title: Insulin causes renal dopamine D1 receptor desensitization via GRK2-mediated receptor phosphorylation involving phosphatidylinositol 3-kinase and protein kinase C.
abstract: The renal dopamine system plays an important role in sodium homeostasis and a defect in dopamine D1 receptor (D1R) function is present in hypertension, diabetes, and aging. Our previous studies in hyperinsulinemic animals and in renal cell cultures treated with insulin showed decrease in D1R number and defective coupling to G proteins; however, the exact mechanisms remained unknown. Therefore, we investigated insulin-mediated D1R desensitization and underlying molecular mechanism in opossum kidney (OK) cells. Chronic exposure (24 h) of OK cells to 10 nM insulin caused significant decrease in D1R number and agonist affinity. The D1R was hyperserine phosphorylated, uncoupled from G proteins and SKF38393, a D1R agonist, failed to stimulate G proteins and inhibit Na-K-ATPase activity. Insulin increased protein kinase C (PKC) activity and caused G protein-coupled receptor kinase 2 (GRK2) translocation to the membranes. Tyrosine kinase inhibitor genistein and phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked insulin-mediated PKC activation and GRK2 membranous translocation. In addition to genistein and wortmannin, GRK2 membranous tranlocation was also blocked by PKC inhibitor chelerythrine chloride and GRK2-specific siRNA. Genistein, wortmannin, chelerythrine chloride, and GRK2 siRNA abrogated D1R serine phosphorylation and normalized D1R expression and affinity in insulin-treated cells. Furthermore, these inhibitors and siRNA restored D1R G protein coupling and ability of SKF38393 to inhibit Na-K-ATPase activity. In conclusion, insulin-induced D1R desensitization involves PI3K, PKC, and GRK2. Insulin activates PI3K-PKC-GRK2 cascade, causing D1R serine phosphorylation, which leads to D1R downregulation and uncoupling from G proteins, and results in the failure of SKF38393 to stimulate G proteins and inhibit Na-K-ATPase activity.
authors.affiliation: Heart and Kidney Institute, College of Pharmacy, University of Houston, 4800 Calhoun Rd, S & R-2 Bldg., Houston, TX 77204, USA.
authors.forename: Anees Ahmad
authors.initials: AA
authors.lastname: Banday
authors.forename: Fatima Rizwan
authors.initials: FR
authors.lastname: Fazili
authors.forename: Mustafa F
authors.initials: MF
authors.lastname: Lokhandwala
mesh_terms: D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D007328:Insulin / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology; D009893:Opossums; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D017447:Receptors, Dopamine D1 / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases / Q000378:metabolism*
chemical_list: D007328:Insulin; D017447:Receptors, Dopamine D1; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D051552:beta-Adrenergic Receptor Kinases