N-glycosylation plays a role in biosynthesis and internalization of the adenylate cyclase stimulating vasopressin V₂-receptor of LLC-PK₁ renal epithelial cells: An effect of concanavalin A on binding and expression

The role of N-glycosylation in the function and biosynthesis of the vasopressin V₂-receptor in LLC-PK₁ renal epithelial cells was examined using various lectins and inhibitors operating at different steps of the glycosidic pathway. Tunicamycin, which blocks all N-glycosylation, and castanospermine, which inhibits glycosidase I and hence blocks formation of high-mannose-type N-glycosylated intermediates, resembled one another in affecting V₂-receptor biosynthesis and internalization in a concentration-dependent manner. In contrast, swainsonine, an inhibitor of mannosidase II and hence of complex-type oligosaccharide formation, had no effect. Interestingly, the α-d-mannose/α-d-glucose-specific lectin concanavalin A, (Con A), in contrast to the β-d-galactose-specific lectin ricin, had a marked effect on the V₂-receptor in LLC-PK₁ cells, increasing both receptor numbers up to twofold in vivo and specific [³H]AVP binding up to 50% in vitro in a concentration-dependent manner. The concentrations inducing half-maximal response were about 0.2 and 20 gmg/ml for the in vivo and in vitro responses, respectively, implying distinct effects on V₂-expression and ligand binding. That the in vitro effect on binding was due to a direct effect on the V₂-receptor could be shown by the lack of a Con A effect on [³H]AVP binding in membranes prepared from LLC-PK₁ cells down-regulated for the V₂receptor or from cells of the LLC-PK₁ V₂-receptor deficient mutant M18. All results were consistent with a functional role for N-glycosylation of the V₂-receptor in LLC-PK₁ cells.