Forskolin has been demonstrated to interact directly with the adenylyl cyclase catalytic subunit in diverse tissues. However, the ability of forskolin to bind and activate adenylyl cyclase is different depending upon the source of the tissue. Previous work had established that bovine brain adenyl cyclase binds forskolin with high affinity and has a nucleophilic group at the forskolin binding site. Many different types of adenylyl cyclase have recently been cloned. The expression of the different types of adenylyl cyclase in a recombinant sf9 expression system provides an opportunity to study effects of forskolin on specific adenylyl cyclases. The recombinant type I adenylyl cyclase binds forskolin with high affinity. Reaction of type I adenylyl cyclase with an isothionyl derivative of forskolin (6-NCS-Fsk) causes irreversible inhibition of forskolin binding and inhibition of activation by forskolin, suggesting covalent binding of the forskolin derivative to an amino or thiol group at the forskolin binding site. We plan to use the isothionyl derivative of forskolin to identify the nucleophilic group at the forskolin binding site 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. Similar experiments may be performed with other types of adenylyl cyclases to compare the localization of the forskolin binding sites among the individual types of adenylyl cyclase.
