Robert Jessica, Clauser Eric, Petit Patrice Xavier, Ventura Maria Angeles
Départements d'Endocrinologie and Génétique Développement et Pathologies Moléculaires, Institut Cochin, INSERM U567, CNRS UMR8104, Université René Descartes, Paris 75014, France.
J Biol Chem. 2005 Jan 21;280(3):2300-8. doi: 10.1074/jbc.M410655200. Epub 2004 Nov 4.
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.
关于内质网（ER）输出信号，尤其是G蛋白偶联受体家族成员的输出信号，我们了解甚少。我们研究了人垂体加压素V1b/V3受体膜输出所涉及的结构基序。一系列携带缺失和点突变的V3受体在AtT20促肾上腺皮质激素细胞中表达。我们通过监测放射性配体结合以及采用一种基于新型流式细胞术的方法分析同时标记有绿色荧光蛋白和Myc表位的V3受体，来分析这些受体的输出。这种新方法使我们能够量化总受体表达和膜结合受体表达。缺少C末端的受体未在细胞表面表达，这表明该结构域中存在一个输出基序。C末端远端包含两个双酸性（DXE）内质网输出基序；然而，突变这两个基序对V3受体输出均无影响。C末端近端包含一个双亮氨酸（345）LL（346）基序，其周围有疏水性残基苯丙氨酸（341）、天冬酰胺（342）和亮氨酸（350）。这五个残基中一个或多个的突变使受体输出最多减少100%。此外，这些突变体与钙连接蛋白共定位，表明它们被保留在内质网中。最后，该基序足以赋予CD8α糖蛋白-V3受体嵌合体输出特性。总之，我们在V3受体的C末端鉴定出了一个新的输出基序，即FN(X)(2)LL(X)(3)L。
