A Model for cAMP-mediated cGMP Response in Dictyostelium discoideum

In Dictyostelium discoideum extracellular cyclic AMP (cAMP), as shown by previous studies, induces a transient accumulation of intracellular cyclic guanosine-5'-monophosphate (cGMP), which peaks at 10 s and recovers basal levels at 30 s after stimulation, even with persistent cAMP stimulation. Additional investigations have shown that the cAMP-mediated cGMP response is built up from surface cAMP receptor-mediated activation of guanylyl cyclase and hydrolysis of cGMP by phosphodiesterase. The regulation of these activities was measured in detail on a seconds time-scale, demonstrating complex adaptation of the receptor, allosteric activation of cGMP-phosphodiesterase by cGMP, and potent inhibition of guanylyl cyclase by Ca2+. In this paper we present a computer model that combines all experimental data on the cGMP response. The model is used to investigate the contribution of each structural and regulatory component in the final cGMP response. Four models for the activation and adaptation of the receptor are compared with experimental observations. Only one model describes the magnitude and kinetics of the response accurately. The effect of Ca2+ on the cGMP response is simulated by changing the Ca2+ concentrations outside the cell (Ca2+ influx) and in stores (IP3-mediated release) and changing phospholipase C activity. The simulations show that Ca2+ mainly determines the magnitude of the cGMP accumulation; simulations are in good agreement with experiments on the effect of Ca2+ in electropermeabilized cells. Finally, when cGMP-phosphodiesterase activity is deleted from the model, the simulated cGMP response is elevated and prolonged, which is in close agreement with the experimental observations in mutant stmF that lacks this enzyme activity. We conclude that the computer model provides a good description of the observed response, suggesting that the main structural and regulatory components have been identified

Inhibition of Receptor-Stimulated Guanylyl Cyclase by Intracellular Calcium Ions in Dictyostelium Cells

In Dictyostelium discoideum extracellular cAMP stimulates guanylyl cyclase and phospholipase C; the latter enzyme produces Ins(1,4,5)P3 which releases Ca2+ from internal stores. The following data indicate that intracellular Ca2+- ions inhibit guanylyl cyclase activity. 1) In vitro, Ca2+ inhibits guanylyl cyclase with IC50=41 nM Ca2+ and Hill-coefficient of 2.1. 2) Extracellular Ca2+ does not affect basal cGMP levels of intact cells. In electro-permeabilized cells, however, cGMP levels are reduced by 85% within 45 s after addition of 10-6 M Ca2+ to the medium; halfmaximal reduction occurs at 200 nM extracellular Ca2+. 3) Receptor-stimulated activation of guanylyl cyclase in electro-permeabilized cells is also inhibited by extracellular Ca2+ with half-maximal effect at 200 nM Ca2+. 4) In several mutants an inverse correlation exists between receptor-stimulated Ins(1,4,5)P3 production and cGMP formation. We conclude that receptor-stimulated cytosolic Ca2+ elevation is a negative regulator of receptor-stimulated guanylyl cyclase.

The G Protein β Subunit Is Essential for Multiple Responses to Chemoattractants in Dictyostelium

Increasing evidence suggests that the βγ-subunit dimers of heterotrimeric G proteins play a pivotal role in transducing extracellular signals. The recent construction of Gβ null mutants (gβ-) in Dictyostelium provides a unique opportunity to study the role of βγ dimers in signaling processes mediated by chemoattractant receptors. We have shown previously that gβ- cells fail to aggregate; in this study, we report the detailed characterization of these cells. The gβ- cells display normal motility but do not move towards chemoattractants. The typical GTP-regulated high affinity chemoattractant-binding sites are lost in gβ- cells and membranes. The gβ- cells do not display chemoattractant-stimulated adenylyl cyclase or guanylyl cyclase activity. These results show that in vivo Gβ links chemoattractant receptors to effectors and is therefore essential in many chemoattractant-mediated processes. In addition, we find that Gβ is required for GTPγS stimulation of adenylyl cyclase activity, suggesting that the βγ-dimer activates the enzyme directly. Interestingly, the gβ- cells grow at the same rate as wild-type cells in axenic medium but grow more slowly on bacterial lawns and, therefore, may be defective in phagocytosis.