Dopamine D3 receptor inhibits the ubiquitin‐specific peptidase 48 to promote NHE3 degradation

Abstract

The dopamine D3 receptor (D3R) is crucial in the regulation of blood pressure and sodium balance, in that Drd3 gene ablation in mice results in hypertension and failure to excrete a dietary salt load. The mechanism responsible for the renal sodium retention in these mice is largely unknown. We now offer and describe a novel mechanism by which D3R decreases sodium transport in the long term by inhibiting the deubiquitinylating activity of ubiquitin-specific peptidase 48 (USP48), thereby promoting Na(+)-H(+) exchanger (NHE)-3 degradation. We found that stimulation with the D3R-specific agonist PD128907 (1 μM, 30 min) promoted the interaction and colocalization among D3R, NHE3, and USP48; inhibited USP48 activity (-35±6%, vs. vehicle), resulting in increased ubiquitinylated NHE3 (+140±10%); and decreased NHE3 expression (-50±9%) in human renal proximal tubule cells (hRPTCs). USP48 silencing decreased NHE3's half-life (USP48 siRNA t1/2=6.1 h vs. vehicle t1/2=12.9 h), whereas overexpression of USP48 increased NHE3 half-life (t1/2=21.8 h), indicating that USP48 protects NHE3 from degradation via deubiquitinylation. USP48 accounted for ∼30% of the total deubiquitinylating activity in these cells. Extending our studies in vivo, we found that pharmacologic blockade of D3R via the D3R-specific antagonist GR103691 (1 μg/kg/min, 4 d) in C57Bl/6J mice increased renal NHE3 expression (+310±15%, vs. vehicle), whereas an innovative kidney-restricted Usp48 silencing via siRNA (3 μg/d, 7 d) increased ubiquitinylated NHE3 (+250±30%, vs. controls), decreased total NHE3 (-23±2%), and lowered blood pressure (-24±2 mm Hg), compared with that in control mice that received either the vehicle or nonsilencing siRNA. Our data demonstrate a crucial role for the dynamic interaction between D3R and USP48 in the regulation of NHE3 expression and function.

Dopamine D3Receptor Antagonist (GSK598809) Potentiates the Hypertensive Effects of Cocaine in Conscious, Freely-Moving Dogs

“…cells (Zeng et al, 2008;Yu et al, 2009). Recent evidence suggests dopamine D 3 receptor activation in the kidneys ultimately stimulates secretion of a sodium load (to reduce hypertension) by promoting degradation of the Na 1 -H 1 exchanger in renal proximal tubules (Armando et al, 2014). The pressor effect of the dopamine D 3 receptor antagonist in this study and its augmentation of cocaine's pressor effects are consistent with this body of evidence.…”

The deubiquitinating enzyme USP48 stabilizes TRAF2 and reduces E‐cadherin‐mediated adherens junctions

“…However, the physiologic function of USP48 remains largely unknown. One recent study demonstrates that USP48 deubiquitinates the Na + -H + exchanger (NHE3) and plays a role in blood pressure regulation (47). Tzimas et al (31) have shown that USP48 is involved in the inhibition of NF-kB activation, a theme also presented in the study by Schweitzer and Naumann (32) in which the COP9 signalosome (CSN) and USP48 cooperatively control the nuclear turnover of RelA.…”

Unique Regulation of Human Na+/H+ Exchanger 3 (NHE3) by Nedd4-2 Ligase That Differs from Non-primate NHE3s

“…However, "cycloheximide chase" has an intrinsic limitation on determining degradation of long-lived proteins due to the potential effects of prolonged protein synthesis inhibition on overall cell function (40), and hence, we cannot completely rule out the role of ubiquitination in hNHE3 degradation based on the current results. While this work was being pursued, Armando et al (41) reported that dopamine inhibited NHE3 in hRPTC human renal proximal tubule cells by inhibition of the deubiquitinating activity of Ub-specific ligase 48, USP48. Inhibition of USB48, which removes Ub from NHE3, decreased the half-life of NHE3 and enhanced NHE3 degradation.…”