Fan G, Goldsmith P K, Collins R, Dunn C K, Krapcho K J, Rogers K V, Spiegel A M
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Endocrinology. 1997 May;138(5):1916-22. doi: 10.1210/endo.138.5.5131.
The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.
人钙离子受体（hCaR）是G蛋白偶联受体超家族的成员。其较大的（约600个残基）氨基末端胞外结构域含有9个潜在的N-连接糖基化位点。对稳定转染了hCaR的HEK-293细胞来源的细胞膜进行免疫印迹分析，结果显示分别有两条主要的免疫反应条带，大小约为150 kDa和130 kDa。用PN-糖苷酶F对细胞膜进行完全消化后，得到一条单一的主要免疫反应条带，大小约为115 kDa，证实了N-连接糖基化的存在。用衣霉素处理这些细胞，衣霉素可阻断N-连接糖基化，结果抑制了对Ca2+的信号转导。流式细胞术分析显示，经衣霉素处理的细胞中hCaR在细胞膜上的表达降低。对经衣霉素处理的细胞进行免疫印迹分析，结果显示150 kDa条带的量减少，且130 kDa条带转变为推测的非糖基化115 kDa形式。用衣霉素处理转染了突变型hCaR互补DNA的细胞，该互补DNA在第1个跨膜结构域之前的第599位含有一个无义密码子，结果阻断了一种95 kDa蛋白（代表氨基末端胞外结构域）分泌到培养基中。这些结果表明，N-连接糖基化是hCaR在细胞表面正常表达所必需的。
