INCORPORATION OF AN ADDITIONAL GLYCOSYLATION SITE ENHANCES EXPRESSIONOF FUNCTIONAL HUMAN GONADOTROPIN-RELEASING-HORMONE RECEPTOR

Mutation of N-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 glyc osylation site to the human gonadotropin-releasing hormone receptor, a s a means for increasing its expression. Covalent labeling of the muta nt receptor expressed in COS-1 cells with a gonadotropin-releasing hor mone (GnRH) photoreactive analog demonstrated a shift in apparent mole cular weight, indicating that the new site was in fact glycosylated. T he receptor with extra glycosylation site displayed normal binding aff inities for agonists buserelin and [D-Ala(6)-Pro(9)-NHEt]-GnRH, and th e antagonist antide, and a slightly increased affinity for GnRH. Recep tor number was increased by 1.7-fold in membrane preparations from cel ls expressing the mutant receptor, compared with wild-type. Photoaffin ity labeling of cell-surface receptors in intact cells demonstrated a 1.8-fold increase in binding sites on the cell surface. The GnRH recep tor (GnRHR) with extra glycosylation site conferred a markedly enhance d signaling response to agonist. Dose-response curves for GnRH-stimula ted inositol phosphate production were left-shifted by an average of 4 .4-foId, and maximal inositol phosphate responses were increased by 1. 2 fold, in cells transfected with mutant compared with wild-type recep tor, indicating that the increase in binding sites represented functio nal receptors. These results demonstrate that addition of an extra gly cosylation site enhances expression of the human GnRHR, a strategy tha t may be applicable to other cell-surface receptors.