A Novel C-terminal Motif Is Necessary for the Export of the Vasopressin V1b/V3 Receptor to the Plasma Membrane

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. M. J.P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google Scholar). of these motifs have been in the C terminus of a M. C. Zhou Nat. Cell Biol. 2001; PubMed Scopus Google Scholar) that the proximal C terminus of the receptor contains a motif that is required for the export of this receptor from the ER. This motif export properties on a CD8α that is retained and to the ER chaperone protein M. C. Zhou Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). These the by the C-terminal of GPCRs in the folding and export of these receptors. This in is of a conserved hydrophobic proximal an and a T. T. T. M. M. Science. 2000; PubMed Scopus Google Scholar). of this C-terminal in the retention of GPCRs in the ER M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, A. M. Schulein R. Rosenthal W. PubMed Scopus Google Scholar, M. C. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google the of this is not and the the transport of GPCRs from the ER to the plasma membrane to be In this we investigated and cell surface expression of the human pituitary vasopressin V1b/V3 receptor This receptor is involved in and the A. T. G. J. 2004; PubMed Scopus Google Scholar). The C-terminal of this receptor contains two distal di-acidic motifs (DXE) and a proximal di-leucine motif surrounded by hydrophobic We identified a novel ER export signal in the hydrophobic of the proximal C terminus of the V3 receptor. This signal of a impaired ER export and transport of the receptor to the cell this motif was sufficient to confer transport properties on a CD8α glycoprotein-V3 receptor chimera. were from or for molecular were from were by was from and was from was from and glycopeptide N-glycosidase were from The were and and and and were the of the C. P. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). This was the and the signal of the receptor of the and of the Myc of the by the of this was by of the and of a expression for an wild human V3 receptor was in the as was used to and the and transmembrane sequences of the V3 receptor and to and the C terminus of the receptor. were then the in the of the Several were as the lacking C-terminal was by of the and transmembrane sequences of the V3 and of this the of the The C-terminal C-terminal C-terminal and C-terminal were by the of the The was by a the of the The was by of the C-terminal and of this the of the The was by of and of the the of the mutations of the proximal C-terminal and the two distal C-terminal motifs were from the or of two of these mutants and and of the wild receptor were the of the and the The and were by of the CD8α extracellular and transmembrane domains from the L. G. P. C. R. 2000; PubMed Scopus Google Scholar) to the C terminus of the or V3 were by and by Cell and corticotroph AtT20 were in with and were the to the were A cell was for the were by were by fluorescence-activated cell were to in with with BSA, and by with cell were with in with for at on a were then and with for at on a and were with for at on a were by of by was to cells. were then analyzed by a flow for were analyzed for was by AtT20 with The of to was from of as is the and is the were to in were then with with BSA, and and with in the with PBS, were in a and and to for binding was in the presence of were from as P. J. Mol. 22: PubMed Scopus Google Scholar). membrane from AtT20 were and with were as M. M. G. M. 2002; PubMed Scopus Google Scholar). binding and flow were both on the and binding were for the total expression of receptor by selective of cell surface or were to in with PBS, and with for were then and with for at with PBS, were then with for the receptors colocalized with internal were and at with and with ER or for at or were used to and of the and and were with for and then with for was a and were in the were with PBS, and in of for at on a and then at for the total cell were with of protein and of a with The were then and in the were and for with and then at for with either PNGase or Endo H, to the of the The was by proteins were to and then binding was by of the with were then with an at and for with The proteins were the are expressed as means We used to When significant were by ANOVA, a means were the least significant difference were the of by an and tagged the N terminus of the wild and V3 receptors with both and Myc epitopes to of the C-terminal a process to be involved in receptor export to the cell This allowed us to quantify and to receptor by flow receptor were by and surface receptor were by of the extracellular Myc epitopes We the of to to the membrane export of the receptor an was and binding and and A and ligand binding that the of the and receptors for were and and analysis of binding that the of plasma membrane expression was for the receptor for the of the V3 receptor. of the V3 receptor. receptor signal by and Myc epitopes were to the N terminus of the V3 receptor to the binding in AtT20 the receptor and the receptor binding were of in the presence or of of the from of a series of C-terminal deletions on V3 receptor export. was to the N of the C-terminal and AtT20 these were analyzed by flow The of these receptors to was investigated are with those for the receptor as in the to ANOVA, LSD test, mutants are to their expression of significant were for di-acidic motifs and are not required for ER export of the V3 receptor. the C-terminal residues of the and vasopressin V3 receptors. The the distal DXE was to the N of the and AtT20 these were analyzed by flow The of these receptors to was investigated are with those for the receptor as in the to no significant and and and of the and the were expressed in AtT20 and analyzed by flow by binding are expressed as the of the of the receptor are means of no significant di-leucine motif is for ER export of the V3 receptor. was to the N of and and or and AtT20 these were analyzed by flow The of these receptors to was investigated are with those for the receptor as in the to ANOVA, LSD test, mutants are to their expression of or significant were for cell surface of AtT20 the receptor. were receptor with and residues are in the V3 receptor export. was to the N of the AtT20 these were analyzed by flow The of these receptors to was investigated are with those for the receptor as in the to ANOVA, LSD test, mutants are to their expression of significant were for and and and of the and the were expressed in AtT20 and analyzed by flow by binding are expressed as the of the of the receptor are means of cell surface of AtT20 the receptor. were of and AtT20 and were with the F, analysis of AtT20 with the were either or with Endo or PNGase AtT20 were used as a of the V3 a C-terminal The of from the C terminus of the V3 receptor cell surface expression of the receptor and ligand binding A and The of the with that of the was no the and the In contrast, we that the receptor to the plasma membrane A and and the internal particularly the We these by analysis of Endo and PNGase from the wild one to the receptor was to Endo and one receptor was by this to a at The of from PNGase wild only a In contrast, the of the one This molecular is with the of from the wild with Endo and PNGase to a that this protein was only and to the the of the C terminus for ER we a series of mutants We that the C-terminal was required for ER export and that the of the of cell surface expression was to the of the export was in the receptor lacking the residues in wild The receptor was in both the ER and the in this not This contains two di-acidic motifs (DXE) (14Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (398) Google Scholar). The for ER of the V3 di-acidic residues are conserved in and vasopressin V3 receptors We investigated the by these residues in receptor export by mutating and either or in to and no in the plasma membrane expression and ligand binding the wild and the receptors and DXE mutants were expressed at the cell surface These that is required for ER export. The for ER of the V3 of the proximal at the of the transmembrane and receptor expression at the plasma membrane and to retention in the ER and not This contains a di-leucine export motif R. Hermosilla R. Oksche A. M. G. Rosenthal W. Mol. Pharmacol. PubMed Scopus Google Scholar). We investigated this di-leucine motif 345LL346 was involved in V3 receptor by receptors in residues had been for was and for the and and for the wild The mutations had a effect on ligand binding the of the receptors for was for wild for for and for These receptors to the ER and however, were at the plasma membrane and were in with receptors carrying or and deletions and In contrast, export was wild for mutants in the residues of the di-leucine motif had been with not This that the of the is more for ER export of the receptor. These the of the di-leucine motif in correct export of the V3 receptor. A for ER of the V3 The proximal C-terminal the di-leucine motif of the V3 receptor contains hydrophobic This may to the export motif identified in the receptor. We investigated the by these hydrophobic residues in the V3 receptor export by to receptor mutants in these residues were to Cell surface expression and ligand binding were wild for the This mutation to ER of the and no was in the or plasma membrane not binding was in membrane not suggesting that the mutation in a folding were for the In contrast, cell surface expression and ligand binding were and wild for the and in the effect of mutation on the cell surface expression was the mutation that an in V3 receptor export. Cell surface expression of the V3 receptor was wild for the In addition, we that export of the receptor was wild for mutants in and had been to suggesting that the of these is more their for correct export of the receptor not These studies that the residues are required for V3 receptor transport from the ER to the cell Phe341, Asn342, and Leu350. These either or in are for the of the V3 receptor and a export motif, We then investigated the effect of mutating the motif on receptor the residues in the motif were to to this was not at the plasma membrane A and and of this were not expressed at the plasma membrane that the of export is not to the presence of the and with that was retained within the ER These were by analysis The protein an molecular of the molecular molecular to Endo and PNGase that the transport of this protein was impaired and that was to the our that the motif is for ER export of the V3 receptor. The V3 C terminus on the CD8α the proximal C-terminal motif as an ER then these residues confer export properties on a protein that is retained in the ER. We investigated this by in the of the CD8α was by the C terminus of the V3 receptor CD8α was to the ER in the of L. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). and analysis that a the extracellular and transmembrane domains of CD8α and the C-terminal of the V3 receptor was to the cell surface and of this was the residues of the motif were to and This protein was in the ER These that the motif is sufficient to ER export. vasopressin by have been identified in the the the and the pituitary V3 receptors A. Nature. PubMed Scopus Google Scholar, M. A. M. C. A. P. Rosenthal W. Nature. PubMed Scopus Google Scholar, C. F. C. M. PubMed Scopus Google Scholar). The vasopressin V3 receptor is expressed in pituitary corticotroph with the protein and A. T. G. J. 2004; PubMed Scopus Google Scholar, P. J. Mol. 22: PubMed Scopus Google Scholar, C. F. C. M. PubMed Scopus Google Scholar). The sequences required for desensitization, G-protein coupling, and are for the and V2 receptors G. M. M. Mol. Pharmacol. 1997; PubMed Scopus Google Scholar, M. Trends 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. L. J. Physiol. 2001; Scholar, C. Bouvier M. Sci. A. 2004; PubMed Scopus Google Scholar). Several mutations of the V2 receptor have been they are for nephrogenic diabetes These mutations result in ER retention of the V2 receptor (11Krause G. Hermosilla R. Oksche A. Rutz C. Rosenthal W. Schulein R. Mol. Pharmacol. 2000; 57: 232-242PubMed Google Scholar, R. Hermosilla R. Oksche A. M. G. Rosenthal W. Mol. Pharmacol. PubMed Scopus Google Scholar, J.P. A. U.E. Laperriere A. M. Bichet D.G. Bouvier M. 2001; PubMed Scopus Google Scholar). In contrast, only are on the V3 receptor M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Google Scholar). In this we that the C-terminal of the V3 receptor is for protein transport to the plasma of this receptor export from the ER. This result that a in the C-terminal of the V3 is for export from the ER. This is with those from studies of the export of including the Sci. A. 1997; PubMed Scopus Google vasopressin V2 G. M. Mol. 1997; PubMed Scopus Google Mol. Google M. C. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google M. C. Zhou Nat. Cell Biol. 2001; PubMed Scopus Google and α2B-adrenergic and angiotensin AT1 M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) receptors. the C-terminal is required for total ER export of the receptor the of this to of ER Cell surface expression of the vasopressin V2 receptor is by in the of the protein A. M. Schulein R. Rosenthal W. PubMed Scopus Google however, in protein not the expression of the vasopressin receptor M. L. J. Physiol. 2001; Scholar). motifs in membrane proteins are in the C terminus of the V3 two distal di-acidic motifs (DXE) and a proximal di-leucine motif surrounded by hydrophobic We investigated the of these motifs in ER export by mutants by The C-terminal of the V3 receptor is one of the in the vasopressin receptor family and the only one to di-acidic motifs These motifs are involved in ER export of several transmembrane proteins, such as vesicular stomatitis virus glycoprotein, and (14Nishimura N. Balch W.E. Science. 1997; 277: 556-558Crossref PubMed Scopus (398) Google Scholar). our that the DXE motifs are not for V3 receptor ER export. the C terminus of contains a di-acidic motif that not the ER export of this transmembrane protein N. G. Biochem. J. 2004; PubMed Scopus Google Scholar). is this DXE is an export signal for some proteins and not for The proximal of the C terminus contains a di-leucine motifs have been W. C. J. 13: PubMed Scopus Google Scholar) to and to as signals for cell surface in the We that mutation or of the di-leucine motif ER suggesting that the di-leucine a in this The E(X)3LL motif of the vasopressin V2 receptor is for correct folding of the protein; the di-leucine residues are involved in a hydrophobic with of the (11Krause G. Hermosilla R. Oksche A. Rutz C. Rosenthal W. Schulein R. Mol. Pharmacol. 2000; 57: 232-242PubMed Google Scholar). the di-leucine residues of the V3 receptor are not at the in the C terminus. the V3 the in this V2 receptor This di-leucine of a more complex and hydrophobic motif, This is to that of several motifs that are for GPCR such as the M. C. Zhou Nat. Cell Biol. 2001; PubMed Scopus Google Scholar) and motifs M.T. Zhou F. Wu G. J. Biol. Chem. 2004; 279: 30741-30750Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The export motif, was identified in the to an ER-associated more studies have that is a chaperone to the set of chaperones J. Wu J. Cao G. J. 2003; PubMed Scopus Google Scholar, Sci. 2001; PubMed Scopus Google Scholar). of the receptor motif with this protein is to be of a quality control The motif identified in our is from the motif as of or an motif, to the to ER export the α2B-adrenergic and the angiotensin AT1 receptors an export motif in the C terminus. or of and ER export of these that the of the hydrophobic residues in the is for the of this The motif of the V3 receptor is from the motif as of the di-leucine and an motif, to the α2B-adrenergic and the angiotensin AT1 impaired V3 receptor export. of these motifs were to ER export of the V3 the of the specific motif for export. In addition, the of the in the motif to be more their we that V3 receptor export was wild for mutants in the and residues had been with or this motif to as a transport signal as the ER export properties of this motif were to the CD8α our that is a novel motif involved in V3 receptor export from the ER. The novel export motif may with COPII-associated proteins or cargo receptors. This motif may the hydrophobic of the that with chaperones to ensure correct folding of the receptor. The of this V3 receptor motif in one or both mechanisms not been is that the mutations this motif an ER retention however, this is as ER retention signals The motif may be involved in interactions with proteins as for receptors as for the receptor L.M. A. J. N. R. M.G. Nature. PubMed Scopus Google Scholar) and and for Cell. 1991; Full Text PDF PubMed Scopus Google Scholar, Nature. PubMed Scopus Google Scholar). Finally, is that is a motif of GPCRs for their correct folding and export A. M. Bouvier M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus (262) Google Scholar). studies are including of the interacting with this These studies us to the mechanisms involved in GPCR export. We and from the and from the of the We are to for with the for the CD8α and for