title: Incorporation of an additional glycosylation site enhances expression of functional human gonadotropin-releasing hormone receptor.
abstract: Mutation ofN-glycosylation sites in the mouse gonadotropin-releasing hormone receptor was previously shown to impair its expression in COS-1 cells. We therefore investigated the effects of adding an extra glycosylation site to the human gonadotropin-releasing hormone receptor, as a means for increasing its expression. Covalent labeling of the mutant receptor expressed in COS-1 cells with a gonadotropin-releasing hormone (GnRH) photoreactive analog demonstrated a shift in apparent molecular weight, indicating that the new site was in fact glycosylated. The receptor with extra glycosylation site displayed normal binding affinities for agonists buserelin and [D: -Ala(6)-Pro(9)-NHEt]-GnRH, and the antagonist antide, and a slightly increased affinity for GnRH. Receptor number was increased by 1.7-fold in membrane preparations from cells expressing the mutant receptor, compared with wild-type. Photoaffinity labeling of cell-surface receptors in intact cells demonstrated a 1.8-fold increase in binding sites on the cell surface. The GnRH receptor (GnRHR) with extra glycosylation site conferred a markedly enhanced signaling response to agonist. Dose-response curves for GnRH-stimulated inositol phosphate production were left-shifted by an average of 4.4-fold, and maximal inositol phosphate responses were increased by 1.2 fold, in cells transfected with mutant compared with wild-type receptor, indicating that the increase in binding sites represented functional receptors. These results demonstrate that addition of an extra glycosylation site enhances expression of the human GnRHR, a strategy that may be applicable to other cell-surface receptors.
authors.affiliation: M. R. C. Regulatory Peptides Research Unit, Department of Chemical Pathology, University of Cape Town Medical School, 7925, Observatory, South Africa.
authors.forename: J S
authors.initials: JS
authors.lastname: Davidson
authors.forename: C A
authors.initials: CA
authors.lastname: Flanagan
authors.forename: P D
authors.initials: PD
authors.lastname: Davies
authors.forename: J
authors.initials: J
authors.lastname: Hapgood
authors.forename: D
authors.initials: D
authors.lastname: Myburgh
authors.forename: R
authors.initials: R
authors.lastname: Elario
authors.forename: R P
authors.initials: RP
authors.lastname: Millar
authors.forename: W
authors.initials: W
authors.lastname: Forrest-Owen
authors.forename: C A
authors.initials: CA
authors.lastname: McArdle