2Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, 20 Penn St., HSF II, Ste. S003C, Baltimore, MD, USA 21201, Email:
FASEB J. 2014 Mar;28(3):1422-34. doi: 10.1096/fj.13-243840. Epub 2013 Dec 5.
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.
多巴胺 D3 受体（D3R）在调节血压和钠平衡中起着至关重要的作用，因为 Drd3 基因敲除小鼠会导致高血压和无法排泄膳食盐负荷。这些小鼠中肾钠潴留的机制在很大程度上尚不清楚。我们现在提供并描述了一种新的机制，即 D3R 通过抑制泛素特异性肽酶 48（USP48）的去泛素化活性，从而促进钠-氢交换体（NHE）-3 降解，从而长期抑制钠转运。我们发现，用 D3R 特异性激动剂 PD128907（1 μM，30 分钟）刺激可促进 D3R、NHE3 和 USP48 之间的相互作用和共定位；抑制 USP48 活性（-35±6%，与载体相比），导致 NHE3 的泛素化增加（+140±10%）；并减少人肾近端小管细胞（hRPTCs）中的 NHE3 表达（-50±9%）。USP48 沉默减少了 NHE3 的半衰期（USP48 siRNA t1/2=6.1 h，与载体 t1/2=12.9 h），而 USP48 的过表达增加了 NHE3 的半衰期（t1/2=21.8 h），表明 USP48 通过去泛素化保护 NHE3 免受降解。USP48 占这些细胞中总去泛素化活性的约 30%。在体内研究中，我们发现通过 D3R 特异性拮抗剂 GR103691（1 μg/kg/min，4 天）在 C57Bl/6J 小鼠中阻断 D3R 可增加肾 NHE3 的表达（+310±15%，与载体相比），而通过 siRNA 进行创新性的肾脏限制性 USP48 沉默（3 μg/d，7 天）可增加 NHE3 的泛素化（+250±30%，与对照相比），降低总 NHE3（-23±2%），降低血压（-24±2mmHg），与接受载体或非沉默 siRNA 的对照小鼠相比。我们的数据表明，D3R 和 USP48 之间的动态相互作用在调节 NHE3 表达和功能中起着至关重要的作用。
