Most of the large family of G-protein-coupled receptors (GPCRs) possess putative N-glycosylation sites within their N-termini. However, for the vast majority of GPCRs, it has not been determined which, if any, of the consensus glycosylation sites are actually utilized or what the functional ramifications are of modification by oligosaccharide. The occurrence and function of glycosylation of the V1a vasopressin receptor (V1aR) has been investigated in this study. Using a combination of translation systems that are either glycosylation-competent or do not support glycosylation, we established that of the four putative N-glycosylation sites at Asn14, Asn27, Asn198 and Asn333 only the first three sites are actually modified by carbohydrate. This was confirmed by disruption of consensus sites by site-directed mutagenesis, individually and in combination. The V1aR is not O-glycosylated. The functionality of a series of glycosylation-defective V1aR constructs was characterized after expression in HEK 293T cells. It was found that carbohydrate moieties are not required for the receptor to bind any of the four classes of ligand available, or for intracellular signalling. The glycosylation status of the V1aR did, however, regulate the level of total receptor expression and also the abundance of receptor at the cell surface. Furthermore, the nature of this regulation (increased or decreased expression) was dictated by the locus of the oligosaccharide modification. Modification of any one of the consensus sites alone, however, was sufficient for wild-type expression, indicating a redundancy within the glycosylation sites. A role for the carbohydrate in the correct folding or stabilization of the V1aR is indicated. Glycosylation is not required, however, for efficient trafficking of the receptor to the cell surface. This study establishes the functional importance of N-glycosylation of the V1aR.
Abbreviations used: AVP, [arginine8]vasopressin; GPCR, G-protein-coupled receptor; OTR, oxytocin receptor; PhAc, phenylacetyl; VPR, vasopressin receptor; V1aR, V1a vasopressin receptor; rV1aR, rat V1aR; V1bR, V1b vasopressin receptor; V2R, V2 vasopressin receptor; ACTH, adrenocorticotrophic hormone; GnRH, gonadotropin-releasing hormone; VIP-1, vasoactive intestinal peptide; DMEM, Dulbecco's modified Eagle's medium; NYT, acetyl-Asn-Tyr-Thr-amide.