Characterization of forskolin binding sites in rat brain membranes using [14,15-3H]14,15-dihydroforskolin as a ligand.

[14,15-3H]14,15-Dihydroforskolin [( 3H]DHF) has been used as a radioactive ligand to identify forskolin binding sites in rat brain membranes. The binding was saturable and reversible. The binding sites showed positive cooperative properties as evident from an upward convex Scatchard plot and a Hill coefficient of 1.6. The equilibrium dissociation constants (KD) were in the range between 10 microM and 10 nM as estimated from the limiting slopes of the curved Scatchard plot. Half-maximal saturation of the binding sites was observed at a ligand concentration of 225 nM. The binding kinetics were very rapid: Binding equilibrium was reached in less than 2 min and a large excess of cold forskolin displaced 80% of the radioligand within 2 min. The dissociation reaction was not first order, characterized by a decreasing dissociation rate constant. Bound [3H]DHF could be displaced with forskolin (IC50 0.3 microM), 14,15-dihydroforskolin (IC50 0.8 microM) and 7-desacetylforskolin (IC50 3 microM). However, nucleotides (ATP, GTP) and other receptor ligands (adenosine, isoproterenol) had no effect on the binding. Although the density of the forskolin binding sites (3.2 pmole/mg protein) is similar to those of other adenylate cyclase linked receptors, discrepancies between the KD and the ED50 obtained in adenylate cyclase studies and the finding that activation of the enzyme by forskolin is negative cooperative makes it difficult to clearly relate the binding sites to adenylate cyclase.