Protein phosphatase 2A subunit B55 alpha is required for angiotensin type 2 receptor elicited natriuresis

Angiotensin type 2 receptor (AT₂R) activation promotes natriuresis, thereby contributing to sodium balance and blood pressure regulation. In this study, we explored a novel intermediate in AT₂R signaling, protein phosphatase 2A (PP2A) regulatory subunit B55α. Probing for PP2A subunit-AT₂R interactions in vivo using proximity ligation assays on kidney sections prepared from rats after renal interstitial (RI) infusion of vehicle or the AT₂R agonist compound 21 (C21), we observed a sixfold increase in AT₂R-B55α interaction in apical brush border membranes of renal proximal tubule cells (RPTCs) with C21 stimulation. In vitro binding of purified AT₂R and B55α supported a direct interaction between these two proteins. To test whether B55α is required for renal AT₂R signaling, we administered siRNA targeting B55α to rats in vivo by RI infusion, which resulted in a ∼70% decrease in B55α in proximal but not distal tubules. Remarkably, RPTC B55α knockdown abolished C21-induced natriuresis and simultaneously prevented C21-mediated AT₂R redistribution to apical brush border membranes and sodium transporter Na⁺/H⁺ exchanger-3 (NHE-3) retrieval. Furthermore, B55α knockdown prevented cellular Src (c-Src) phosphorylation with C21 stimulation, increased AT₂R colocalization with lysosomal marker lysosomal-associated membrane protein 1 (LAMP1) by four to sixfold, and reduced AT₂R colocalization with early and late endosomal markers early endosome antigen 1 (EEA1) and Rab7 by 50%. In conclusion, our results show that RPTC PP2A B55α binds to activated AT₂R and is required for AT₂R signaling to natriuresis and AT₂R intracellular trafficking. We thus establish RPTC PP2A B55α as a key AT₂R signaling intermediate and potential therapeutic target to promote sodium excretion in hypertensive individuals.<b>NEW & NOTEWORTHY</b> Protein phosphatase 2A (PP2A) B55α is required for angiotensin type 2 receptor (AT₂R) natriuretic signaling and AT₂R intracellular trafficking in renal proximal tubule cells (RPTCs). RPTC PP2A B55α is thus a key AT₂R signaling intermediate and potential therapeutic target to promote sodium excretion in hypertensive individuals. This study introduces knocking down B55α in vivo specifically in RPTCs using renal interstitial infusion of siRNA as a novel and unique approach to investigate physiological protein function in the kidney.