Nuclear GRK5 binds Class I HDACs and the transcriptional repressor Sin3A

Katrina Lester1, George Baillie2, Julie Pitcher1. 1University College London, London, UK, 2University of Glasgow, Glasgow, UK

G-protein coupled receptor kinase 5 (GRK5), known for its role in G-protein coupled receptor (GPCR) desensitisation, can also adopt a nuclear localisation. Nuclear GRK5 has been shown to have a causative role in pathological cardiac hypertrophy when overexpressed in the hearts of transgenic (TG) mice. In certain cardiovascular diseases, ventricular hypertrophy may be considered a milestone in the progression to heart failure. At the cellular level, GRK5–dependent pathological cardiac hypertrophy ensues following GRK5-mediated phosphorylation of histone deacetylase 5 (HDAC5), a class II HDAC, which relieves the inhibition of the prohypertrophic transcription factor, MEF2. Here we describe an alternative potential mechanism whereby GRK5 may induce hypertrophy via transcriptional repression.

We demonstrate a novel and direct interaction between GRK5 and the class I HDACs, HDAC1, HDAC3 and HDAC8. Additionally, we show that GRK5 can also interact indirectly with both HDAC1 and HDAC2 via its direct binding to the transcriptional repressor protein Sin3A. Considering that HDAC1 and GRK5 function to activate hypertrophy, we hypothesize that both proteins may function in the same prohypertrophic signalling pathway, potentially via the Sin3A repressor complex. Indeed, the overexpression of a GRK5 construct fused to the Gal4-DNA binding domain (DBD) sequence represses Gal4-mediated transcription relative to the Gal4-DBD control. Screening GRK5 mutants for their ability to bind HDAC1 and Sin3A demonstrates that mutation of the nuclear export sequence (NES) of GRK5 inhibits Sin3A/HDAC1 binding. Conversely, mutation of residues located in the C-terminal polybasic domain (CTPB) of the kinase potentiates Sin3A/HDAC1 binding. It is anticipated that these GRK5 mutant constructs will allow an assessment of the role of these interactions in mediating pathological hypertrophy both in neonatal rat ventricular myocytes (NRVM) in vitro and TG mice in vivo.

Our results suggest that in addition to the regulation of MEF2 activity via GRK5-mediated phosphorylation and nuclear export of HDAC5, GRK5 may also be potentiating cardiac hypertrophy via a GRK5/HDAC1/Sin3A transcriptional repressor complex. Patients with ventricular overload disease have high cardiac levels of GRK5 expression. Elucidation of the molecular pathways by which GRK5 is causing cardiac hypertrophy could potentially lead to the development of novel specific inhibitors.