GRK5 regulates NFkB transcription activity in endothelial cells

Abstract

Recent studies suggest that G protein-coupled receptor kinases (GRKs) play multiple roles in regulating signalling. In particular, GRK5 is able to enter the nucleus through nuclear localization (NLS) and exporting (NES) sequences. Our hypothesis is that GRK5 regulates transcription events in the nucleus, in particular NFkB activity. In bovine aortic endothelial cells GRK5 associates to IkB, a NFkB inhibitor and such interaction increases in presence of isoproterenol, β adrenergic agonist, 10 M. GRK5 overexpression causes nuclear accumulation of IkB, suggesting a role for GRK5 to inhibit NFkB transcriptional activity. To confirm the functional implication of such interaction, we used luciferase as reporter gene under the control of a NFkB-dependent promoter. GRK5 overexpression inhibits NFkB activity both in presence and absence of isoproterenol, TNFα and LPS. These data suggest an universal role of GRK5 to inhibit NFkB activity. To evaluate the role of NLS and NES in GRK5IkB interaction, we overexpressed GRK5 mutants lacking either NLS or NES sequences (GRK5ΔNLS and GRK5ΔNES). Both GRK5ΔNLS and GRK5ΔNES overexpression results in inhibition of NFkB activity (GRK5: −73±16%; GRK5ΔNLS: 42±6%; GRK5ΔNES: −67±12%; p<0.01 vs. control) and nuclear accumulation. Immunoprecipitation studies with overexpression of small peptides reproducing single domains of GRK5 (N-CAM, RGS, Catalytic domain, C-CAM) suggest that RGS is the GRK5 domain that associates to IkB. In conclusion, these results reveal a novel role of GRK5 as modulator of NFkB transcriptional activity that will be object of future investigations in cardiovascular disease models.