IRREVERSIBLE INHIBITION OF FORSKOLIN INTERACTIONS WITH TYPE-I ADENYLYL-CYCLASE BY A 6-ISOTHIOCYANATE DERIVATIVE OF FORSKOLIN

Forskolin (Fsk) has been demonstrated to interact directly with the en zyme adenylyl cyclase (EC 4.6.1.1) in diverse tissues. However, the ab ility of Fsk to bind to and activate adenylyl cyclase varies depending on the tissue being studied. Different adenylyl cyclase subtypes have been cloned and expressed in a recombinant Sf9 expression system. Thi s provides an opportunity to study the effects of chemically reactive derivatives of Fsk on individual adenylyl cyclase subtypes in the abse nce of G(s alpha). Reaction of type I adenylyl cyclase with an isothio cyanate derivative of Fsk N-(2-isothiocyanatoethyl)amino]carbonyl]fors kolin) causes irreversible inhibition of Fsk binding with an IC50 of 3 00 nM and irreversible inhibition of Fsk activation with an IC50 of 10 mu M, suggesting that there are two sites of [N-(2-isothiocyanatoethy l)amino]carbonyl]forskolin interaction. These studies establish the us efulness of the isothiocyanate derivative of Fsk in localizing the sit e(s) of Fsk interaction with type I adenylyl cyclase.