Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis.
Clathrin-dependent endocytosis of Na(+),K(+)-ATPase in response to dopamine regulates its catalytic activity in intact cells. Because fission of clathrin-coated pits requires dynamin, we examined the mechanisms by which dopamine receptor signals promote dynamin-2 recruitment and assembly at the site of Na(+),K(+)-ATPase endocytosis. Western blotting revealed that dopamine increased the association of dynamin-2 with the plasma membrane and with phosphatidylinositol 3-kinase. Dopamine inhibited Na(+),K(+)-ATPase activity in OK cells and in those overexpressing wild type dynamin-2 but not in cells expressing a dominant-negative mutant. Dephosphorylation of dynamin is important for its assembly. Dopamine increased protein phosphatase 2A activity and dephosphorylated dynamin-2. In cells expressing a dominant-negative mutant of protein phosphatase 2A, dopamine failed to dephosphorylate dynamin-2 and to reduce Na(+),K(+)-ATPase activity. Dynamin-2 is phosphorylated at Ser(848), and expression of the S848A mutant significantly blocked the inhibitory effect of dopamine. These results demonstrate a distinct signaling network originating from the dopamine receptor that regulates the state of dynamin-2 phosphorylation and that promotes its location (by interaction with phosphatidylinositol 3-kinase) at the site of Na(+),K(+)-ATPase endocytosis.
Department of Molecular Medicine, The Rolf Luft Center for Diabetes Research, Karolinska Institutet, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Riad
R
Efendiev
Guillermo A
GA
Yudowski
Jean
J
Zwiller
Barbara
B
Leibiger
Adrian I
AI
Katz
Per-Olof
PO
Berggren
Carlos H
CH
Pedemonte
Ingo B
IB
Leibiger
Alejandro M
AM
Bertorello
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism*; D034281:Dynamins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D002966:Clathrin; D004298:Dopamine; D034285:Dynamin II; D034281:Dynamins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D000254:Sodium-Potassium-Exchanging ATPase