ABSTRACT
BACKGROUND: Recently is has been shown that α- and ß-hederin increase the ß2-adrenergic responsiveness of alveolar type II cells (A549) and human airway smooth muscle cells (HASM), respectively, by inhibiting the internalization of ß2-adrenergic receptors (ß2AR) under stimulating conditions. Internalization of ß2AR 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 ß2AR. STUDY DESIGN: To study this process we performed In-Cell Western using isoprenaline stimulated HEK293 cells overexpressing ß2AR 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 Förster 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 ß2AR-GFP in HEK293 cells.
