N-Linked Glycosylation of the Human Ca²⁺Receptor Is Essential for Its Expression at the Cell Surface

Abstract

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.

The Ca2+-sensing Receptor Activates Cytosolic Phospholipase A2 via a Gqα-dependent ERK-independent Pathway

“…Blots with both the transiently and stably transfected cells revealed bands of the appropriate molecular mass, ϳ125 and 140 kDa, and demonstrated similar levels of CaR protein expression. The two bands of the CaR result from differential glycosylation (24,25). Bands were not present in membranes from the G418-resistant cells.…”

Prevalence of calcium sensing receptor autoantibodies in patients with sporadic idiopathic hypoparathyroidism

“…The presence of antibodies against the calcium sensing receptor was determined using Western blot analysis (11,18). Briefly, parathyroid adenoma tissues were obtained at surgery from patients with primary hyperparathyroidism who underwent parathyroidectomy.…”

The calcium-sensing receptor and related diseases

“…The native CASR, as well as recombinant receptors transfected in HEK293 cells, present as three forms: 1) a 120 kDa band, which represents the non glycosylated species; 2) a 140 kDa band, which represents the immature glycosylated receptor; and 3) a 160 kDa band, which is the mature fully glycosylated receptor (18). Although the immature glycosylated receptor can reach the plasma membrane to a low extent, only the fully glycosylated receptor is functional (19,20). …”