G protein-coupled receptors regulate Na+,K+-ATPase activity and endocytosis by modulating the recruitment of adaptor protein 2 and clathrin

Inhibition of Na+,K+-ATPase (NKA) activity in renal epithelial cells by act ivation of G protein-coupled receptors is mediated by phosphorylation of th e catalytic alpha-subunit followed by endocytosis of active molecules, We e xamined whether agonists that counteract this effect do so by dephosphoryla tion of the alpha-subunit or by preventing its internalization through a di rect interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by pr eventing alpha-subunit phosphorylation, but by impaired endocytosis of alph a-subunits into clathrin vesicles and early and late endosomes, Dopamine-in duced inhibition of NKA activity and alpha-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic alpha-subunit, Phosphorylation of the alpha-subunit is essential because dopamine failed t o promote such interaction in cells racking the protein kinase C phosphoryl ation residue (S18A). Confocal microscopy confirmed that oxymetazoline prev ents incorporation of NKA molecules into clathrin Vesicles by inhibiting th e ability of dopamine to recruit clathrin to the plasma membrane. Dopamine decreased the basal levels of inositol hexakisphosphate (InsP(6)), whereas oxymetazoline prevented this effect. Similar increments (above basal) in th e concentration of InsP(6) induced by oxymetazoline prevented AP-2 binding to the NKA alpha-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without alte ring the state of alpha-subunit phosphorylation; increased InsP(6) in respo nse to G protein-coupled receptor signals blocks the recruitment of AP-2 an d thereby clathrin-dependent endocytosis of NKA.