GLYCOSYLATION IS NOT ESSENTIAL FOR VASOPRESSIN-DEPENDENT ROUTING OF AQUAPORIN-2 IN TRANSFECTED MADIN-DARBY, CANINE KIDNEY-CELLS

Glycosylation has been shown to be important for propel routing and me mbrane insertion of a number of proteins. In the collecting duct, aqua porin-2 (AQP2) is inserted into the apical membrane after stimulation of vasopressin type-2 receptors and retrieved into an endosomal compar tment after withdrawal of vasopressin. The extent of glycosylation of AQP2 in human kidney and urine and the effects of deglycosylation on r outing of AQP2 in an AQP2-transfected Madin-Darby canine kidney cell l ine (clone WT10) were investigated. Semiquantitative immunoblotting of human kidney membranes and urine showed an AQP2 glycosylation of 35 t o 45% for medulla, papilla, and urine, with low Variation among indivi duals. The 1-desamino-8-D-arginine vasopressin-induced transcellular o smotic water permeability (P-f) of WT10 cells by a factor of 2.6 +/- 0 .2 was reduced to 1.5 +/- 0.1 after pretreatment with the glycosylatio n inhibitor tunicamycin. However, when WT10 cells were incubated with 8-br-cAMP, the P-f increased by a factor 2.8 +/- 0.2 and by 2.9 +/- 0. 2 after prior incubation with tunicamycin. Immunoblot analyses reveale d that in WT10 cells, 34% of AQP2 is glycosylated, which was reduced t o 2% after tunicamycin treatment. Surface biotinylation and subsequent semiquantitative immunoblotting revealed that stimulation by cAMP inc reased the level of AQP2 in the apical membrane of WT10 cells 1.5-fold , independent of the presence of tunicamycin. However, in tunicamycin- treated WT10 cells, all AQP2 in the apical membrane was unglycosylated , whereas in untreated cells 30% of AQP2 in the apical membrane was gl ycosylated. These results prove that glycosylation has no function in the routing of AQP2 in Madin-Darby canine kidney cells.