Dopamine-induced endocytosis of Na+,K+-ATPase is initiated by phosphorylation of Ser-18 in the rat alpha subunit and Is responsible for the decreased activity in epithelial cells.
Dopamine inhibits Na+,K+-ATPase activity in renal tubule cells. This inhibition is associated with phosphorylation and internalization of the alpha 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 alpha1 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 alpha1 subunit and expressed the mutated forms in a renal epithelial cell line. Dopamine inhibited Na+,K+-ATPase activity and increased alpha subunit phosphorylation and clathrin-dependent endocytosis into endosomes in cells expressing the wild type alpha1 subunit or the S11A alpha1 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 alpha1 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 alpha 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 alpha subunit into defined intracellular compartments and that phosphorylation of Ser-18 is essential for this process.
Chibalin
A V
AV
Department of Molecular Medicine, Karolinska Institutet, The Rolf Luft Center for Diabetes Research, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Ogimoto
G
G
Pedemonte
C H
CH
Pressley
T A
TA
Katz
A I
AI
Féraille
E
E
Berggren
P O
PO
Bertorello
A M
AM
D000818:Animals; D002384:Catalysis; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D004298:Dopamine / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D004847:Epithelial Cells / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002966:Clathrin; D012694:Serine; D000254:Sodium-Potassium-Exchanging ATPase; D004298:Dopamine