N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface

G. Fan, P. K. Goldsmith, R. Collins, C. K. Dunn, K. J. Krapcho, K. V. Rogers, A. M. Spiegel

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Abstract

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (~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.

Original languageEnglish (US)
Pages (from-to)1916-1922
Number of pages7
JournalEndocrinology
Volume138
Issue number5
DOIs
StatePublished - Jan 1 1997
Externally publishedYes

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ASJC Scopus subject areas

  • Endocrinology

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Fan, G., Goldsmith, P. K., Collins, R., Dunn, C. K., Krapcho, K. J., Rogers, K. V., & Spiegel, A. M. (1997). N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface. Endocrinology, 138(5), 1916-1922. https://doi.org/10.1210/endo.138.5.5131