Intracellular and secreted cAMPs play crucial roles in controlling cell movement and gene regulation throughout development of the social amoeba Dictyostelium discoideum. cAMP is produced by three structurally distinct ACs (adenylate cyclases), ACA, ACG and ACB, which have distinctive but overlapping patterns of expression and, as concluded from gene disruption studies, seemingly overlapping functions. In addition to gene disruption, acute pharmacological abrogation of protein activity can be a powerful tool to identify the protein's role in the biology of the organism. We analysed the effects of a range of compounds on the activity of ACA, ACB and ACG to identify enzyme-specific modulators. Caffeine, which was previously used to specifically block ACA function, also inhibited cAMP accumulation by ACB and ACG. IPA (2',3'-O-isopropylidene adenosine) specifically inhibits ACA when measured in intact cells, without affecting ACB or ACG. All three enzymes are inhibited by the P-site inhibitor DDA (2',5'-dideoxyadenosine) when assayed in cell lysates, but not in intact cells. Tyrphostin A25 [alpha-cyano-(3,4,5-trihydroxy)cinnamonitrile] and SQ22536 [9-(tetrahydro-2'-furyl)adenine] proved to be effective and specific inhibitors for ACG and ACA respectively. Both compounds acted directly on enzyme activity assayed in cell lysates, but only SQ22536 was also a specific inhibitor when added to intact cells.
- 3',5'-Cyclic-AMP Phosphodiesterases
- Adenylate Cyclase
- Cell Line
- Gene Expression Profiling
- Protozoan Proteins