Forskolin, a diterpene natural product, interacts directly with adenylyl cyclase in diverse tissues to cause an increase in intracellular cyclic AMP. However, the ability of forskolin to bind to and activate adenylyl cyclase varies depending upon the tissue being studied. Several isotypes of adenylyl cyclase have been cloned and expressed in a recombinant Sf9 expression system. Dr. Wei-Jen Tang has made available membranes from cells expressing the recombinant adenylyl cyclases and we have compared the interactions of forskolin with the individual enzyme subtypes. The recombinant type I adenylyl cyclase binds forskolin with high affinity (in the nM range) and is activated by forskolin with an EC50 of approximately 1 micromolar. Therefore, we chose this enzyme subtype to study the relationship between forskolin binding and activation in more detail. We used an alkylating (isothionyl) derivative of forskolin (6-NCS-Fsk), which reacts irreversibly with a nucleophilic group at the site of interaction, to determine whether forskolin binding and activation occur at the same site. 6-NCS-Fsk causes irreversible inhibition of forskolin binding with an IC50 of approximately 300 nM, suggesting covalent binding of the forskolin derivative to an amino or thiol group at the forskolin binding site. 6-NCS-Fsk also causes a concentration-dependent loss of forskolin-stimulated activation of adenylyl cyclase. However, this irreversible inhibition of forskolin activation occurs with an IC50 of approximately 10 micromolar. The difference in the concentration ranges at which 6-NCS-Fsk causes irreversible inhibition of forskolin binding and activation (nM vs. micromolar) suggests that there are two sites of 6-NCS-Fsk interaction. The concentration ranges of the inhibitory effects of 6-NCS-Fsk are consistent with the concentration ranges of forskolin binding and activation. We are examining the usefulness of the isothionyl derivative of forskolin to identify the reactive nucleophilic group at the forskolin interaction site(s) of type I adenylyl cyclase. This will involve reacting the isothionyl derivative with purified type I adenylyl cyclase, generating proteolytic fragments, and using mass spectrometry to identify the chemically modified peptide. We are establishing the conditions for generating proteolytic fragments using recombinant type I adenylyl cyclase which has been tagged with a radioactive forskolin photoaffinity derivative.
