α-Hederin inhibits G protein-coupled receptor kinase 2-mediated phosphorylation of β2-adrenergic receptors

Abstract Background Recently is has been shown that α- and β-hederin increase the β 2 -adrenergic responsiveness of alveolar type II cells (A549) and human airway smooth muscle cells (HASM), respectively, by inhibiting the internalization of β 2 -adrenergic receptors (β 2 AR) under stimulating conditions. Internalization of β 2 AR is initiated by phosphorylations of certain serines and threonines by cAMP dependent protein kinase A (PKA) and G protein-coupled receptor kinases (GRK). Purpose To evaluate the effect of α-hederin on PKA and GRK2 mediated phosphorylation of GFP-tagged β 2 AR. Study design To study this process we performed In-Cell Western using isoprenaline stimulated HEK293 cells overexpressing β 2 AR as GFP fusion protein and specific antibodies against PKA (Ser345/346) and GRK2 (Ser355/356) phosphorylation sites. Results There was no effect found on the PKA mediated phosphorylation ( n = 14) but we could show that α-hederin (1 µM, 12 h) significantly inhibits GRK2 mediated phosphorylation at Ser355/356 by 11 ± 5% ( n ≥ 29, p ≤ 0.01) under stimulating conditions compared to the positive control. In Forster resonance energy transfer (FRET) experiments using the isolated kinases in solution α-hederin did not show any influence neither to GRK2 nor to PKA. Conclusion Taken together, these results indicate that α-hederin acts as an indirect GRK2 inhibitor leading to a reduced homologous desensitization of β 2 AR-GFP in HEK293 cells