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Phosphorylation of Adaptor Protein–2 µ2 Is Essential for Na⁺,K⁺-ATPase Endocytosis in Response to Either G Protein–Coupled Receptor or Reactive Oxygen Species

Department of Medicine, Atherosclerosis Research Unit, Membrane Signaling Networks; Department of Molecular Medicine, Rolf Luft Center for Diabetes Research, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden; Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University; Department of Medicine, University of Chicago, Chicago, Illinois; and Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, Texas

Correspondence and requests for reprints should be addressed to Dr. Alejandro M. Bertorello, Membrane Signaling Networks, King Gustaf V Research Institute M1:01, Karolinska University Hospital-Solna, S-171 76 Stockholm, Sweden. E-mail: alejandro.bertorello{at}medks.ki.se

Activation of G protein–coupled receptor by dopamine and hypoxia-generated reactive oxygen species promote Na⁺,K⁺-ATPase endocytosis. This effect is clathrin dependent and involves the activation of protein kinase C (PKC)- and phosphorylation of the Na⁺,K⁺-ATPase -subunit. Because the incorporation of cargo into clathrin vesicles requires association with adaptor proteins, we studied whether phosphorylation of adaptor protein (AP)-2 plays a role in its binding to the Na⁺,K⁺-ATPase -subunit and thereby in its endocytosis. Dopamine induces a time-dependent phosphorylation of the AP-2 µ2 subunit. Using specific inhibitors and dominant-negative mutants, we establish that this effect was mediated by activation of the adaptor associated kinase 1/PKC- isoform. Expression of the AP-2 µ2 bearing a mutation in its phosphorylation site (T156A) prevented Na⁺,K⁺-ATPase endocytosis and changes in activity induced by dopamine. Similarly, in lung alveolar epithelial cells, hypoxia-induced endocytosis of Na⁺,K⁺-ATPase requires the binding of AP-2 to the tyrosine-based motif (Tyr-537) located in the Na⁺,K⁺-ATPase -subunit, and this effect requires phosphorylation of the AP-2 µ2 subunit. We conclude that phosphorylation of AP-2 µ2 subunit is essential for Na⁺,K⁺-ATPase endocytosis in response to a variety of signals, such as dopamine or reactive oxygen species.

Key Words: clathrin • dopamine • hypoxia • kidney tubule cells • lung alveolar cells

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