Abstract 362: G Protein-Linked Receptor Kinase 4 is Palmitoylated and Acetylated and Regulates Subcellular Localization in Human Renal Proximal Tubule Cells
G protein-linked receptor kinase 4 (GRK4) is a kinase expressed in human renal proximal tubule cells (RPTC). Three single nucleotide polymorphisms in this gene are associated with hyper-phosphorylation and inactivation of the dopamine-1 receptor (D1R), and are associated with both hypertension and salt sensitivity. GRK4 is a prime therapeutic target for hypertension so we sought to discover potential post-translational modifications that could affect both localization and function. We identified two unique posttranslational modifications of GRK4 that we hypothesize are associated with protein localization. GRK4 is found at the plasma membrane (PM), in the cytoplasm and in the nucleus. We determined that GRK4 is both acetylated and palmitoylated. Using cell fractionation and immunoprecipitation, we determined that GRK4 was found in the nucleus, but acetylated GRK4 was only found in the cytoplasmic fraction using six different human RPTC cell lines (>10 fold n=6, p<0.01). We previously reported that PM GRK4 internalized following fenoldopam (FEN, dopamine agonist) stimulation, but the internalization only occurred in normally coupled human renal proximal tubule cells (nRPTC), not uncoupled cells (uRPTC). GRK4 is palmitoylated at the C-Terminal end of the protein (cystein 563, GGCPalmLTMVP). Using the palmitic acid tracer, 17-octadecynoic acid (17-ODYA) and Huisgen’s cycloaddition reaction (Click Reaction Chemistry) and biotin-azide for detection, we determined that GRK4 is depalmitoylated in a FEN (1μM, 30 min) and cAMP (forskolin, 10 μM, 30 min.) dependent manner. The amount of palmitoylated GRK4 was decreased following treatment with FEN (28.4%±3.6, n=3, p<0.01 vs VEH) or forskolin (35.3%±6.7, n=3, p<0.05 vs VEH) in nRPTC. No significant change in GRK4 palmitoylation was observed in uRPTC. The depalmitoylation of GRK4 correlates with its change in plasma membrane localization in both nRPTC and uRPTC. In conclusion we have determined for the first time that GRK4 is acetylated and the acetylated GRK4 is found only in the cytoplasmic fraction. In addition we have determined for the first time that GRK4 is depalmitoylated in nRPTC (not in uRPTC) when stimulated with FEN and this effect was mimicked by increasing intracellular cAMP using forskolin.
- © 2012 by American Heart Association, Inc.