Premium
Tandem Requirement for Renal D 1 R and D 5 R Functionality
Author(s) -
Rozyyev Selim,
Crusan Annabelle P,
Tiu Andrew C,
Jurgens Julie A,
Quion Justin Michael B,
Asico Laureano D,
Felder Robin A,
Jose Pedro A,
Villar Van Anthony M
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.862.18
Subject(s) - receptor , agonist , colocalization , hek 293 cells , fenoldopam , g protein coupled receptor , nephron , chemistry , lipid raft , kidney , microbiology and biotechnology , medicine , transfection , endocrinology , biology , biophysics , biochemistry , gene
The peripheral dopaminergic system assists in maintaining blood pressure homeostasis by engendering natriuresis mainly through the D 1 R and D 5 R receptors. These receptors share structural features and pharmacological profiles; there is no specific agonist that can discriminate between these two receptors. Genetic ablation of one receptor in the kidney results in the loss of activity of the other. Therefore, we evaluated the mechanisms for the cooperative and co‐dependent interaction between these two receptors. Both receptors share the same anatomical distribution, specifically at the brush border and luminal membranes of renal proximal tubules and collecting ducts, respectively. These receptors dimerized (~110 kDa) and coimmunoprecipitated each other, basally and after fenoldopam (D 1 R/D 5 R agonist, 1 μM/15 min) treatment. Bimolecular fluorescence complementation showed that heterologously expressed D 1 R and D 5 R tagged with the C and N termini of the fluorescent protein EYFP, respectively, heterodimerized basally in HEK‐293 cells. This was corroborated by confocal microscopy of HEK‐293 cells that were transiently transfected with D 1 R::RFP and D 5 R::GFP, which showed colocalization basally at the plasma membrane, most abundantly at the apical membrane in polarized cells. The D 1 R and D 5 R partitioned to lipid and non‐lipid rafts; agonist treatment enhanced the lipid raft segregation of D 1 R and D 5 R. Both receptors were abundantly expressed in renal proximal tubules of human and C57Bl/6 mouse kidneys, where they colocalized basally that was increased by fenoldopam (1 μM/15 min). D 1 R/D 5 R stimulation is known to increase cAMP production and inhibit renal sodium transport. Receptor gene silencing via antisense technology in human renal proximal tubule cells (hRPTCs) decreased the cAMP response to fenoldopam (−71±2% for D 1 R, vs . −38±7% for D 5 R, vs . −90±2% for both, *P<0.05). In hRPTCs grown in Transwells, fenoldopam or ouabain (500 nM/30 min) given at the basolateral compartment inhibited Na + /K + ‐ATPase (NKA) activity and increased intracellular Na + . siRNA‐mediated gene silencing of DRD1 / DRD5 prevented the inhibition of NKA activity by fenoldopam (*P<0.05). Our observations demonstrate the requirement for D 1 R/D 5 R to form heterodimers to achieve their full functionality. Support or Funding Information R01DK039308, R01HL092196, and P01HL074940 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .