title: 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 β2-adrenergic responsiveness of alveolar type II cells (A549) and human airway smooth muscle cells (HASM), respectively, by inhibiting the internalization of β2-adrenergic receptors (β2AR) under stimulating conditions. Internalization of β2AR is initiated by phosphorylations of certain serines and threonines by cAMP dependent protein kinase A (PKA) and G protein-coupled receptor kinases (GRK).
  
  
  OBJECTIVE
  To evaluate the effect of α-hederin on PKA and GRK2 mediated phosphorylation of GFP-tagged β2AR.
  
  
  METHODS
  To study this process we performed In-Cell Western using isoprenaline stimulated HEK293 cells overexpressing β2AR 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.
  
  
  CONCLUSIONS
  Taken together, these results indicate that α-hederin acts as an indirect GRK2 inhibitor leading to a reduced homologous desensitization of β2AR-GFP in HEK293 cells.
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.
authors.forename: Janka
authors.initials: J
authors.lastname: Schulte-Michels
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Wolf
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Aatz
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Engelhard
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Sieben
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Martinez-Osuna
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Häberlein
authors.affiliation: Institute of Biochemistry and Molecular Biology, Rheinische Friedrich-Wilhelms-University of Bonn, Bonn, Germany. Electronic address: haeberlein@uni-bonn.de.
authors.forename: Hanns
authors.initials: H
authors.lastname: Häberlein
mesh_terms: D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D057809:HEK293 Cells; D035981:Hedera / Q000737:chemistry; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D009828:Oleanolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012503:Saponins / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; D012503:Saponins; C010177:beta-hederin; D009828:Oleanolic Acid; D017868:Cyclic AMP-Dependent Protein Kinases; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D007545:Isoproterenol
keywords: GRK2; homologous desensitization; phosphorylation; α-hederin; β(2)-adrenergic receptor