Abstract 16723: Dopamine D3 Receptor Decreases NHE3 Expression and Function by Inhibiting the Activity of the De-Ubiquitinylating Enzyme, USP48, in Renal Proximal Tubule Cells
Disruption of the dopamine D3 receptor gene in mice (D3R-/-) results in high blood pressure, salt sensitivity, decreased ability to excrete a sodium load and increased renal expression of sodium/hydrogen exchanger isoform 3 (NHE3) protein (180%, P<0.05) but not mRNA, and D3R blockade in wild-type mice increased (2 fold, P<0.04) NHE3 protein expression. We hypothesized that renal D3R regulates NHE3 expression/function. In human renal proximal tubular cells (hRPTCs), the colocalization and co-immunoprecipitation of D3R and NHE3 are increased by D3R stimulation (PD 128907, 1μM), associated to internalization of D3R and NHE3 (confocal microscopy) and inhibition (80%, P<0.05) of NHE3 dependent Na+ transport. D3R stimulation also increases NHE3 ubiquitinylation and internalization of the ubiquitinylated NHE3 in the cytoplasm (150%, P<0.05) suggesting that D3R activation promotes NHE3 ubiquitinylation and internalization. To determine the mechanism of the D3R-mediated NHE3 ubiquitinylation we performed a yeast-two hybrid screening which showed that the 3rd intracytoplasmic loop of human D3R interacts with human ubiquitin-specific peptidase 48 (USP48), a de-ubiquitinylating enzyme. D3R and USP48, and USP48 and NHE3 co-localized and co-immunoprecipitated in hRPTCs. USP48 accounted for ~30% of hRPTCs' total de-ubiquitinylating activity. D3R stimulation increased the interaction between D3R and USP48 and resulted in a 35% reduction in USP48 de-ubiquitinylating activity, indicating that D3R inhibits USP48 activity in hRPTCs. USP48 silencing increased the degradation of NHE3 (vehicle t1/2 = 6.1 hr; USP48 siRNA t1/2 = 12.9 hr; P<0.05) while over-expressing USP48 increased NHE3 half -life (t1/2 = 21.8 hr; P<0.05 vs. vehicle) indicating that USP48 protects NHE3 from degradation via de-ubiquitinylation. Moreover, selective renal USP48 silencing in mice by subcapsular infusion of USP48 siRNA decreases NHE3 expression (25%, P<0.04) and systolic blood pressure measured under anesthesia (vehicle: 102±2 vs. siRNA 78±2 mmHg; P<0.03). Our data demonstrate that agonist activation of D3R promotes the degradation of NHE3 via the inhibition of USP48 and thus provide a novel mechanism by which the D3R engenders natriuresis and regulates blood pressure.
- © 2011 by American Heart Association, Inc.