Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-Al urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-Al phosphorylation at $486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed $486 in roughly the center of the sequence. We also developed two stably transfected mlMCD3 cell lines: one expressing wild-type UT-Al and one expressing a mutated form of UT-Al, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-Al-mIMCD3 cells but not in the S486A/S499A-UT-Al-mIMCD3 cells. The phospbo-S486-UT-Al antibody identified UT-Al protein in the wild-type UT-Al-mIMCD3 cells but not in the S486A/S499A-UT-Al-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospbo-S486-UT-Al (measured using the phospho-S486 antibody) and of total UT-Al phosphorylation (measured by 32p incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-Al in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-Al appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-Al and an S486-UT-Al phospho-specific antibody. We confirmed that vasopressin increases UT-Al accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-Al at $486 in rat IMCDs and that the S486-phospho-UT-Al form is primarily detected in the apical plasma membrane. urea transporter; phospho-specific; IMCD3; vasopressin; forskolin doi: 10.1152/ajprenal.00682.2009.