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.

Renal proximal tubule Na,K-ATPase is controlled by CREB-regulated transcriptional coactivators as well as salt-inducible kinase 1

“…Dopamine was observed to cause a reduction in the level of basolateral Na,K-ATPase, presumably as a consequence of the phosphorylation of the α subunit of Na,K-ATPase by Protein Kinase C (PKC) ζ, and endocytosis [11]. Alpha-adrenergic agonists counteracted these affects of dopamine, by preventing the recruitment of clathrin to the plasma membrane, rather than by preventing α subunit phosphorylation [58]. …”

Inhibition of Activity of GABA Transporter GAT1 byδ-Opioid Receptor

“…Mechanisms have been discussed that protein-protein interaction like interaction with syntaxin A or other adaptor proteins modulates the GABA transporter [63] that may be modulated by PKC [64]. Also other transport proteins, channels, G-protein-coupled receptors, and cytoplasmic proteins [65–68] seem to be affected by protein-protein interactions. …”

Targeting of renal proximal tubule Na,K-ATPase by salt-inducible kinase

“…In contrast, phosphorylation of Thr-81 by ERK1/2 activates the pump. The α subunit also contains a recognition site for Adapter Protein-2 (AP-2) [16], and a PKA phosphorylation site (Fig. 1B).…”

“…In order to exert its inhibitory effect, dopamine activates PKCς, which phosphorylates the Na,K-ATPase α1 subunit (at Ser-18) [16]. The 14-3-3 protein binds to this phospho-Ser motif, which facilitates the binding of PI3K to an adjacent Proline-Rich Domain in α1, resulting in PI3K activation.…”

“…Dephosphorylated Na,K-ATPase has a low affinity for both PI3K and AP-2. However, in the presence of dopamine, AP-2 is phosphorylated by PKCς, and acquires an increased affinity for the basolateral α1 subunit (AP-2 binds to Tyr-537 in the M4/M5 loop) (Figure 1B) [16]. After phosphorylated AP-2 associates with the Na,K-ATPase, AP-2 recruits clathrin to the complex.…”