N-linked glycosylation of the human Ca super(2+) receptor is essential for its expression at the cell surface

• Published in: Endocrinology (Philadelphia) 1997-01, Vol.138 (5), p.1916-1922
• Main Authors: Fan, Gaofeng, Goldsmith, P K, Collins, R, Dunn, C K, Krapcho, K J, Rogers, K V, Spiegel, A M
• Format: Article
• Language: English
• ISSN: 0013-7227

The human Ca super(2+) receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large ( similar to 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 Ca super(2+). 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.