Effect of cholera toxin on the activation of adenylate cyclase by calmodulin in bovine striatum.

The effect of cholera toxin on activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, GTP, dopamine, and forskolin was investigated in bovine striatum. Adenylate cyclase activity was measured in washed membrane fractions prepared from homogenates that had been preincubated with cholera toxin. Pretreatment of striatal membranes with cholera toxin increased the response of adenylate cyclase to GTP, calmodulin, and forskolin as compared to vehicle controls. After cholera toxin pretreatment, the maximal response of adenylate cyclase to GTP was increased 4.7-fold and the apparent Ka for GTP was reduced 3-fold. The apparent Vmax for calmodulin was doubled after cholera toxin pretreatment. The activation of adenylate cyclase by forskolin was increased by cholera toxin, but the effect on kinetic parameters was not determined due to solubility considerations. In contrast, dopamine-stimulated adenylate cyclase activity was abolished after cholera toxin pretreatment. Examination of a concentration-response curve for cholera toxin in altering these activities revealed that calmodulin-stimulated adenylate cyclase was maximally affected at lower concentrations of cholera toxin than was activation by GTP and forskolin. Cholera toxin also affected the interaction between calmodulin and GTP. In the absence of cholera toxin, calmodulin decreased the apparent Ka for GTP nearly 10-fold. After cholera toxin pretreatment, however, calmodulin could not further decrease the apparent Ka for GTP but increased the maximal response to GTP by 30%. Calmodulin could potentiate GTP activation by stabilizing the interaction between Ns and the catalytic subunit, an action which could be negated by prior treatment with cholera toxin. ADP-ribosylation of the striatal homogenates with [32P]NAD demonstrated predominant labeling of a band of Mr 45,000 which corresponds to the known molecular weight of the alpha-subunit of the stimulatory GTP-binding protein, Ns. These results suggest that the activational state of Ns can affect the stimulation of adenylate cyclase by calmodulin and forskolin. Calmodulin and forskolin may act at separate sites on the catalytic subunit that can allosterically interact with Ns.