97- and 117-kDa forms of collecting duct urea transporter UT-A1 are due to different states of glycosylation.
UT-A1 is an extremely hydrophobic 929-amino acid integral membrane protein, expressed in the renal inner medullary collecting duct, with a central role in the urinary concentrating mechanism. Previous immunoblotting studies in rats have revealed that UT-A1 is present in kidney in 97- and 117-kDa monomeric forms and that the relative abundance of the two forms is altered by vasopressin treatment and other treatments that altered urinary inner medullary urea concentration. The present studies were carried out using protein chemistry techniques to determine the origin of the two forms. Peptide-directed polyclonal antibodies targeted to five sites along the polypeptide sequence from the NH2 to the COOH terminus labeled both forms, thus failing to demonstrate a significant deletion in the primary amino acid chain. The 97- and 117-kDa monomeric forms were both reduced to 88 kDa by deglycosylation with N-glycosidase F, indicating that a single polypeptide chain is glycosylated to two different extents. Studies using nonionic detergents for membrane solubilization or using homobifunctional cross-linkers demonstrated that UT-A1 exists as a 206-kDa protein complex in native kidney membranes. The mobility of this complex was also increased by deglycosylation. Both the 97- and 117-kDa proteins, as well as the 206-kDa complex, were immunoprecipitated with UT-A1 antibodies. We conclude that UT-A1 is a glycoprotein and that the two monomeric forms (97 and 117 kDa) in inner medullary collecting duct are the consequence of different states of glycosylation.
Bradford
A D
AD
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Terris
J M
JM
Ecelbarger
C A
CA
Klein
J D
JD
Sands
J M
JM
Chou
C L
CL
Knepper
M A
MA
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004586:Electrophoresis; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D006596:Hexosaminidases / Q000494:pharmacology; D015151:Immunoblotting; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D026901:Membrane Transport Proteins; D011233:Precipitin Tests; D020033:Protein Isoforms / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D014667:Vasopressins / Q000494:pharmacology; D000096982:Urea Transporters
D000906:Antibodies; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D000939:Epitopes; D006596:Hexosaminidases; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D020033:Protein Isoforms; D014667:Vasopressins; D000096982:Urea Transporters