Forskolin, a diterpene with cardiovascular action and potential utility as an antineoplastic agent, activates adenylate cyclase by an unknown biochemical mechanism. In order to study this problem, we propose to synthesize various photoaffinity analogs of forskolin and to investigate the photoinsertion of these probes into proteins related to adenylate cyclase. Our first objective is the development of general photoactive cross-linking reagents suitable for studying a variety of ligand-receptor interactions including the forskolin receptor(s). These reagents, which involve an azide-bearing resorcinol, catechol, and salicylate subunit, will be linked either to the C-7Beta hydroxyl or the Delta14-double bond of forskolin. After confirming that these analogs activate adenylate cyclase and compete effectively with [3H]-forskolin for specific binding sites, radiolabelled forskolin photoprobes will be used to detect the forskolin binding proteins in partially purified membranes from cells that are both activated by forskolin and those which are not activated by forskolin. These studies, in combination with studies of forskolin photoprobe binding to adenylate cyclase purified using an affinity column, will contribute to our understanding of the ubiquitous adenylate cyclase complex as well as the unique features of forskolin itself which has rapidly emerged as a useful tool in many biochemical studies.
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