Di-leucine motifs have been shown previously (41) to mediate endocytosis and to function as sorting signals for basolateral cell surface targeting in the trans-Golgi network. We showed that mutation or deletion of the di-leucine motif significantly reduced ER export, suggesting that the di-leucine sequence plays a key role in this process. The E(X)3LL motif of the vasopressin V2 receptor is important for correct folding of the protein; the di-leucine residues are involved in a hydrophobic interaction with another leucine of the first intracellular loop (11). However, the di-leucine residues of the V3 receptor are not located at the equivalent position in the C terminus. Moreover, the V3 sequence lacks the crucial Glu residue present in this V2 receptor motif.

This di-leucine sequence forms part of a more complex and hydrophobic motif, FN(X)2LL(X)3L. This sequence is similar to that of several motifs that are important for GPCR export, such as the F(X)3F(X)3F (17) and F(X)6I/LL motifs (12). The F(X)3F(X)3F export motif, which was first identified in the dopamine D1 receptor, binds to an ER-associated protein, DRiP78. Most interestingly, more recent studies have shown that DRiP78 is a chaperone protein, belonging to the DnaJ/Hsp 40 set of chaperones (42, 43). Thus, interaction of the D1 receptor motif with this protein is likely to be part of a quality control mechanism. The FN(X)2LL(X)3L motif identified in our study is different from the dopamine D1 F(X)3F(X)3F motif as deletion of Leu345 or Leu346 (generating an F(X)3L(X)3L motif, related to the dopamine D1 motif) led to major ER export defects.

Both the {alpha}2B-adrenergic and the angiotensin AT1 receptors present an F(X)6I/LL export motif in the C terminus. Deletion or insertion of amino acids between F and I/LL prevents ER export of these receptors, indicating that the position of the hydrophobic residues in the {alpha}-helix is crucial for the function of this motif. The FN(X)2LL(X)3L motif of the V3 receptor is different from the F(X)6I/LL motif as deletion of the di-leucine Leu345 and Leu346 (generating an F(X)6L motif, related to the {alpha}2B-adrenergic and the angiotensin AT1 motif) drastically impaired V3 receptor export. Thus, none of these mutant motifs (F(X)3L(X)3LorF(X)6L) were able to rescue ER export of the V3 receptor, highlighting the requirement of the specific FN(X)2LL(X)3L motif for export. In addition, the structure of the amino acids in the FN(X)2LL(X)3L motif appears to be more important than their noncharged nature; we found that V3 receptor export was lower than wild type for mutants in which the Leu345, Leu346, and Leu350 residues had been substituted with valine or phenylalanine.

Moreover, this motif seems to act directly as a transport signal as the ER export properties of this motif were transferable to the CD8{alpha} glycoprotein. Taken together, our data indicate that FN(X)2LL(X)3L is a novel motif involved in V3 receptor export from the ER.

The novel FN(X)2LL(X)3L export motif may interact with COPII-associated proteins or cargo receptors. This motif may also constitute the hydrophobic side of the {alpha}-helix that interacts with chaperones to ensure correct folding of the receptor. The involvement of this V3 receptor motif in one or both mechanisms has not yet been elucidated. It is also possible that the mutations introduced into this motif generated an ER retention signal; however, this is unlikely as ER retention signals generally contain basic residues. The motif may also be involved in interactions with putative escort proteins as reported for other receptors as follows: RAMP for the calcitonin receptor-like receptor (44) and cyclophilin-related ninaA and RanBP2 for opsin (45, 46). Finally, it is possible that FN(X)2LL(X)3L is a dimerization motif since oligomerization of GPCRs seems crucial for their correct folding and export (47). Further studies are underway, including identification of the protein(s) interacting with this motif. These studies should allow us to elucidate the mechanisms involved in GPCR export.


   FOOTNOTES
 
* This work was supported by INSERM, CNRS, and Université René Descartes and by grants from Ligue Nationale Contre le Cancer, ARC, and Fondation de France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 33-153732754; Fax: 33-153732751; E-mail: ventura{at}cochin.inserm.fr.

1 The 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-{beta}-N-acetylglucosaminidase H; LSD test, least significant difference test. Back


   ACKNOWLEDGMENTS
 
We thank Aude Jobart and Meriem Garfa from the confocal facility and Frank Letourneur from the sequencing facility of the Institut Cochin. We are grateful to Colette Auzan for help with the SDS-PAGE, Serge Benichou for the CD8{alpha} plasmid, and Bruno Saubaméa for helpful suggestions.



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 RESULTS
 DISCUSSION
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