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

Alexander V. Chibalin, Goichi Ogimoto, Carlos H. Pedemonte, Thomas A. Pressley, Adrian I. Katz, Eric Féraille, Per Olof Berggren, Alejandro M. Bertorello

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190 Scopus citations

Abstract

Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the α subunit, both events being protein kinase C-dependent. Studies of purified preparations, fusion proteins with site-directed mutagenesis, and heterologous expression systems have identified two major protein kinase C phosphorylation residues (Ser-11 and Ser-18) in the rat α1 subunit isoform. To identify 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 α1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased α subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type α1 subunit or the S11A α1 mutant, and both effects were blocked by protein kinase C inhibition. In contrast, dopamine did not elicit any of these effects in cells expressing the S18A α1 mutant. While Ser-18 phosphorylation is necessary for endocytosis, it does not affect per se the enzymatic activity: preventing endocytosis with wortmannin or LY294009 blocked the inhibitory effect of dopamine on Na+,K+-ATPase activity, although it did not alter the increased α subunit phosphorylation induced by this agonist. We conclude that dopamine-induced inhibition of Na+,K+-ATPase activity in rat renal tubule cells requires endocytosis of the α subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.

Original languageEnglish (US)
Pages (from-to)1920-1927
Number of pages8
JournalJournal of Biological Chemistry
Volume274
Issue number4
DOIs
StatePublished - Jan 22 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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