Identification of the glycosylation sites utilized on the V-1a vasopressinreceptor and assessment of their role in receptor signalling and expression

Most of the large family of G-protein-coupled receptors (GPCRs) possess put ative N-glycosylation sites within their N-termini. However. for the vast m ajority of GPCRs, it has not been determined which, if any, of the consensu s glycosylation sites are actually utilized or what the functional ramifica tions are of modification by oligosaccharide. The occurrence and function o f glycosylation of the V-1a vasopressin receptor (V1aR) has been investigat ed in this study. Using a combination of translation systems that are tithe r glycosylation-competent or do not support glycosylation, we established t hat of the four putative N-glycosylation sites at Asn(14), Asn(27), Asn(198 ) and Asn(333) only the first three sites are actually modified by carbohyd rate. This was confirmed by disruption of consensus sites by site-directed mutagenesis, individually and in combination. The V1aR is not O-glycosylate d. The Functionality of a series of glycosylation-defective V1aR constructs was characterized after expression in HEK 293T cells. It was found that ca rbohydrate moieties are not required for the receptor to bind any of the fo ur classes of ligand available, or for intracellular signalling. The glycos ylation status of the V1aR did, however, regulate the level of total recept or expression and also the abundance of receptor at the cell surface. Furth ermore, the nature of this regulation (increased or decreased expression) w as dictated by the locus of the oligosaccharide modification. Modification of any one of the consensus sites alone, however, was sufficient for wild-t ype expression, indicating a redundancy within the glycosylation sites. A r ole for the carbohydrate in the correct folding or stabilization of the V1a R is indicated. Glycosylation is not required, however, for efficient traff icking of the receptor to the cell surface. This study establishes the func tional importance of N-glycosylation of the V1aR.