Tyrosine 537 within the Na+,K+-ATPase α-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 α1-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 α1-subunit. We identified a conserved sequence (Y537LEL) within the α1-subunit that represents an AP-2 binding site. Binding of AP-2 to the Na+,K+-ATPase α1-subunit in response to dopamine (DA) was increased in OK cells stably expressing the wild type rodent α-subunit (OK-WT), but not in cells expressing the Y537A mutant (OK-Y537A). DA treatment was associated with increased α1-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 estersincrease 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.