G-protein coupled receptor kinase 2 (GRK2) regulates nuclear factor of activated T-cells (NFAT) nuclear activity via glycogen synthase kinase 3β (GSK3β).

<p>(A, B) G-protein coupled receptor (GPCR) induced nuclear NFAT activity is decreased by GRK2 knockdown. (A) Representative immunofluorescence images of neonatal rat ventricular cardiac myocytes (NRVMs) under respective treatment conditions; NFAT subunits are marked using a proximity ligation assay (respective antibodies against each NFAT c1 and c4) resulting in green fluorescence, α-Actinin (red), DAPI (blue). (B) Transcriptional NFAT activity was evaluated using the NFAT-luciferase-reporter assay system, n = 3–4, * P< 0.05; ** P < 0.01. (C-G) GPCR stimulation results in increased GSK3β phosphorylation mediated by GRK2. (C) Representative Western Blots of GSK3β and phosphorylated GSK3β (pGSK3β) under respective treatment conditions, GAPDH served as loading control. Samples shown on each lane are blotted, antibody stained and developed on the same western blot membrane. Vertical dotted line indicates 2 excluded samples with non ANG II/PE GPCR stimulation. Quantification of GSK3β phosphorylation and total GSK3β protein under PE (D, n = 4) and ANG II (E; n = 6) stimulation, all values normalized to scrambled control (Scr), * P< 0.05, ** P < 0.01. (F, G) GRK2 overexpression promotes GSK3β phosphorylation. (F) Representative Western Blots of pGSK3β and GSK3β after transfection with an adenovirus harboring GRK2 (AdGRK2) or β-galactosidase/LacZ as control (AdLacZ) or untreated cells (-), GAPDH served as loading control. (G) Quantification of pGSK3β and total GSK3β with and without GRK2 overexpression, n = 3, * P < 0.05.