MUTAGENESIS OF N-GLYCOSYLATION SITES IN THE HUMAN VASOACTIVE-INTESTINAL-PEPTIDE-1 RECEPTOR - EVIDENCE THAT ASPARAGINE-58 OR ASPARAGINE-69 IS CRUCIAL FOR CORRECT DELIVERY OF THE RECEPTOR TO PLASMA-MEMBRANE

The functional role of N-linked carbohydrates in the human vasoactive intestinal peptide (VIP) 1 receptor was investigated by site-directed mutagenesis (Asn-->Thr) of the four consensus N-glycosylation sites on Asn(58), Asn(69), Asn(100) (N-terminal extracellular domain) and Asn( 293) (second extracellular loop). Mutated receptors were investigated after transient expression in Cos-7 cells, by ligand binding assay, af finity cross-linking, western blotting, and confocal laser microscopy of epitope-tagged receptor proteins. Mutations of each consensus site revealed that Asn(58), Asn(69), and Asn(100) were occupied by a 9-kDa N-linked carbohydrate whereas Asn(293) was not used for glycosylation. Each mutated receptor was expressed (western blot) and delivered at t he plasma membrane (confocal microscopy) of Cos-7 cells. They displaye d a dissociation constant similar to that of the wild-type receptor, i .e., 0.5-1 nM. In contrast, no VIP binding to Cos-7 cells could be obs erved with the mutant devoid of consensus N-glycosylation sites due to a strict sequestration of this mutant in the perinuclear endoplasmic reticulum. However, when solubilized with a zwitterionic detergent, th is mutant bound [I-125]VIP specifically, indicating that it retained i ntrinsic binding activity. The construction of other mutants in which three out of four N-glycosylation sites were altered, demonstrated tha t N-glycosylation at either Asn(58) or Asn(69) is necessary and suffic ient to ensure correct delivery of the receptor to the plasma membrane . Further pharmacological studies involving incubation of Cos-7 cells with castanospermine or deoxymannojirimycin immediately after transfec tion of mutated cDNAs encoding receptors with a single glycosylation s ite at Asn(58) or Asn(69) suggested that carbohydrate at Asn(58) was i nvolved in a calnexin-dependent folding process of the receptor wherea s carbohydrate at Asn(69) was not. These studies highlight the functio nal importance of the N-glycosylation of the human VIP 1 receptor whic h belongs to a new subfamily of seven membrane-spanning receptors.