title: 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.
authors.affiliation: 2Division of Nephrology, Department of Medicine, University of Maryland School of Medicine, 20 Penn St., HSF II, Ste. S003C, Baltimore, MD, USA 21201, Email: vvillar@medicine.umaryland.edu.
authors.forename: Ines
authors.initials: I
authors.lastname: Armando
authors.forename: Van Anthony M
authors.initials: VA
authors.lastname: Villar
authors.forename: John E
authors.initials: JE
authors.lastname: Jones
authors.forename: Hewang
authors.initials: H
authors.lastname: Lee
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Wang
authors.forename: Laureano D
authors.initials: LD
authors.lastname: Asico
authors.forename: Peiying
authors.initials: P
authors.lastname: Yu
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
authors.forename: Crisanto S
authors.initials: CS
authors.lastname: Escano
authors.forename: Annabelle M
authors.initials: AM
authors.lastname: Pascua-Crusan
authors.forename: Robin A
authors.initials: RA
authors.lastname: Felder
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Jose
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D010450:Endopeptidases / Q000502:physiology*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000502:physiology; D016133:Polymerase Chain Reaction; D059748:Proteolysis; D050637:Receptors, Dopamine D3 / Q000502:physiology*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D017931:DNA Primers; D050637:Receptors, Dopamine D3; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D010450:Endopeptidases; C091939:ubiquitin isopeptidase
keywords: C57Bl/6 mice; blood pressure; kidney; renal proximal tubule cells