Alpha-hederin, but not hederacoside C and hederagenin from Hedera helix, affects the binding behavior, dynamics, and regulation of beta 2-adrenergic receptors.

Hederacoside C, alpha-hederin, and hederagenin are saponins of dry extracts obtained from the leaves of ivy (Hedera helix L.). Internalization of beta(2)-adrenergic receptor-GFP fusion proteins after stimulation with 1 microM terbutaline was inhibited by preincubation of stably transfected HEK293 cells with 1 microM alpha-hederin for 24 h, whereas neither hederacoside C nor hederagenin (1 microM each) influenced this receptor regulation. After incubation of A549 cells with 5 nM Alexa532-NA, two different diffusion time constants were found for beta(2)AR-Alexa532-NA complexes by fluorescence correlation spectroscopy. Evaluation of the autocorrelation curve revealed diffusion time constants: tau(bound1) = 1.4 +/- 1.1 ms (n = 6) found for receptor-ligand complexes with unrestricted lateral mobility, and tau(bound2) = 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 tau(bound1) and 8.7 +/- 4.3% for tau(bound2) (n = 6). A549 cells pretreated with 1 microM alpha-hederin for 24 h showed dose-dependent alterations in this distribution with 37.1 +/- 5.5% for tau(bound1) and 4.1 +/- 1.1% for tau(bound2). Simultaneously, the level of Alexa532-NA binding was significantly increased from 33.0 +/- 6.8 to 41.2 +/- 4.6%. In saturation experiments, alpha-hederin did not influence the beta(2)-adrenergic receptor density (B(max)), whereas the K(D) value for Alexa532-NA binding decreased from 36.1 +/- 9.2 to 24.3 +/- 11.1 nM. Pretreatment of HASM cells with alpha-hederin (1 microM, 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 beta(2)AR or the intracellular cAMP level.