Activation of GPCR by dopamine as well as hypoxia- generated reactive oxygen species promote Na⁺,K⁺-ATPase endocytosis. This effect is clathrin dependent and involves the activation of PKC- and phosphorylation of the Na⁺,K⁺-ATPase -subunit. Because incorporation of cargo into clathrin vesicles required its association with adaptor proteins, we studied whether phosphorylation of adaptor protein-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 adaptor protein -2 µ2 subunit. Using specific inhibitors and dominant negative mutants it was established that this effect was mediated by activation of the AAK1/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. It is therefore concluded that phosphorylation of AP-2 µ2 subunit is essential for Na⁺,K⁺-ATPase endocytosis in response to a variety of signals, such as dopamine and reactive oxygen species.