Loss of N-linked glycosylation reduces urea transporter UT-A1 response to vasopressin.
The vasopressin-regulated urea transporter (UT)-A1 is a transmembrane protein with two glycosylated forms of 97 and 117 kDa; both are derived from a single 88-kDa core protein. However, the precise molecular sites and the function for UT-A1 N-glycosylation are not known. In this study, we compared Madin-Darby canine kidney cells stably expressing wild-type (WT) UT-A1 to Madin-Darby canine kidney cell lines stably expressing mutant UT-A1 lacking one (A1m1, A1m2) or both glycosylation sites (m1m2). Site-directed mutagenesis revealed that UT-A1 has two glycosylation sites at Asn-279 and -742. Urea flux is stimulated by 10 nM vasopressin (AVP) or 10 microM forskolin (FSK) in WT cells. In contrast, m1m2 cells have a delayed and significantly reduced maximal urea flux. A 15-min treatment with AVP and FSK significantly increased UT-A1 cell surface expression in WT but not in m1m2 cells, as measured by biotinylation. We confirmed this finding using immunostaining. Membrane fractionation of the plasma membrane, Golgi, and endoplasmic reticulum revealed that AVP or FSK treatment increases UT-A1 abundance in both Golgi and plasma membrane compartments in WT but not in m1m2 cells. Pulse-chase experiments showed that UT-A1 half-life is reduced in m1m2 cells compared with WT cells. Our results suggest that mutation of the N-linked glycosylation sites reduces urea flux by reducing UT-A1 half-life and decreasing its accumulation in the apical plasma membrane. In vivo, inner medullary collecting duct cells may regulate urea uptake by altering UT-A1 glycosylation in response to AVP stimulation.
Chen
Guangping
G
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA. gchen3@emory.edu
Fröhlich
Otto
O
Yang
Yuan
Y
Klein
Janet D
JD
Sands
Jeff M
JM
D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism* / Q000502:physiology*; D011134:Polysaccharides / Q000737:chemistry; D014508:Urea / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology*; D000096982:Urea Transporters
D005576:Colforsin; D026901:Membrane Transport Proteins; D011134:Polysaccharides; D014508:Urea; D014667:Vasopressins; D000096982:Urea Transporters