Bai M, Trivedi S, Brown E M
Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1998 Sep 4;273(36):23605-10. doi: 10.1074/jbc.273.36.23605.
The extracellular calcium (Ca2+o)-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface cross-linking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.
细胞外钙（Ca2+o）-传感受体（CaR）是一种G蛋白偶联受体，在钙稳态中发挥重要作用。在本研究中，我们采用表位标记、细胞表面生物素化和免疫沉淀技术，证明CaR在转染的人胚肾（HEK293）细胞表面主要以二聚体形式表达。在非还原条件下对细胞表面蛋白进行Western分析表明，CaR以几种分子量大于200 kDa的形式存在。在还原条件下，这些受体的大多数高分子量形式可转化为160 kDa的单一单体形式。这一结果表明，CaR通过对还原剂敏感的分子间二硫键在细胞表面形成二聚体甚至更高的寡聚体。与这一假设一致，在还原条件下，使用细胞表面交联剂可使280 kDa的假定二聚体CaR相对于160 kDa的单体形式受体的比例大幅增加。当我们共转染并共免疫沉淀野生型全长受体和缺少其胞质尾的CaR截短形式时，进一步证明了完整细胞中CaR的二聚化。综上所述，我们从这些结果得出结论，功能性CaR以二聚体形式存在于转染的HEK293细胞的细胞表面。
