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

Abstract

Inhibition of Na ؉ ,K ؉ -ATPase (NKA) activity in renal epithelial cells by activation of G protein-coupled receptors is mediated by phosphorylation of the catalytic ␣-subunit followed by endocytosis of active molecules. We examined whether agonists that counteract this effect do so by dephosphorylation of the ␣-subunit or by preventing its internalization through a direct interaction with the endocytic network. Oxymetazoline counteracted the action of dopamine on NKA activity, and this effect was achieved not by preventing ␣-subunit phosphorylation, but by impaired endocytosis of ␣-subunits into clathrin vesicles and early and late endosomes. Dopamine-induced inhibition of NKA activity and ␣-subunit endocytosis required the interaction of adaptor protein 2 (AP-2) with the catalytic ␣-subunit. Phosphorylation of the ␣-subunit is essential because dopamine failed to promote such interaction in cells lacking the protein kinase C phosphorylation residue (S18A). Confocal microscopy confirmed that oxymetazoline prevents incorporation of NKA molecules into clathrin vesicles by inhibiting the 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 the concentration of InsP 6 induced by oxymetazoline prevented AP-2 binding to the NKA ␣-subunit in response to dopamine. In conclusion, inhibition of NKA activity can be reversed by preventing its endocytosis without altering the state of ␣-subunit phosphorylation; increased InsP6 in response to G protein-coupled receptor signals blocks the recruitment of AP-2 and thereby clathrin-dependent endocytosis of NKA.

Relevance of Dopamine Signals Anchoring Dynamin-2 to the Plasma Membrane during Na+,K+-ATPase Endocytosis

“…The experimental protocols were carried out in isolated PCT from rat kidney (9) or in a cell line derived from opossum proximal tubules (OK cells) which expresses the DA receptor and utilizes the same signaling pathways as PCT during regulation of Na ϩ ,K ϩ -ATPase activity and endocytosis (31). For consistency with previous observations, all the experiments were performed at 23°C.…”

Genetics of arterial hypertension and hypotension

“…Catecholamines, especially dopamine, are involved in the regulation of Na + excretion during salt load or deprivation (Aperia 2000). Dopamine stimulates the phosphorylation of Na + /K + ATPase α-subunits in kidney tubules (Chibalin et al 1999) and activates PI3 kinases, the products of which, such as phosphatidyl-inositol-3-phosphate, are essential for the clathrin-dependent endocytosis of the Na + /K + ATPase by mediating the interaction of the adaptor protein adaptin (AP2) with a tyrosine motif in the Na + /K + ATPase α-subunit (Ogimoto et al 2000;Done et al 2002).…”

Analysis of Na+,K+-ATPase Motion and Incorporation into the Plasma Membrane in Response to G Protein–coupled Receptor Signals in Living Cells

“…The effect of dopamine on Na ϩ ,K ϩ -ATPase activity as well as the signaling mechanisms involved is similar to type-II cells isolated from rat lung (Guerrero et al, 2001;Ridge et al, 2002). Selection of stable clones of A549 cells expressing the GFP-tagged rat Na ϩ ,K ϩ -ATPase ␣ 1 -subunit was performed as described previously (Pedemonte et al, 1997;Ogimoto et al, 2000). LysoTracker Green DND-26 (LTG) was purchased from Molecular Probes (Eugene, OR).…”