Abstract. Increasing evidence suggests that the 13`/-subunit dimers of heterotrimeric G proteins play a pivotal role in transducing extracellular signals. The recent construction of G13 null mutants (gfl-) in Dictyostelium provides a unique opportunity to study the role of 13

In several G-protein-coupled signaling systems, ligandinduced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss of ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that for chemoattractant receptors agonistinduced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation.

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 Ca21 on the cGMP response is simulated by changing the Ca21 concentrations outside the cell (Ca2+ influx) and in stores (IP3-mediated release) and changing phospholipase C activity. The simulations show that Ca21 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.

In Dictyostelium discoideum extracellular cAMP stimulates guanylyl cyclase and phospholipase C; the latter enzyme produces Ins(1,4,5)P~ which releases Ca 2+ from internal stores. The following data indicate that intracellular Ca 2~-ions inhibit guanylyl cyclase activity. 1) In vitro, Ca 2+ inhibits guanylyl cyclase with IC50=41 nM Ca 2+ and Hillcoefficient of 2.1.2) Extracellular Ca 2+ 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 Ca 2+ to the medium; halfmaximal reduction occurs at 200 nM extracellular Ca 2+. 3) Receptor-stimulated activation of guanylyl cyclase in electropermeabilized cells is also inhibited by extracellular Ca 2+ with half-maximal effect at 200 nM Ca 2+. 4) In several mutants an inverse correlation exists between receptor-stimulated Ins(1,4,5)P 3 production and cGMP formation. We conclude that receptor-stimulated cytosolic Ca 2+ elevation is a negative regulator of receptor-stimulated guanylyl cyclase. ~ ~992 Academic Press, Inc.The cellular slime mold D.discoideum uses extracellular cAMP for cell-cell communication during chemotaxis and differentiation (1-3). cAMP binds to surface receptors, activates G-proteins and stimulates several second messenger systems, including adenylyl cyclase, guanylyl cyclase and phospholipase C. The produced cAMP is secreted in the medium where it can diffuse and activate neighboring cells. The produced cGMP remains largely intracellular where it activates cGMP receptors or is degraded by a cGMP-stimulated cGMP-phosphodiesterase (4). The produced Ins(1,4,5)P 3 (5,6) liberates Ca 2+ ions from nonmitochondrial stores (7).The activation of adenylyl cyclase and phospholipase C are most likely mediated by GTPbinding regulatory proteins (8); the mechanism by which guanylyl cyclase is activated is less well understood. Earlier experiments with saponin treated calls revealed stimulation of guanylyl cyclase activity by Ca 2+ ions (9-11). Recently, a Mg2+-dependent guanylyl cyclase 263