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 rol e in the urinary concentrating mechanism. Previous immunoblotting studies i n rats have revealed that UT-A1 is present in kidney in 97- and 117-kDa mon omeric forms and that the relative abundance of the two forms is altered by vasopressin treatment and other treatments that altered urinary inner medu llary urea concentration. The present studies were carried out using protei n chemistry techniques to determine the origin of the two forms. Peptide-di rected polyclonal antibodies targeted to five sites along the polypeptide s equence 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 deglycosylat ion with N-glycosidase F, indicating that a single polypeptide chain is gly cosylated to two different extents. Studies using nonionic detergents for m embrane 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 t he 97- and 117-kDa proteins, as well as the 206-kDa complex, were immunopre cipitated with UT-A1 antibodies. We conclude that UT-A1 is a glycoprotein a nd that the two monomeric forms (97 and 117 kDa) in inner medullary collect ing duct are the consequence of different states of glycosylation.