Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction
Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction
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DOI：
10.1074/jbc.273.51.34558
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发表时间：
1998-12-18
影响因子：
4.8
通讯作者：
Spiegel, AM
中科院分区：
文献类型：
--
作者：
Ray, K;Clapp, P;Spiegel, AM
人钙受体（hCaR）是一种G蛋白偶联受体，在其大的胞外结构域中含有11个潜在的N-连接糖基化位点。通过定点诱变检查实际修饰的潜在N-连接糖基化位点的数量以及阻断这些位点的糖基化对细胞表面表达和信号转导的影响。将用于N-连接糖基化的共有序列（Asn-Xaa-Ser/Thr）的天冬酰胺残基单独突变为谷氨酰胺，并以各种组合突变为谷氨酰胺，以破坏全长受体背景下的潜在N-连接糖基化位点。将cDNA构建体瞬时转染到缺乏内源性hCaR的HEK-293细胞中，并通过免疫印迹、糖苷酶消化、完整细胞酶联免疫测定和细胞外钙刺激的磷酸肌醇水解测定的迁移率差异来分析表达的受体。野生型与突变型受体的免疫印迹分析和糖苷酶消化研究表明，在11个潜在的N-连接糖基化位点中，（Asn-90、-130、-261、-287、-446、-468、-488和-541）被糖基化;其余三个位点（Asn-386、-400和-594）在天然受体中可能未被有效糖基化。多个N-连接的糖基化位点的顺序诱变和通过免疫印迹、免疫荧光、细胞表面蛋白的生物素化和完整细胞酶联免疫测定的分析表明，少至三个糖基化位点的破坏损害了受体在细胞表面的适当加工和表达，五个糖基化位点的破坏使细胞表面表达降低50-90%，这取决于哪五个位点被破坏。各种糖基化缺陷突变受体的磷酸肌醇水解测定结果一般与细胞表面表达水平相关。我们的研究结果表明，在11个潜在的N-连接的糖基化位点上的hCaR，8个网站实际上是利用的，糖基化的至少三个网站是至关重要的细胞表面表达的受体，但糖基化似乎并不重要的信号转导。
The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site-directed mutagenesis. Asparagine residues of the consensus sequences (Asn-Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on irnmunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild type versus mutant receptors demonstrate that, of the 11 potential sites for N-linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, -488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface, Disruption of five glycosylation sites reduced cell surface expression by 50-90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction.