Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction

Kausik Ray, Peter Clapp, Paul K. Goldsmith, Allen M. Spiegel

Research output: Contribution to journalArticle

119 Citations (Scopus)

Abstract

The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site- directed mutagenesis. Asparagine residues of the consensus sequences (Asn- Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild type versus mutant receptors demonstrate that, of the 11 potential sites for N- linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, - 488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50-90% depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction.

Original languageEnglish (US)
Pages (from-to)34558-34567
Number of pages10
JournalJournal of Biological Chemistry
Volume273
Issue number51
DOIs
StatePublished - Dec 18 1998
Externally publishedYes

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Glycosylation
Calcium-Sensing Receptors
Signal transduction
Signal Transduction
Mutagenesis
Glycoside Hydrolases
Cell Surface Receptors
Phosphatidylinositols
Immunoenzyme Techniques
Digestion
Hydrolysis
Assays
Biotinylation
HEK293 Cells
Asparagine
Consensus Sequence
Enzymes
G-Protein-Coupled Receptors
Site-Directed Mutagenesis
Glutamine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification of the sites of N-linked glycosylation on the human calcium receptor and assessment of their role in cell surface expression and signal transduction. / Ray, Kausik; Clapp, Peter; Goldsmith, Paul K.; Spiegel, Allen M.

In: Journal of Biological Chemistry, Vol. 273, No. 51, 18.12.1998, p. 34558-34567.

Research output: Contribution to journalArticle

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abstract = "The human calcium receptor (hCaR) is a G-protein-coupled receptor containing 11 potential N-linked glycosylation sites in the large extracellular domain. The number of potential N-linked glycosylation sites actually modified, and the effect on cell surface expression and signal transduction of blocking glycosylation at these sites, was examined by site- directed mutagenesis. Asparagine residues of the consensus sequences (Asn- Xaa-Ser/Thr) for N-linked glycosylation were mutated to glutamine individually and in various combinations to disrupt the potential N-linked glycosylation sites in the context of the full-length receptor. The cDNA constructs were transiently transfected into HEK-293 cells lacking endogeneous hCaR, and expressed receptors were analyzed by mobility differences on immunoblots, glycosidase digestion, intact cell enzyme-linked immunoassay, and extracellular calcium-stimulated phosphoinositide hydrolysis assay. Immunoblot analyses and glycosidase digestion studies of the wild type versus mutant receptors demonstrate that, of the 11 potential sites for N- linked glycosylation, eight sites (Asn-90, -130, -261, -287, -446, -468, - 488, and -541) are glycosylated; the three remaining sites (Asn-386, -400, and -594) may not be efficiently glycosylated in the native receptor. Sequential mutagenesis of multiple N-linked glycosylation sites and analyses by immunoblotting, immunofluorescence, biotinylation of cell surface proteins, and intact cell enzyme-linked immunoassay indicated that disruption of as few as three glycosylation sites impairs proper processing and expression of the receptor at the cell surface. Disruption of five glycosylation sites reduced cell surface expression by 50-90{\%} depending on which five sites were disrupted. Phosphoinositide hydrolysis assay results for various glycosylation-defective mutant receptors in general correlated well with the level of cell surface expression. Our results demonstrate that among 11 potential N-linked glycosylation sites on the hCaR, eight sites are actually utilized; glycosylation of at least three sites is critical for cell surface expression of the receptor, but glycosylation does not appear to be critical for signal transduction.",
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