Down-regulation by extracellular ATP of rat hepatocyte organic anion transport is mediated by serine phosphorylation of oatp1

Recent studies implicate a role in hepatocyte organic anion transport of a plasma membrane protein that has been termed oatp1 (organic anion transport protein 1), Little is known regarding mechanisms by which its transport ac tivity is modulated in vivo, In previous studies (Campbell, C. G., Spray, D , C,, and Wolkoff, A W, (1993) J, BioL Chem. 268, 15399-15404), we demonstr ated that hepatocyte uptake of sulfobromophthalein was down-regulated by ex tracellular ATP, We have now found that extracellular ATP reduces the V-max for transport of sulfobromophthalein by rat hepatocytes; K-m remains unalt ered. Reduced transport also results from incubation of hepatocytes with th e phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the c ell surface after exposure of cells to ATP or phosphatase inhibitor, sugges ting that loss of transport activity is not caused by transporter internali zation. Exposure of P-32-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryp tic phosphopeptide; oatp1 from control cells is not phosphorylated, This ph osphopeptide comigrates with one of four phosphopeptides resulting from inc ubation of cells with okadaic acid. These studies indicate that the phospho rylation state of oatp1 must be an important consideration when assessing a lterations of its functional expression in pathobiological states.