Association of gonadotropin receptor precursors with the protein folding chaperone calnexin.
Association of gonadotropin receptor precursors with the protein folding chaperone calnexin.
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DOI：
10.1210/endo.139.4.5881
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发表时间：
1998-04
期刊：
影响因子：
4.8
通讯作者：
T. Rozell;D. Davis;Y. Chai;D. Segaloff
中科院分区：
文献类型：
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作者：
T. Rozell;D. Davis;Y. Chai;D. Segaloff
促黄体激素/绒毛膜促性腺激素受体（LHR）和促卵泡激素受体（FSHR）是G蛋白偶联受体超家族的成员。每个受体的羧基一半由通过细胞内和细胞外环连接的经典的七个跨膜区组成。此外，每个受体都含有一个大的胞外结构域。尽管G蛋白偶联受体的结构很复杂，但人们对这些受体在生物合成过程中如何呈现正确的构象知之甚少。虽然伴侣蛋白在其他蛋白质的折叠中的作用已经有了很好的记录，但它们在G蛋白偶联受体的折叠中的作用一直是个谜。为了更好地了解LH和FSH受体的折叠，我们研究了它们与一般伴侣蛋白钙连接蛋白、结合蛋白（BiP）和94 kDa葡萄糖调节蛋白（GRP 94）的关系。将表达高水平每种受体的克隆293细胞系溶解，并将提取物与结合蛋白A-琼脂糖珠的适当抗体一起孵育。使用两种方法进行实验：1）受体/伴侣复合物与抗受体抗体之一的共免疫沉淀，然后使用抗钙连接蛋白或抗KDEL（其识别BiP和GRP 94）抗体的SDS-PAGE和蛋白质印迹;或2）受体/伴侣复合物与抗钙连接蛋白或抗KDEL的共免疫沉淀，然后使用抗受体抗体之一的蛋白质印迹。使用这些方案，我们发现rLHR和rFSHR两者的未成熟形式与钙连接蛋白相关，但几乎没有观察到受体与BiP或GRP 94的相关性。这些实验表明，野生型LHR和FSHR的前体形式可以与钙连接蛋白结合，提高了这种伴侣蛋白可能促进促性腺激素受体折叠的可能性。
The lutropin/choriogonadotropin receptor (LHR) and follitropin receptor (FSHR) are members of the superfamily of G protein-coupled receptors. The carboxyl half of each receptor is composed of the classical seven membrane spanning regions connected by intracellular and extracellular loops. In addition, each receptor contains a large extracellular domain. Despite the complexity of the structure of G protein-coupled receptors, little is known about how these receptors assume their correct conformations during biosynthesis. Although the role of chaperone proteins in the folding of other proteins has been well documented, their role in the folding of G protein-coupled receptors has been an enigma. To better understand the folding of the LH and FSH receptors, we examined their association with the general chaperone proteins calnexin, binding protein (BiP), and the 94-kDa glucose-regulated protein (GRP94). Clonal 293 cell lines expressing comparably high levels of each receptor were solubilized, and the extracts were incubated with the appropriate antibody bound to Protein A-sepharose beads. Experiments were performed using two approaches: 1) coimmunoprecipitation of receptor/chaperone complexes with one of the antireceptor antibodies, then SDS-PAGE and Western blotting using either anticalnexin or anti-KDEL (which recognizes BiP and GRP94) antibodies; or 2) coimmunoprecipitation of receptor/chaperone complexes with anticalnexin or anti-KDEL, then Western blotting with one of the antireceptor antibodies. Using these protocols, we found that the immature forms of both the rLHR and rFSHR are associated with calnexin, but little or no association was observed for either receptor with BiP or GRP94. These experiments show that the precursor forms of the wild-type LHR and FSHR can associate with calnexin, raising the possibility that this chaperone protein may facilitate in the folding of the gonadotropin receptors.