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

Background: Recently is has been shown that (alpha-and beta-hederin increase the beta(2)-adrenergic responsiveness of alveolar type II cells (A549) and human airway smooth muscle cells (HASM), respectively, by inhibiting the internalization of beta(2)-adrenergic receptors (r32AR) under stimulating conditions. Internalization of beta(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 cx-hederin on PKA and GRK2 mediated phosphorylation of GFP-tagged beta(2)AR. Study design: To study this process we performed In-Cell Western using isoprenaline stimulated HEK293 cells overexpressing beta(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 alpha-hederin (1 mu M, 12 h) significantly inhibits GRK2 mediated phosphorylation at Ser355/356 by 11 503 (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 alpha-hederin did not show any influence neither to GRK2 nor to PKA. Conclusion: Taken together, these results indicate that cc-hederin acts as an indirect GRK2 inhibitor leading to a reduced homologous desensitization of beta(2)AR-GFP in HEK293 cells. (C) 2015 The Authors. Published by Elsevier GmbH. This is an open access article tinder the CC BY license.