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 activity 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 demonstrated that hepatocyte uptake of sulfobromophthalein was down-regulated by extracellular ATP. We have now found that extracellular ATP reduces the V(max) for transport of sulfobromophthalein by rat hepatocytes; K(m) remains unaltered. Reduced transport also results from incubation of hepatocytes with the phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the cell surface after exposure of cells to ATP or phosphatase inhibitor, suggesting that loss of transport activity is not caused by transporter internalization. Exposure of (32)P-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryptic phosphopeptide; oatp1 from control cells is not phosphorylated. This phosphopeptide comigrates with one of four phosphopeptides resulting from incubation of cells with okadaic acid. These studies indicate that the phosphorylation state of oatp1 must be an important consideration when assessing alterations of its functional expression in pathobiological states.
Glavy
J S
JS
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wu
S M
SM
Wang
P J
PJ
Orr
G A
GA
Wolkoff
A W
AW
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D027321:Anion Transport Proteins; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D010768:Phosphoserine; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D013448:Sulfobromophthalein / Q000493:pharmacokinetics
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D010748:Phosphopeptides; D013448:Sulfobromophthalein; D010768:Phosphoserine; D019319:Okadaic Acid; C059041:calyculin A; D000255:Adenosine Triphosphate; D017027:Protein Tyrosine Phosphatases