A novel C-terminal motif is necessary for the export of the vasopressin V1b/V3 receptor to the plasma membrane
A novel C-terminal motif is necessary for the export of the vasopressin V1b/V3 receptor to the plasma membrane
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DOI：
10.1074/jbc.m410655200
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发表时间：
2005-01-21
影响因子：
4.8
通讯作者：
Ventura, MA
中科院分区：
文献类型：
--
作者：
Robert, J;Clauser, E;Ventura, MA
关于内质网（ER）输出信号，特别是g蛋白偶联受体家族成员的输出信号知之甚少。我们研究了人垂体后叶加压素V1b/V3受体参与膜输出的结构基序。在at20皮质细胞中表达了一系列携带缺失和点突变的V3受体。我们通过监测放射性配体结合，并通过一种新的基于流式细胞术的方法分析带有绿色荧光蛋白和Myc表位标记的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和膜结合受体的表达。缺乏C末端的受体不在细胞表面表达，表明在该区域存在输出基序。远端C端含有两个二酸（DXE） ER输出基序；然而，这两个基序的突变对V3受体的输出没有影响。近端C端包含一个二亮氨酸(LL346)-L-345基序，由疏水残基Phe（341）、Asn（342）和Leu（350）包围。这五种残基中的一种或多种发生突变，可使受体输出量100%丧失。此外，这些突变体与钙连蛋白共定位，表明它们保留在内质网中。最后，该基序足以赋予cd8 α糖蛋白- v3受体嵌合体输出特性。总之，我们在V3受体的C端发现了一个新的输出基序FN(X)(2)LL(X)(3)L。
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 (LL346)-L-345 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.