a-Hederin, but Not Hederacoside C and Hederagenin from Hedera helix, Affects the Binding Behavior, Dynamics, and Regulation of ß2-Adrenergic Receptors

Authors: Anne Sieben, Lars Prenner, Thomas Sorkalla, Anne Wolf, Daniel Jakobs, Frank Runkel, Hanns Haberlein
Publisher/Year: Biochemistry, 2009, 48 (15), pp 3477–3482
Pub Med ID/Journal ID:

Abstract

Hederacoside C, a-hederin, and hederagenin are saponins of dry extracts obtained from the leaves of ivy (Hedera helix L.). Internalization of ß2-adrenergic receptor-GFP fusion proteins after stimulation with 1 µM terbutaline was inhibited by preincubation of stably transfected HEK293 cells with 1 µM a-hederin for 24 h, whereas neither hederacoside C nor hederagenin (1 µM each) influenced this receptor regulation. After incubation of A549 cells with 5 nM Alexa532-NA, two different diffusion time constants were found for ß2AR-Alexa532-NA complexes by fluorescence correlation spectroscopy. Evaluation of the autocorrelation curve revealed diffusion time constants: tbound1 = 1.4 ± 1.1 ms (n = 6) found for receptor-ligand complexes with unrestricted lateral mobility, and tbound2 = 34.7 ± 14.1 ms (n = 6) for receptor-ligand complexes with hindered mobility. The distribution of diffusion time constants was 24.3 ± 2.5% for tbound1 and 8.7 ± 4.3% for tbound2 (n = 6). A549 cells pretreated with 1 µM a-hederin for 24 h showed dose-dependent alterations in this distribution with 37.1 ± 5.5% for tbound1 and 4.1 ± 1.1% for tbound2. Simultaneously, the level of Alexa532-NA binding was significantly increased from 33.0 ± 6.8 to 41.2 ± 4.6%. In saturation experiments, a-hederin did not influence the ß2-adrenergic receptor density (Bmax), whereas the KD value for Alexa532-NA binding decreased from 36.1 ± 9.2 to 24.3 ± 11.1 nM. Pretreatment of HASM cells with a-hederin (1 µM, 24 h) revealed an increased intracellular cAMP level of 13.5 ± 7.0% under stimulating conditions. Remarkably, structure-related saponins like hederacoside C and hederagenin did not influence either the binding behavior of ß2AR or the intracellular cAMP level.