René J.W. de Wit

Ligand binding properties of the cytoplasmic cAMP-binding protein of Dictyostelium discoideum

In the cellular slime mold Dictyostelium discoideum cyclic AMP (CAMP) mediates chemotaxis [ 1,2] and differentiation [3,4]. Extracellular CAMP is detected by membrane-bound receptors after which an adenylate cyclase is transiently activated [5]. Part of the newly synthesized CAMP is secreted, part of it probably acts intracellularly. Intracellular CAMP is detected by a high-affinity CAMP binding protein, which has a Mr of 40 000 [6121. This protein is present in much higher concentrations in differentiating than in vegetative cells [9,12]. 2.1. D. Discoideum cyclic AMP binding protein

Binding of folates to Dictyostelium discoideum cells. Demonstration of five classes of binding sites and their interconversion

Abstract Studies of the binding of four folate derivatives to the cell surface of Dictyostelium discoideum indicate the existence of five types of sites. About 99% of the total number of binding sites (160 000 per cell) belongs to the ‘non-selective’ type, which recognizes folate, 2-deaminofolate and methotrexate with equal affinity. As judged by the kinetics of association and dissociation this class consists of two distinct subtypes; a high-affinity site, designated by AH, and a low-affinity site AL. Upon addition of ligand a number of the low-affinity sites is converted to the high-affinity state. Prolonged dissociation revealed the presence of extremely slowly dissociating sites. While the A-sites released bound ligand within 5 s, the slow (B) type yielded a half-time of about 6 min. This class (550 sites per cell) showed a clear selectivity for the four folates, with N10-methylfolate being the best ligand. From the kinetics of association and dissociation it is concluded that the B-sites are interconvertible with another binding type. In addition a class of sites was detected, which binds N10-methylfolate and folate with high affinity but 2-deaminofolate and methotrexate with approx. 100-fold lower affinity. Kinetic studies reveal that this C-class is also composed of two subtypes; a fast equilibrating site (within 1 s) designated as CF, and a slower site CS. It is proposed that before binding of ligand only CF exists, while after binding this binding type is converted into CS. At equilibrium more than 90% of the C-sites have attained the CS state.

Antagonists of chemoattractants reveal separate receptors for cAMP, folic 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.

Guanine nucleotides modulate the ligand binding properties of cell surface folate receptors in Dictyostelium discoideum

Dictyostelium discoideum cells show 2 distinct classes of cell surface binding sites for folates. One type is non‐specific, i.e., binds folic acid (FA), 2‐deaminofolic acid (DAFA), and methotrexate (MTX) with similar affinity (K 0.5 ⋍ 140 nM). Scatchard analysis of this non‐specific binding type suggests either heterogeneity or negative cooperativity. Isolated D. discoideum membranes show similar binding characteristics. Guanine nucleotides changed the binding levels of [3H]MTX. In the presence of 0.1 mM GTP, the number of binding sites remains unchanged, while the affinity decreases. GDP and guanylyl imidodiphosphate (GPPNP) are required at about 20‐fold higher concentration than GTP, which elicits a half‐maximal effect at 15μM. Other guanine and adenine nucleotides are ineffective up to 1 mM. These results suggests that the non‐specific cell surface receptor for folic acid interacts with a guanine nucleotide regulatory (G‐) protein.

Cyclic AMP induces a transient alkalinization in Dictyostelium

In a wide range of cell types, stimulus‐response coupling is accompanied by a rise in cytoplasmic pH (pHi). It is shown that stimulation of developing Dictyostelium discoideum cells with the chemoattractant cAMP also results in a rise in pHi. About 1.5 min after stimulation, pHi starts increasing from pHi∼7.45 to pHi∼7.60, as is revealed independently by two different pH null‐point methods. The rise in pHi is transient, also with a persistent stimulus, and effectively inhibited by diethylstilbestrol (DES), strongly suggesting that the rise in pHi is accomplished by the DES‐sensitive plasma membrane proton pump which has been demonstrated in D. discoideum.

Pertussis toxin inhibits cAMP surface receptor‐stimulated binding of [35S]GTPγS to Dictyostelium discoideum membranes

GTP‐binding activity to Dictyostelium discoideum membranes was investigated using various guanine nucleotides. Rank order of binding activities was: GTPγS>GTP>8‐N3‐GTP; the binding of GTPγS and GTP, but not of 8‐N3‐GTP, was stimulated by receptor agonists. [3H]GTP binding to D. discoideum membranes has been described previously by a single binding type (K d = 2.6 μM, B max = 85 nM). More detailed studies with [35S]GTPγS showed heterogeneous binding composed of two forms of binding sites with respectively high (K d = 0.2 μM) and low (K d = 6.3 μM) affinity. cAMP derivatives enhanced GTPγS binding by increasing the affinity and the number of the high‐affinity sites, while the low‐affinity sites were not affected by cAMP. The specificity of cAMP derivatives for stimulation of GTPγS binding showed aclose correlation with the specificity for binding to the cell surface cAMP receptor. Pretreatment of D. discoideum cells with pertussis toxin did not affect basal GTP and GTPγS binding, but eliminated the cAMP stimulation of GTP and GTPγS binding. These results indicate that D. discoideum cells have a pertussis toxin‐sensitive GTP‐binding protein that interacts with the surface cAMP receptor, suggesting the functional interaction of surface receptor with a G‐protein in D. discoideum.

Ca2+- or phorbol ester- dependent effect of ATP on a subpopulation of cAMP cell-surface receptors in membranes from D. discoideum. A role for protein kinase C.

Abstract D. discoideum cells contain surface receptors for the chemoattractant cAMP which are composed of fast and slowly dissociating binding sites with half-lifes of respectively about 1 s and 15 s (Van Haastert and De Wit, J. Biol. Chem. 259 , 13321–13328). In membranes prepared by shearing the cells through a Nucleopore filter, ATP has no effect on cAMP-binding at equilibrium, but the number of slowly dissociating sites is increased about 2-fold by ATP while their apparent affinity and off-rate are not altered by ATP. The effect of ATP is stimulated about 3-fold by Ca2+ with a half maximal effect at 100 μM Ca2+. The tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), increases this Ca2+-sensitivity of the ATP effect to about 0.2 μM Ca2+. These data suggest that a specific subpopulation of cAMP receptors in membranes from D. discoideum is altered by the action of protein kinase C.

A novel chemotaxis regulating enzyme that splits folic acid into 6-hydroxymethylpterin and P-aminobenzoylglutamic acid

After sta~ation, amoebae of the cellular slime molds secrete acrasin [l] by which they attract each other and form an aggregate that differentiates into stalk cells and spores. In a favourable environment spores germinate yielding amoebae which feed on bacteria and m~tiply. Vegetative amoebae are also sensitive to chemotactic stimuli [4,6], which differ from acrasins. They are attracted to bacteria, which apparently secrete chemoattractants for amoebae [2,3]. Folic acid was reported a chemoattractant for vegetative amoebae of the cellular slime molds [4]. Folic acid is also, although less ~hemotactic~y active in the aggregative stage except in D~c~oste~~um discoideum [5,7 J. The function of folic acid in the cellular slime molds is thought to be a signal for food detection [4,7]. The inactivation of the chemotactic signal is regulated by enzymatic degradation. One way to inactivate folic acid is by folic acid deaminase, as studied in D. discoideum and Po~ys~hondyZium violaceum [8,9]. We examined the chemotactic activity of the deaminated folic acid in several species of the cellular slime molds and discovered that in some species the ehemotactic activity is not lost by deamination. This indicates that another chemotactic regulator enzyme exists. Folic acid metabolism has been

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).