Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis.
In renal epithelial cells endocytosis of Na(+),K(+)-ATPase molecules is initiated by phosphorylation of its alpha(1)-subunit, leading to activation of phosphoinositide 3-kinase and adaptor protein-2 (AP-2)/clathrin recruitment. The present study was performed to establish the identity of the AP-2 recognition domain(s) within the Na(+),K(+)-ATPase alpha(1)-subunit. We identified a conserved sequence (Y(537)LEL) within the alpha(1)-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na(+),K(+)-ATPase alpha(1)-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent alpha-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased alpha(1)-subunit abundance in clathrin vesicles from OK-WT but not from OK-Y537A cells. In addition, this mutation also impaired the ability of DA to inhibit Na(+),K(+)-ATPase activity. Because phorbol esters increase Na(+),K(+)-ATPase activity in OK cells, and this effect was not affected by the Y537A mutation, the present results suggest that the identified motif is specifically required for DA-induced AP-2 binding and Na(+),K(+)-ATPase endocytosis.
Doné
Stefania Cotta
SC
Department of Molecular Medicine, Karolinska Institutet, The Rolf Luft Centrum for Diabetes Research, Karolinska Hospital, 171 76 Stockholm, Sweden.
Leibiger
Ingo B
IB
Efendiev
Riad
R
Katz
Adrian I
AI
Leibiger
Barbara
B
Berggren
Per-Olof
PO
Pedemonte
Carlos H
CH
Bertorello
Alejandro M
AM
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D004298:Dopamine / Q000494:pharmacology; D004705:Endocytosis; D008565:Membrane Proteins / Q000378:metabolism*; D018613:Microscopy, Confocal; D008956:Models, Chemical; D009154:Mutation; D009893:Opossums; D010703:Phorbol Esters / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D002966:Clathrin; D008565:Membrane Proteins; D010703:Phorbol Esters; D014443:Tyrosine; D000254:Sodium-Potassium-Exchanging ATPase; D004298:Dopamine