Pim M.W. Janssens

Did vertebrate signal transduction mechanisms originate in eukaryotic microbes

Abstract Current understanding of transmembrane signal transduction is based mainly on studies of vertebrate cells and bacteria. Vertebrate and bacterial signal transduction appear to operate in entirely different ways but similarities do exist between the systems of vertebrates and invertebrates. This raises the question: at what stage in evolution did eukaryotic transduction systems evolve?

A magnesium-dependent guanylate cyclase in cell-free preparations of Dictyostelium discoideum.

Receptor-mediated regulation of guanylate cyclase is well-studied in intact Dictyostelium discoideum cells, but study of the enzyme in cell-free preparations has hampered. A major obstacle has been that in vitro guanylate cyclase activity could be detected only in the presence of unphysiological concentrations of Mn2+-ions. In this paper we report the identification of a guanylate cyclase in D.discoideum cell homogenates that has high activity with Mg2+-GTP. The enzyme is activated by non-hydrolyzable ATP and GTP analogues and inhibited by submicromolar concentrations of Ca2+-ions. We suggest that the presently identified enzyme is regulated in intact cells via cell surface receptors. The compounds that modulated the enzyme activity in vitro may reflect physiologically relevant regulation mechanisms.

The evolutionary origin of eukaryotic transmembrane signal transduction

1. A comparison was made of transmembrane signal transduction mechanisms in different eukaryotes and prokaryotes. 2. Much attention was given to eukaryotic microbes and their signal transduction mechanisms, since these organisms are intermediate in complexity between animals, plants and bacteria. 3. Signal transduction mechanisms in eukaryotic microbes, however, do not appear to be intermediate between those in animals, plants and bacteria, but show features characteristic of the higher eukaryotes. 4. These similarities include the regulation of receptor function, adenylate cyclase activity, the presence of a phosphatidylinositol cycle and of GTP-binding regulatory proteins. 5. It is proposed that the signal transduction systems known to operate in present-day eukaryotes evolved in the earliest eukaryotic cells.

Cell fractionation, detergent sensitivity and solubilization of Dictyostelium adenylate cyclase and guanylate cyclase

SummaryCell fractionation studies have been performed, in order to obtain insight into the subcellular distribution of Dictyostelium adenylate cyclase and guanylate cyclase and also to provide a starting point for further study and isolation of these enzymes and their regulatory components.Adenylate cyclase and cAMP receptors were found in the same membrane fractions, but were distributed different from the plasma membrane marker alkaline phosphatase. Guanylate cyclase was partially soluble, partially particulate. In isopycnic gradients, particulate guanylate cyclase was present in other fractions than cAMP receptors and adenylate cyclase, but in similar ones to alkaline phosphatase. These observations are consistent with the hypothesis that cell-surface cAMP receptors and adenylate cyclase interact via a membrane-bound G-protein, whereas the receptors activate guanylate cyclase via a cytosolic factor.The adenylate cyclase activity in membranes obtained by sucrose gradient centrifugation was retained in the presence of various detergents, while with the same detergents the activity of particulate guanylate cyclase was lost. This adenylate cyclase was solubilized as assessed by gel filtration and centrifugation experiments, and it behaved heterogeneous in fractionation studies. In gel filtration, the major component eluted at a position corresponding to a Stokes radius of 4–7 nm. A purification of about 70-fold as compared to the cell homogenate was obtained by affinity chromatography of adenylate cyclase on ATP-Sepharose. We conclude that cell fractionation provides useful starting material for isolation and further study of Dictyostelium adenylate cyclase.

Adaptation of Dictyostelium Discoideum Cells to Chemotactic Signals

The cellular slime mold Dictyostelium discoideum lives in the soil where it feeds on bacteria. Exhaustion of the food supply induces cell aggregation. Subsequently, cells differentiate to two cell types; spores embedded in a slime droplet on top of a tubular stalk of vacuolized cells. Cell aggregation is mediated by Chemotaxis. Upon starvation some cells start to secrete a chemoattractant which has been identified as cAMP (1). Extracellular cAMP induces two responses, which are both mediated by cell surface receptors. First, cAMP activates adenylate cyclase; the produced cAMP is secreted and may trigger other cells, thus relaying the signal. Second, cAMP induces a chemotactic response by which cells move in the direction of the cAMP source. The combined effects of cAMP relay and Chemotaxis may lead to the accumulation of as many as 100,000 cells in a central collecting point derived from an area of about 1 cm2 (see 2,3 for recent reviews).

Cis-unsaturated fatty acids modulate the function of the cell-surface cAMP receptor of Dictyostelium discoideum

The binding of cAMP to the chemotactic cAMP receptor in intact Dictyostelium discoideum cells and isolated membranes is strongly inhibited by unsaturated fatty acids. In isolated membranes, cis-unsaturated fatty acids decreased the number of accessible cAMP binding sites, without significantly altering their affinity. Most potent were C18 and C20 cis-poly unsaturated fatty acids, like arachidonic acid, linoleic acid and linolenic acid. Trans-unsaturated fatty acid was less potent than its cis isomer, while saturated fatty acids did not affect the binding of cAMP to receptors at all. Oxidation reactions were not important for the effect of unsaturated fatty acids. When membranes were preincubated with millimolar concentrations of Ca2+, the effect of unsaturated fatty acids was strongly diminished. Mg2+ was ineffective. Ca2+, if presented after the incubation of membranes with unsaturated fatty acids, did not reverse the inhibitory effect. The specificity of the fatty acid effect, and the interference with Ca2+, but not Mg2+, suggest that the properties of the cAMP receptor are changed as a result of alterations in the lipid bilayer structure of the membrane.