Ray K, Clapp P, Goldsmith P K, Spiegel A M
Metabolic Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1998 Dec 18;273(51):34558-67. doi: 10.1074/jbc.273.51.34558.
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 immunoblots, 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.
人钙受体（hCaR）是一种G蛋白偶联受体，在其大的细胞外结构域中含有11个潜在的N-连接糖基化位点。通过定点诱变研究了实际被修饰的潜在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N-连接糖基化共有序列（Asn-Xaa-Ser/Thr）中的天冬酰胺残基单独或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，并通过免疫印迹上的迁移差异、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，有8个位点（Asn-90、-130、-261、-287、-446、-468、-488和-541）发生了糖基化；其余3个位点（Asn-386、-400和-594）在天然受体中可能没有被有效地糖基化。对多个N-连接糖基化位点进行连续诱变，并通过免疫印迹、免疫荧光、细胞表面蛋白生物素化和完整细胞酶联免疫测定进行分析，结果表明，破坏少至3个糖基化位点会损害受体在细胞表面的正确加工和表达。破坏5个糖基化位点会使细胞表面表达降低50%-90%，具体降低幅度取决于被破坏的是哪5个位点。各种糖基化缺陷突变型受体的磷酸肌醇水解测定结果总体上与细胞表面表达水平密切相关。我们的结果表明，在hCaR上11个潜在的N-连接糖基化位点中，有8个位点实际被利用；至少3个位点的糖基化对受体的细胞表面表达至关重要，但糖基化似乎对信号转导并不关键。
