α-Hederin, but Not Hederacoside C and Hederagenin from Hedera helix, Affects the Binding Behavior, Dynamics, and Regulation of β2-Adrenergic Receptors

Hederacoside C, α-hederin, and hederagenin are saponins of dry extracts obtained from the leaves of ivy (Hedera helix L.). Internalization of β2-adrenergic receptor−GFP fusion proteins after stimulation with 1 μM terbutaline was inhibited by preincubation of stably transfected HEK293 cells with 1 μM α-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 β2AR−Alexa532-NA complexes by fluorescence correlation spectroscopy. Evaluation of the autocorrelation curve revealed diffusion time constants: τbound1 = 1.4 ± 1.1 ms (n = 6) found for receptor−ligand complexes with unrestricted lateral mobility, and τbound2 = 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 τbound1 and 8.7 ± 4.3% for τbound2 (n = 6). A549 cells pretreated with 1 μM α-hederin for 24 h showed dose-dependent alterations in this distribution with 37.1 ± 5.5% for τbound1 and 4.1 ± 1.1% for τbound2. Simultaneously, the level of Alexa532-NA binding was significantly increased from 33.0 ± 6.8 to 41.2 ± 4.6%. In saturation experiments, α-hederin did not influence the β2-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 α-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 β2AR or the intracellular cAMP level.