Abstract 532: GRK4 Palmitoylation is Necessary for Membrane Association and Dopamine-1 Receptor Activity
The renal dopaminergic system regulates a large percentage of renal sodium excretion under sodium loaded conditions; however, not much is known about intracellular dopaminergic regulatory mechanisms. The expression of the principal sodium regulatory dopaminergic receptor (the D1R) is negatively modulated by the G protein linked receptor kinase type 4 (GRK4) through phosphorylation while the D1R is expressed in the plasma membrane. Therefore, we hypothesized that mechanisms associated with GRK4 membrane localization might be associated with its activity. Using human renal proximal tubule cell lines (RPTC), we previously demonstrated that GRK4 is palmitoylated using 17-octadecynoic acid (17-ODYA) and click chemistry and that fenoldopam (FEN, D1R agonist) decreased palmitoylation only in normal cAMP-coupled RPTC. Here we expand on those studies by showing that GRK4 palmitoylation was inhibited by 2-bromopalmitate (2BP, 100 μmol/L, palmitoylation inhibitor) which shifted GRK4 expression from the membrane to the cytoplasm. We used two different techniques to show loss of GRK4 membrane association when GRK4 palmitoylation was inhibited. The first technique involved classic cell fractionation using ultracentrifugation and western blot identification of GRK4 in membrane protein fractions (VEH 0.174±0.023 RFU, 2BP 0.091±0.013 RFU, P<0.05, N=3) The second technique utilized a novel membrane-associated fluorescent linked immunosorbent assay (mFLISA) to demonstrate the decrease in GRK4 membrane association (VEH 13,568±664 RFU, 2BP 5,155±974 RFU, P<0.05, N=12). Additionally, we showed that inhibiting GRK4 palmitoylation ledto D1-like plasma membrane recruitment using a non-cell permeable fluorescent D1-like antagonist bodipy630 SKF83566 (VEH 58,242±3858 RFU, 2BP 80,153±4061 RFU, P<0.01, N=6). In summary, we demonstrated a novel D1R regulatory mechanism through GRK4 palmitoylation, affecting its localization and activity.
Author Disclosures: J.J. Gildea: None. Z. Huang: None. D. Bigler Wang: None. H. Tran: None. R.A. Felder: None.
- © 2014 by American Heart Association, Inc.