N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface.
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.
Fan
G
G
Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Goldsmith
P K
PK
Collins
R
R
Dunn
C K
CK
Krapcho
K J
KJ
Rogers
K V
KV
Spiegel
A M
AM
D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D014415:Tunicamycin; D006026:Glycoside Hydrolases; D002118:Calcium