RenéJ.W. De Wit

Identification of the acrasin of Dictyostelium minutum as a derivative of folic acid.

The acrasin of the slime mold Dictyostelium minutum was isolated from aggregating cells and purified. The compound was species specific and more active in the aggregative than in the vegetative stage. Three observations strongly suggest a structural relationship between the acrasin and folic acid. (1) Folic acid inhibited acrasin degradation by D. minutum. (2) Methotrexate, an antagonist of chemotaxis towards folic acid, also inhibited the response to the acrasin. (3) The chemotactic response to an excess of folic acid was delayed. The response was also delayed to simultaneously tested low amounts of a related compound, but not to unrelated compounds (Van Haastert, 1982). The response to the acrasin was observed to be delayed by excess of folic acid. The acrasinase was identified as a folic acid C9-N10 splitting enzyme. Based on chromatographic properties and biological activity of the acrasin and folate derivatives, the chemical structure of the acrasin is discussed.

Relationships between the ligand specificity of cell surface folate binding sites, folate degrading enzymes and cellular responses in Dictyostelium discoideum

The affinity of four distinct types of folate binding sites and of two membrane-bound folate degrading enzymes for 15 folic acid derivatives was monitored. Apart from two types (AH and AL of binding sites, all binding classes or enzymes show different affinity patterns. This strongly suggests the observed binding sites to be nonidentical to the membrane-bound folate deaminase or folate C9-N10 cleaving enzyme. The analog specificity of the chemotactic response towards folates shows a strong resemblance to the specificity of binding to one of the binding classes: ‘B-sites’ (p < 0.01%). Less or no correlation was observed between the other classes or the enzymes and the chemotactic response. This may indicate that the B-sites are involved in the transduction of an extracellular signal to chemotactic cell movement. Folates elicit secretion of cAMP. Recently, the activity of several folate derivatives to evoke a cAMP response was studied (Devreotes, P.N. (1983) Dev. Biol. 95, 154–162). Comparison of this activity and the specificity of the binding sites in this study, suggests that the ‘A-sites’ are involved in the cAMP response.

Folate and cAMP modulate GTP binding to isolated membranes of Dictyostelium discoideum. Functional coupling between cell surface receptors and G-proteins

In membrane preparations from D. discoideum cells GTP-binding activity is observed. The lack of GTP binding to intact cells suggests that the binding sites are localized inside the cell. The GTP-binding activity also remains in the particulate fraction in the presence of 1 mM Ca++. This excludes membrane-associated microtubuli to be responsible for the observed GTP binding. Scatchard analysis suggests the existence of one type of binding site (Kd = 2.6 microM and 3.6 X 10(5) sites per cell). The kinetics of association as well as dissociation, however, suggest that GTP binding is more complex than binding to a single type of site. GDP and guanylyl imidodiphosphate are potent competitors of GTP binding (respectively 5- and 10-fold worse than GTP) while GMP, cGMP and several adenine nucleotides are ineffective up to 1 mM. The chemoattractants cAMP and folic acid both increase the equilibrium binding level of GTP, while dissociation of GTP is accelerated. These data indicate the functional coupling between cell surface receptors and G-proteins.

Negative chemotaxis in Dictyostelium and Polysphondylium.

Abstract A method has been described to measure negative chemotaxis in the cellular slime molds directly and to purify the repellents. Conclusive evidence is given that negative chemotaxis exists in the cellular slime molds and that it occurs generally in Dictyostelium and Polysphondylium. Amoebae respond shortly after their exposure to repellents, which are secreted by vegetative and not by preaggregative cells. The amoebae are sensitive to repellents in both development stages and contain enzyme(s) to inactivate them. Cross reactions of different species indicate that there is more than one repellent, although it cannot be excluded that the variability in response depends on the balancing effect of attractants and repellents.

Folate chemotactic receptors in Dictyostelium discoideum II. Guanine nucleotides alter the rates of interconversion and the proportioning of four receptor states

Abstract The ligand binding properties of folate chemotactic receptors on isolated membranes of Dictyostelium discoideum were analyzed. Three out of the four receptor states (B F , B S and B SS ) were detected, showing rate constants and K d values similar to those obtained for intact cells. Guanine nucleotides changed the proportioning of the receptor states as well as the rates of several conversions. (i) The transformation of B F into B S was inhibited by GDP but not by guanylyl imidodiphosphate (Gua PP [NH] P ) or GTP. (ii) The number of B S sites was lowered by GTP and Gua PP [NH] P . (iii) The binding to B SS was lowered by GTP and GDP, but increased by Gua PP [NH] P . (iv) The rate of disappearance of B SS was increased by GTP, but not by Gua PP [NH] P . Effects of guanine nucleotides were not observed after treatment of the membrane preparations with 15 mg/ml bovine serum albumin. This treatment caused the detection of a binding type different from the types described previously. The affinity of this binding site was extremely high ( K d ≤ 0.2 nM for N 10 -methylfolic acid), while the dissociation was relatively slow ( k −1 ≤ 3·10 −4 s −1 ). It is proposed that bovine serum albumin uncouples the folate receptor from a guanine nucleotide regulatory (G) protein in an irreversible manner. A model is presented in which the four receptor states correspond to distinct interactions with a G protein and GDP or GTP.

Developmental regulation of the folic acid chemosensory system in Dictyostelium discoideum

Abstract The developmental regulation of three classes of cell surface folate receptors, of cell surface folate deaminase, and of three folate-stimulated cellular responses (cGMP and cAMP synthesis, chemotaxis) have been monitored. All of these parameters showed a comparable decline during cell aggregation between 4 and 8 hr after the onset of starvation. Many of the parameters showed an increase during the first 3 hr of starvation (A, B S , and B SS receptors, and the cGMP and cAMP responses) while others were constant during this time period (deaminase, chemotaxis). Moreover, some kinetic properties of A and B SS receptors changed substantially during early development. After cell aggregation (at about 8 hr) all parameters except B SS sites were reduced to less than 10% of their maximal values. Cellular responses to folic acid were absent during the postaggregative development. When aggregating cells were removed from the agar and shaken in suspension for an additional 3 hr, sensitivity to folic acid as judged by cGMP and cAMP synthesis reappeared. Additionally, the further disappearance of cell surface B receptors was inhibited, A sites disappeared more rapidly, and the B SS sites regained the kinetic properties of these sites characteristic of unstarved cells. The changes in receptor kinetics are discussed in terms of coupling to/uncoupling from effector proteins.

Folate chemotactic receptors in Dictyostelium discoideum I. Ligand-induced conversion between four receptor states

Abstract The presence of three distinct populations of folate binding sites on the surface of Dictyostelium discoideum cells has been reported recently. The A sites (consisting of two subtypes) recognize folic acid, 2-deaminofolic acid and methotrexate with equal affinity and appear to be linked to cAMP synthesis. B and C sites (the latter is composed of two interconvertable subtypes; rapidly equilibrating, CF, and more slowly equilibrating, CS) preferentially bind N10-methylfolic acid and folic acid and may both be involved in the chemotactic response (De Wit, R.J.W. and Van Haastert, P.J.M. (1985) Biochim. Biophys. Acta 814, 199–213; De Wit, R.J.W., Bulgakov, R., Pinas, J.E. and Konijn, T.M. (1985) Biochim. Biophys. Acta 814, 214–226). The present study gives two lines of evidence that the B and C sites are interconvertable. (i) Occupancy of the B-sites by N10-methylfolic acid proceeds at a rate identical to that of the association of this ligand to CS at all concentrations tested. This suggests that association to CS and formation of occupied B sites share a common pathway. (ii) After preincubation with ligand, removal of free ligand results in the reappearance of unoccupied CF sites with kinetics identical to the disappearance of occupied B sites. Furthermore, the existence of a third type of C site, which is formed out of CS and may be converted to B, is proposed. This is based on competition studies using folate analogs with a different affinity for CS and B.

Studies of cell-surface glorin receptors, glorin degradation, and glorin-induced cellular responses during development of Polysphondylium violaceum.

The chemoattractant mediating cell aggregation in the slime mold Polysphondylium violaceum is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethylester (glorin). Here we examine the binding properties of tritiated glorin to intact P. violaceum cells. Scatchard analysis of binding data yielded slightly curvilinear plots with Kd values in the range of 20 and 100 nM. The number of glorin receptors increased from 35,000 in the vegetative stage to 45,000 per cell during aggregation. Later, during culmination receptor numbers decreased to undetectable levels (less than 1000). The receptor binding kinetics show binding equilibrium within 30 s at 0 degrees C, and ligand dissociation occurs from two kinetically distinct receptors whose half-times were 2 s for 72% of the bound glorin and 28 s for the remainder. The enzymatic degradation of glorin did not affect binding data during incubations of up to 1 min at 0 degrees C. Two glorinase activities were observed. An ornithine delta-lactam cleaving activity with a Km of ca. 10(-4) M and a propionic acid removing activity (Km 10(-5) M), both of which were detected mainly on the cell surface. Cleavage of the lactam occurred at a higher rate than removal of propionic acid. Lactam-cleaved glorin showed no chemotactic activity nor did it bind to cell-surface glorin receptors. Cell-surface-bound glorinase activity and glorin-induced cGMP synthesis were developmentally regulated, peaking at aggregation. In the most sensitive stage half-maximal responses (cGMP synthesis, chemotaxis, light-scattering) were elicited in the 10-100 nM range. Neither cAMP synthesis nor glorin-induced glorin synthesis was observed. Guanine nucleotides specifically modulated glorin receptor binding on isolated membranes, and, conversely, glorin modulated GTP gamma S binding to membrane preparations. Our results support the notion that glorin mediates chemotactic cell aggregation in P. violaceum acting via cell-surface receptors, G-proteins, and cGMP accumulation.

2-Deaminofolic acid elicits desensitization without excitation of the cyclic GMP response in Dictyostelium discoideum

Abstract Folic acid induces a rapid transient increase in cellular cGMP levels in D. discoideum . A half-maximal cGMP response is effected by about 0.2 μM folate. In addition, extracellular folate is rapidly inactivated by deaminases. The main product 2-deaminofolic acid generates no measurable cGMP response at concentrations of up to 0.1 mM. At 1 mM a 10% response is observed. In contrast, 1 mM deaminofolic acid inhibits the cGMP accumulation as induced by 0.3 μM folic acid by 90%. This antagonism by deaminofolate is competitive with a K I value of 20 μM. When the cells are preincubated with deaminofolic acid before stimulation with folic acid, the inhibition changes to a non-competitive type ( K I = 20 μ M). It is concluded that although deaminofolate does not elicit cGMP accumulation, some cellular process is activated resulting in a diminished cGMP response to saturating folate stimuli. This process of desensitization is characterized by a first-order rate constant of 0.04 s −1 , while folic acid-induced desensitization proceeds with a higher rate of 0.6 s −1 . We discuss that the latter rate constant is more likely to reflect the rate of detection of a desensitizing signal, rather than the rate of the desensitization process itself.

Antagonists of chemoattractants reveal separate receptors for cAMP, folk acid and pterin in Dictyostelium

Abstract Adenosine 3′,5′-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3′-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3′-amino-cAMP on the chemotactic activity of cAMP is competitive, which suggests that 3′-amino-cAMP antagonizes cAMP via the chemotactic receptor for cAMP. 3′-Amino-cAMP does not antagonize folic acid or pterin. The binding of folic acid to post-vegetative Dictyostelium discoideum cells is inhibited by low concentrations of 2-deamino-2-hydro folic acid (DAFA [7]). DAFA is neither chemotactically active, nor does it inhibit a chemotactic reaction to folic acid. This questions the involvement of the main folic acid cell surface-binding sites in the chemotactic response to folic acid. The pterin analog, 6-aminopterin, is an antagonist of pterin, but not of cAMP or folic acid. Our results show that cAMP, folic acid and pterin are detected by different receptors. Furthermore, they suggest that the antagonistic action of 3′-amino-cAMP and 6-aminopterin is localized in the signal transduction pathway at a step before the signals from the separate receptors have arrived at a single pathway.