High affinity binding sites for [3H]forskolin have been identified in rat brain membranes. These sites have a K(d) of 15 nM and a B(max) of about 200 fmol/mg protein. The binding of [3H]forskolin to those high affinity sites in rat brain membranes is increased about two-fold by addition of MgCl2 or MnCl2. Smaller increases are observed in the presence of calcium, sodium, or potassium. The binding of [3H]forskolin is also increased in the presence of NaF or GppNHp, agents that are known to activate adenylate cyclase through the stimulatory guanine nucleotide regulatory protein (N(s)). The increase in [3H]forskolin binding in the presence of NaF or GppNHp is due to an increase in the number of binding sites with no change in the apparent K(d) for the binding sites. The NaF- and GppNHp-stimulated binding requires the presence of magnesium or manganese. The binding of [3H]forskolin to rat brain membranes is reduced in membranes that are heated or pretreated with chymotrypsin, trypsin, or N-ethylmaleimide. NaF stabilizes the binding sites to thermal denaturation. The data demonstrate that the number of high affinity forskolin binding sites are increased under conditions that promote the activation of the catalytic protein of adenylate cyclase by the N(s) protein. It is suggested that the high affinity forskolin binding sites are associated with a complex of the catalytic protein and the activated N(s) protein.
|Original language||English (US)|
|Number of pages||15|
|Journal||Journal of Cyclic Nucleotide and Protein Phosphorylation Research|
|State||Published - 1985|
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