Abstract WP549: GRK5-Mediated Inhibitory Phosphorylation of the Mineralocorticoid Receptor Protects Against Aldosterone`s Stroke-Promoting Vascular Effects

Abstract

Introduction: Stroke is one of the leading causes of death in the Western world. Mineralocorticoid receptor (MR) antagonism is a potential therapy for ischemic and hemorrhagic stroke patients, since aldosterone exerts deleterious effects in the cerebral vasculature via the MR. Prominent among these is activation of epidermal growth factor (EGF)-dependent vessel wall remodeling. GRK2 and GRK5 are the most abundant G protein-coupled receptor (GPCR)-kinases (GRKs) in the heart and vessels and both phosphorylate GPCRs but also non-GPCR substrates at Ser/Thr residues. The human MR gets phosphorylated at various Ser residues, including Ser-843 in the ligand binding domain (LBD) and Ser-601 in the N-terminal domain (NTD). These Ser phosphorylations occur in the cytoplasm and result in MR inhibition via direct transcriptional activity repression or cytosolic retention, respectively. Hypothesis: GRK5 phosphorylates and inhibits the MR in the heart and vessels. Methods: We used rat cardiomyocytes (H9c2) and aortic smooth muscle cells (ASMCs). We checked for GRK interactions with the MR via co-immunoprecipitation. We measured MR phosphorylation via phospho-Ser immunoblotting and MR transcriptional activity via the luciferase reporter assay. We also measured EGF and EGF receptor (EGFR) expression via real time-PCR. Results: GRK5, but not GRK2, phosphorylates the MR in H9c2 cardiomyocytes. Beta2-adrenoceptor activation stimulates this non-canonical effect of GRK5. The GRK5-phosphorylated MR is incapable of activating gene transcription, since aldosterone-induced MR transcriptional activity is markedly suppressed upon GRK5 overexpression in both cardiomyocytes and ASMCs. Conversely, CRISPR-mediated GRK5 gene deletion augments cardiac and ASMC aldosterone-dependent MR transcriptional activity. Finally, beta2-adrenoceptor-stimulated GRK5 phosphorylates and inhibits the MR in ASMCs, thereby suppressing aldosterone-induced EGF and EGFR mRNA expression. Conclusions: GRK5 blocks the toxic MR-dependent effects of aldosterone in the heart and vascular smooth muscles. Thus, GRK5 stimulation (e.g. via beta2-adrenoceptor activation) may protect against the stroke-promoting effects of aldosterone in the cerebral vasculature.