title: Binding of [14,15-3H]14,15-dihydroforskolin to rat liver membranes: comparison with the stimulatory effect of forskolin on adenylate cyclase.
abstract: The binding of [14,15-3H]14,15-dihydroforskolin ([3H]DHF) to rat liver membranes has been further characterized and was compared with the stimulatory effect of forskolin on adenylate cyclase. The binding equilibrium dissociation constant (KD) for 14,15-dihydroforskolin obtained in inhibition experiments was 0.6 microM, with a maximal binding capacity (Bmax) of 114 pmol/mg protein. A similar KD value (0.5 microM) was derived from kinetics studies that revealed very rapid association and dissociation reactions. For structure-activity relationship studies several forskolin derivatives were synthesized and tested for their ability to inhibit [3H]DHF binding and increase adenylate cyclase activity. Among the tested compounds, forskolin itself was the most potent agonist (K1 = 0.2 microM). Further modification of the molecule in position 7 and (or) 1 decreased or abolished its agonist properties in both adenylate cyclase and binding studies. [3H]DHF binding was not affected by several nucleotides, carbohydrates, lectins, and hormone receptor agonists including isoproterenol, glucagon, and adenosine, but the steroids 17-beta-estradiol, progesterone, and testosterone showed slight inhibitory effects at unphysiologically high concentrations. [3H]DHF binding and forskolin-stimulated adenylate cyclase were sensitive to heat and N-ethylmaleimide treatment. Forskolin protected adenylate cyclase against inactivation by heat but not by N-ethylmaleimide. Preincubation of the membrane with trypsin decreased [3H]DHF binding. The results presented in this study demonstrate that the binding sites identified with [3H]DHF have a high specificity for forskolin and provide evidence that these binding sites are involved in the stimulation of adenylate cyclase by forskolin.
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D005576:Colforsin / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006358:Hot Temperature; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D014357:Trypsin / Q000494:pharmacology
chemical_list: D005419:Flavonoids; D005576:Colforsin; C040028:14,15-dihydroforskolin; D014357:Trypsin; D000262:Adenylyl Cyclases; D005033:Ethylmaleimide