N-LINKED GLYCOSYLATION OF THE HUMAN CA2-SURFACE( RECEPTOR IS ESSENTIAL FOR ITS EXPRESSION AT THE CELL)

The human Ca2+ receptor (hCaR) is a member of the superfamily of G pro tein-coupled receptors. Its large (similar to 600 residue) amino-termi nal extracellular domain contains 9 potential N-linked glycosylation s ites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of ap proximately 150 and 130 kDa, respectively. Complete digestion of tile membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked gly cosylation. 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 tunicam ycin-treated cells showed a reduction in the amount of the 150-kDa ban d and conversion of the 130-kDa band to the presumptively nonglycosyla ted 115-kDa form. Tunicamycin treatment of cells, transfected with a m utant hCaR complementary DNA containing a nonsense codon at position 5 99 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.