Abstract 479: A novel Interaction Between G protein-Coupled Receptor Kinase 4 and Sorting Nexin 5 in the Regulation of Dopamine D1 Receptor Phosphorylation

Abstract

A dysfunction of the renal dopaminergic system is implicated in the pathogenesis of essential hypertension. Independent of renal nerves, the kidney synthesizes dopamine to regulate salt and water homeostasis by inhibiting ion transport in the nephron. The dopamine D 1 receptor (D 1 R) plays a pivotal role in regulating salt metabolism and blood pressure. The regulatory mechanisms or proteins involved in D 1 R trafficking are not completely understood, although our group has reported how G protein-coupled receptor kinase 4 (GRK4) promotes agonist-activated D 1 R desensitization and plasma membrane localization. We have recently identified the sorting nexin 5 (SNX5)_a protein involved in protein trafficking and a member of the mammalian retromer_as a novel binding partner of D 1 R using a yeast two-hybrid screen and have endeavored to elucidate how GRK4 and SNX5 cooperatively interact and regulate the D 1 R. Using a series of biological, biophysical and immunological approaches, we showed that GRK4 and SNX5 colocalize with the D 1 R and that these proteins co-segregate in lipid rafts in human renal proximal tubule cells (hRPTCs). Moreover, endogenous SNX5 co-immunoprecipitated with GRK4, but not with the β-arrestins, another class of proteins involved in D 1 R desensitization; treatment with the D1-like dopamine receptor agonist fenoldopam (1 uM, 15 min) enhanced this process 3-fold (n=3/group). We next determined the functional relevance of this interaction by silencing SNX5 and then evaluating the extent of agonist-activated D 1 R phosphorylation. In control cells that were transfected with non-silencing siRNA, fenoldopam increased D 1 R phosphorylation at 10, 20, and 30 min (137%, 140%, and 148%, respectively, compared to basal; P<0.5 vs. 0 min, n=3/group). Interestingly, SNX5 depletion resulted in 160% increase in phosphorylated D 1 R at the basal state and 145%, 170%, 178% and 200% increase after 5, 10, 20 and 30 min of agonist treatment (P<0.5 vs. 0 min, n=3/group). These indicate that SNX5 restricts the GRK4 from phosphorylating the receptor and may help explain why GRK4 is not involved in early phase of D 1 R desensitization. These data highlight the importance of SNX5 and its interaction with GRK4 in regulating D 1 R function in the control of blood pressure.