Keller, S. R.; Gildea, J. J.; LI, J.; Kemp, B. A.; Conaway, M.; Brautigan, D. L.; Carey, R. M.; Howell, N.

Background: Angiotensin type 2 receptor (AT2R) activation promotes natriuresis in renal proximal tubule cells (RPTCs) counteracting sodium retention stimulated by the AT1R. Early signaling events mediating the natriuretic response with AT2R activation in RPTCs are currently unknown. Our previous research suggested protein phosphatase 2A (PP2A) functions downstream of the AT2R. In this study, we investigated the interaction of PP2A regulatory subunit B55 with the AT2R and requirement for B55 in AT2R signaling and natriuresis. Methods and Results: Rats were subjected to renal interstitial (RI) infusion of vehicle or Compound 21 (C21), a non-peptide specific AT2R agonist, and kidney sections were probed for interactions between PP2A subunits and the AT2R using a proximity ligation assay. A dramatic 6-fold increase in AT2R-B55 interaction in apical brush border membranes of RPTCs was observed with C21 stimulation. In vitro binding of purified AT2R and B55 supported a direct interaction between these two proteins. To determine the requirement for B55 in renal AT2R signaling, siRNA targeting B55 was administered to rats in vivo by RI infusion which resulted in a ~70% decrease in B55 in RPTCs but not distal tubules. In rats with B55 knockdown in RPTCs, natriuresis in response to C21 was abolished. Simultaneously, C21-elicited AT2R redistribution to and sodium transporter Na+/H+ exchanger-3 (NHE3) retrieval from apical brush border membranes was lost, as observed with confocal immunofluorescence microscopy. Consistent with impaired AT2R signaling, B55 knockdown prevented c-Src phosphorylation in response to C21. B55 knockdown also led to a dramatic 4 to 6-fold increase in AT2R co-localization with the lysosomal marker LAMP1, and a 50% reduction in AT2R co-localization with EEA1 and Rab 7, markers for early and late endosomes, respectively. Conclusions: PP2A B55 directly binds to the activated AT2R and is required for AT2R-elicited natriuresis, AT2R signaling and intracellular trafficking in RPTCs.