Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and is responsible for the decreasedactivity in epithelial cells

Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibi tion is associated with phosphorylation and internalization of the a subuni t, both events being protein kinase C-dependent, Studies of purified prepar ations, fusion proteins with site-directed mutagenesis, and heterologous ex pression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat alpha(1) subunit isoform. To ident ify the phosphorylation site(s) that mediates endocytosis of the subunit in response to dopamine, we have performed site-directed mutagenesis of these residues in the rat alpha(1) subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and i ncreased alpha subunit phosphorylation and clathrin-dependent endocytosis i nto endosomes in cells expressing the wild type alpha(1) subunit or the S11 A alpha(1) mutant, and both effects were blocked by protein kinase C inhibi tion. In contrast, dopamine did not elicit any of these effects in cells ex pressing the S18A alpha(1) mutant. While Ser-18 phosphorylation is necessar y for endocytosis, it does not affect per se the enzymatic activity: preven ting endocytosis with wort-mannin or LY294009 Mocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increa sed alpha subunit phosphorylation induced by this agonist. We conclude that dopamine-induced inhibition of Na+,K+-ATPase activity in r at renal tubule cells requires endocytosis of the alpha subunit into define d intracellular compartments and that phosphorylation of Ser-18 is essentia l for this process.