2019 - The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on β-adrenoceptors
CardioExcyte 96 publication in Biochemical and Biophysical Research Communications (2019)
Maimari T., Krasel C., Bünemann M., Lorenz K.
Biochemical and Biophysical Research Communications (2019) In Press, Corrected Proof
• RKIP has a well-tolerated positive inotropic effect via β-adrenoceptor activation.
• RKIP binds N-termini of cardiac GRK2 and GRK3 – but not GRK5.
• GRK2/3 N-termini simulate RKIP effects on β-AR signaling by direct receptor interaction.
• Interference strategy using GRK peptides seems to be a promising tool for receptor regulation.
The Raf kinase inhibitor protein (RKIP) activates β-adrenoceptors (β-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated β-AR activation, chronic activation of β-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated β-AR phosphorylation and desensitization.
Here, we evaluate RKIP-mediated effects on β-AR to explore new strategies for β-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 – not GRK5 – as well as several RKIP binding sites within their N-termini (GRK21−185 and GRK31−185). Overexpression of these N-termini prevented β2-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of β2-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction.
Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on β-AR signaling in HEK293 cells and in cardiomyocytes by their binding to β2-AR and, thus, provide important insights for the development of new strategies to modulate β2-AR signaling.