A novel C-terminal motif is necessary for the export of the vasopressin V1b/V3 receptor to the plasma membrane.
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 (345)LL(346) motif surrounded by the hydrophobic residues Phe(341), Asn(342), and Leu(350). 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 CD8alpha glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)(2)LL(X)(3)L, in the C terminus of the V3 receptor.
Robert
Jessica
J
Départements d'Endocrinologie and Génétique Développement et Pathologies Moléculaires, Institut Cochin, INSERM U567, CNRS UMR8104, Université René Descartes, Paris 75014, France.
Clauser
Eric
E
Petit
Patrice Xavier
PX
Ventura
Maria Angeles
MA
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D017483:Receptors, Vasopressin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017483:Receptors, Vasopressin