title: Tyrosine 537 within the Na+,K+-ATPase alpha-subunit is essential for AP-2 binding and clathrin-dependent endocytosis.
abstract: 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.
authors.affiliation: Department of Molecular Medicine, Karolinska Institutet, The Rolf Luft Centrum for Diabetes Research, Karolinska Hospital, 171 76 Stockholm, Sweden.
authors.forename: Stefania Cotta
authors.initials: SC
authors.lastname: Doné
authors.forename: Ingo B
authors.initials: IB
authors.lastname: Leibiger
authors.forename: Riad
authors.initials: R
authors.lastname: Efendiev
authors.forename: Adrian I
authors.initials: AI
authors.lastname: Katz
authors.forename: Barbara
authors.initials: B
authors.lastname: Leibiger
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Berggren
authors.forename: Carlos H
authors.initials: CH
authors.lastname: Pedemonte
authors.forename: Alejandro M
authors.initials: AM
authors.lastname: Bertorello
mesh_terms: 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
chemical_list: 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