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 alpha-D-mannose/alpha-D-glucose-specific lectin concanavalin A, (Con A), in contrast to the beta-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 micrograms/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 V2-receptor 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.
Jans
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DA
Max-Planck-Institut für Biophysik, Frankfurt am Main, FRG.
Jans
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P
Luzius
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Fahrenholz
F
F
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology*; D004848:Epithelium / Q000378:metabolism; D006031:Glycosylation; D007212:Indolizines / Q000494:pharmacology; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D011945:Receptors, Angiotensin / Q000096:biosynthesis / Q000378:metabolism*; D017483:Receptors, Vasopressin; D012276:Ricin; D013552:Swine; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D000262:Adenylyl Cyclases; D001127:Arginine Vasopressin; D003208:Concanavalin A; D007212:Indolizines; D011945:Receptors, Angiotensin; D017483:Receptors, Vasopressin; D012276:Ricin; D014415:Tunicamycin; C037806:castanospermine; D054836:Indolizidines