Activation of a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein during desensitization of Dictyostelium discoideum cells to chemotactic signals.

The chemoattractant cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum. Upon prolonged incubation with cAMP, cells become desensitized via two distinct processes: a decrease in the number of available cAMP-binding sites (down regulation) and modification of the receptor (presumably via phosphorylation) correlated with adaptation. These processes occur simultaneously, but differ in the cAMP dose dependency and reversibility. In this study we investigated the mechanism of adaptation; cells were incubated with a cAMP analog to induce desensitization mediated by adaptation. The cells were then washed, lysed and the interaction between cAMP, receptor, guanine-nucleotide-binding regulatory proteins (G proteins) and GTP was investigated. (1) cAMP receptors that are phosphorylated in vivo remain phosphorylated for at least 45 min after lysis. (2) Desensitization did not alter basal cAMP binding to the receptor nor the inhibitory effect of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on this binding. (3) The stimulatory effect of cAMP on GTP[S] binding was also unchanged, while basal GTP[S] binding and the kinetics of binding were only slightly different. (4) Basal high-affinity GTPase activity was not altered but cAMP stimulation was reduced from 43 +/- 7% in control lysates to 14 +/- 4% in lysates from desensitized cells. (5) cAMP stimulation of GTPase was decreased by pretreatment of cells with pertussis toxin from 43 +/- 7% to 17 +/- 8% but this was not further altered in lysates from desensitized pertussis-toxin-treated cells. These observations indicate that during desensitization the phosphorylated receptor can still interact with G proteins. Furthermore, desensitization did not affect cAMP stimulation of GTP[S] binding but strongly reduced cAMP stimulation of GTPase, suggesting that a G protein becomes activated. This G protein is pertussis toxin sensitive and may be the inhibitor G protein (Gi). This would imply that adenylate cyclase desensitizes because Gi becomes activated.