A novel Dictyostelium RasGEF required for chemotaxis and development
© Arigoni et al; licensee BioMed Central Ltd. 2005
Received: 16 June 2005
Accepted: 07 December 2005
Published: 07 December 2005
Ras proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM.
RasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein.
The data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.
The ras proto-oncogenes encode membrane-bound small monomeric GTP-binding proteins with molecular masses ranging between 20 to 40 kDa, which are highly conserved in the course of eukaryotic evolution. Ras proteins control fundamental cell processes including proliferation, differentiation, motility and polarity [1–3]. Like heterotrimeric G proteins they act as molecular switches cycling between two interconvertible forms: inactive, when bound to guanosine diphosphate (GDP), and active, when bound to guanosine triphosphate (GTP) . Conversion between GTP and GDP-bound states is tightly regulated by two set of proteins: guanosine-nucleotide exchange factors (GEFs), and GTPase activating proteins (GAPs). GEF proteins cause activation by catalysing the exchange of bound GDP with GTP, whereas GAPs inactivate Ras by increasing their rate of GTP hydrolysis .
Systems composed by GTPases, GAPs and GEFs allow great versatility in the construction of signaling pathways. Signals can be amplified (one GEF could activate several GTPases), integrated (several pathways activate the same GEF and GAP, and the behaviour of one GTPase depends on the total effect of all its GEFs and GAPs), or split (one GTPase induces many effects) . This versatility allows small GTPases to mediate a wide range of different biological functions among different organisms. For example in S. cerevisiae RasGEF CDC25 is required for Ras mediated activation of adenylyl cyclase and it is essential for proliferation and spore germination , whereas in Drosophila, the RasGEF Son-of-sevenless (Sos) functions upstream of Ras in R7 photoreceptor differentiation .
Despite its relatively small genome, Dictyostelium possesses a relatively large number of ras and rasGEF encoding genes. Dictyostelium is a lower eukaryote with a simple life cycle, consisting of growth and multicellular development, the latter being fully completed in approximately 24 hours. The amoebae live as single cells, growing by feeding on bacteria, which are taken up by phagocytosis, and dividing by binary fission. Upon starvation, cells start releasing the chemoattractant cAMP and gather by chemotaxis to form multicellular aggregates. Within each aggregate, cells differentiate into prespore and several classes of prestalk cells, while undergoing a series of morphogenetic changes, which end up in the formation of fruiting bodies. Fruiting bodies consist of slender stalks of vacuolated, dead cells, bearing on top spores encapsulated in sori.
Here we report the identification and characterization of a novel RasGEF, named RasGEFM. To date only four Dictyostelium RasGEFs have been characterized, namely rasGEFA, formerly known as aimless , rasGEFB , gbpC and gbpD . The latter two encode for unconventional RasGEFs. GbpC possesses a RasGEF domain coupled to a cyclic nucleotide-binding domain, which is associated with a MAPKKK-like kinase domain, Leucine Rich Repeats (LRR) and a Ras domain. GbpD is highly similar to GbpC, but it lacks the Ras, MAPKKK-like and the LRR domains . These two proteins control myosin phosphorylation and, as consequence, cell motility and chemotaxis . The GEF protein encoded by the rasGEFA gene is essential for cell aggregation, acting at the level of adenylyl cyclase activation . Dictyostelium cells lacking rasGEFB are defective in early development, although they eventually form tiny but normally proportioned fruiting bodies. Furthermore, these cells move unusually rapidly and show severe impairment in cell growth .
Here we present evidences that RasGEFM is involved in a Ras regulatory network, required for cAMP receptor dependent signal transduction. Mutant cells lacking the rasGEFM gene produce very small, flat aggregates and fail to develop further. Chemotaxis is altered in the mutant, due to inability of the cells to polarize properly. The phenotype can be partially rescued by pulsing cells with cAMP. We show that RasGEFM is involved in controlling cAMP relay and cell motility.
Identification, cloning and sequence analysis of D.d.rasGEFM
To identify Ras regulator proteins in the Dictyostelium genome a bioinformatic approach was taken, based on a tBLASTn algorithm, to search the Dictyostelium genome project databases  and . Using known RasGEFs domains as query, we identified approximately 30 genes encoding for proteins with significant homology to putative RasGEF proteins (unpublished results). Among the putative RasGEF encoding genes, one of these, designated as Dd rasGEFM, was isolated for functional studies. The rasGEFM gene, located on the chromosome 2, is organized in 4 exons, which are interrupted by 3 introns. Southern blot analyses performed under high and low stringency conditions, and extensive analysis of the Dictyostelium genomic sequence databases, indicated that the gene is present as single locus (data not shown). The rasGEFM gene encodes a relatively large multi-domain protein of 929 amino acids, with a calculated molecular mass of approximately 102 kDa, whose domain structure is displayed in Fig. 1. The amino-terminal region of the protein contains six short poly-proline stretches, which are putative consensus docking site for SH3 or WW domains  whereas the carboxy-terminal region possesses the putative RasGEF catalytic domain. BLAST analysis shows that the catalytic domain, with evolutionary identical amino acid residues conserved, shares high homology with the murine p140GRF (30% identity, 59% homology). Among Dictyostelium RasGEF proteins, the most similar to RasGEFM are two putative uncharacterized RasGEFs (RasGEFE and RasGEFJ) (Fig. 1).
rasGEFM gene expression is developmentally regulated and partially controlled by the G protein
We examined rasGEFM expression in the LW6 mutant lacking the β subunit of heterotrimeric G protein . Although two Gβ genes are present in the Dictyostelium genome, disruption of one of them blocks all G protein-dependent pathways in LW6 mutant cells, which fail to respond to and to relay cAMP, to express aggregation specific genes and to develop. In this strain only the rasGEFM upper transcript is detected during starvation, while the lower transcript fails to accumulate (Fig. 2B). This indicates that up-regulation of the lower transcript is under control of the heterotrimeric G protein. Gene expression was also analyzed in the HSB1 and HSB50 mutants. HSB1 is defective in cAMP relay, due to a temperature-sensitive mutation in the adapter protein PIA, which is essential for adenylyl cyclase activation [21, 22]. HSB1 cells thus fail to aggregate but, in contrast to LW6, express at moderate level aggregation-specific genes and are sensitive to exogenous cAMP pulses. The HSB50 mutant undergoes chemotaxis and aggregation, but is blocked at mound stage . In both mutants, the rasGEFM expression pattern was similar to that observed in the parental strain (Fig. 2B), suggesting that transcription of the lower transcript does not require cAMP relay, even though it may be enhanced by cAMP pulses, similarly to other aggregation-specific genes . It is worth mentioning that the two transcripts may arise from differential splicing or different degree of polyadenylation. Screening 24 cDNA clones obtained by using mRNA from 6-h starved cells resulted in a signle sized cDNA to be present. Sequencing three such clones gave rise to a transcript containing all four exons. Assuming this transcript to correspond to the most abundant mRNA species at this time point, namely the lower band, then we should conclude that differential splicing is unlikely and the upper band is the result of extensive polyadenylation. Additional experiments are required to confirm this hypothesis and to understand this intriguing developmentally regulated changes in rasGEFM gene expression.
rasGEFM null mutant is delayed in the acquisition of aggregation competence and blocked at the aggregation stage
To examine whether rasGEFM null cells were impaired in their ability to chemotax towards cAMP, a micropipette-based assay was used. HSB61 cells resulted strongly impaired in chemotaxis, as will be described further below, but their behaviour improved significantly when pulsed with cAMP. Taken together these results suggest that HSB61 cells fail to aggregate properly, due to impaired periodic cAMP signalling, which is required for optimal expression of aggregation-stage specific genes.
Cyclic AMP receptor and G protein dependent activation of adenylyl and guanylyl cyclases is altered in rasGEFM null cells
The finding that the HSB61 mutant was delayed in the acquisition of the aggregation competence but partially responded to cAMP pulses suggested that the RasGEFM protein might be involved in cAMP receptor-mediated signaling. We therefore monitored cAMP accumulation during the early hours of development and tested adenylyl and guanylyl cyclase activities in vitro.
These data strengthen the hypothesis that the strongly reduced adenylyl cyclase activation in HSB61 cells is G protein independent, and suggest that Ras GEFM may be located upstream of the heterotrimeric G protein and downstream of the cAMP receptor. If this hypothesis is correct, cAMP binding to membranes upon treatment with GTPγS should be altered in the mutant compared to the wild type, since the affinity of cAMP receptors differs when they are complexed with G proteins . Consistent with this hypothesis, cAMP binding to HSB61 membranes was not affected by GTPγS, whereas it was inhibited cAMP binding approximately 50% in wild type membranes (Fig. 10B).
Taken together, these results lead us to conclude that Ras GEFM very likely acts between the cAMP receptor and the heterotrimeric G protein.
RasGEFM is not the activator of RasC or RasG
Ca2+ influx in rasGEFM null mutant is reduced
In addition to the effects on cAMP and cGMP levels, the second messenger Ca2+ is rapidly regulated by cAMP binding to the receptor . The finding that chemotaxis and cGMP response were altered in the mutant, prompted us to test chemoattractant-induced Ca2+ entry in HSB61 cells. Ca2+ entry is negatively regulated by cGMP , and both Ca2+ and cGMP have been implicated in regulating chemotactic motility .
As shown in Fig. 12Bd, AX2 cells at 8 hours of development, which corresponds to late aggregation stage, displayed a rapid Ca2+ influx in response to cAMP, followed by a very slow efflux. Later on in development, no efflux could be detected . In HSB61 cells, even when pulsed for as long as 12 hours, the kinetics of efflux was similar to 4–6 hours AX2 pulsed cells (Fig. 11B, b and c). Thus, these data further confirm that the mutant development is delayed even after 12 hours pulsing with cAMP. In addition, the finding that maximal Ca2+ entry in response to cAMP is reduced in the mutant may account, at least in part, for the chemotactic defect of the mutant.
Mutant HSB61 is impaired in chemotactic, but not in spontaneous cell motility
When cell motility was analysed, differences between mutant and parental strains were only detected during chemotaxis. Spontaneous cell motility was indistinguishable between both cell types. Directional cell migration in response to external chemoattractant gradients implies at least three steps: a) sensing the chemoattractant, b) formation of a leading front and c) cell polarization, with suppression of lateral pseudopods, followed by forward movement and uropod detachment .
Additional File 1: Chemotaxis of AX2 after 5 hours of starvation.mov: 6.5 MB. Cells were seeded onto 35 mm/glass base dish (Iwaki) and subjected to a cAMP filled micropipette assay, as described in Material and Methods. Images were recorded in a Panasonic videorecorder (AG-TL700) with a ZVS-47 DE camera (Zeiss) mounted on an Axiovert HAL100, using Neofluar 100X/1.3 oil immersion objective and DIC filter. The recorded time-lapse movie was transferred to a computer using USB Istant Video (ADS Technologies). (MOV 7 MB)
Additional File 2: Chemotaxis of HSB61 after 5 hours of starvation.mov: 6.9 MB (MOV 7 MB)
Additional File 3: Chemotaxis of HSB61 starved and pulsed with cAMP for 10 hours.mov: 9.9 MB (MOV 10 MB)
Cyclic AMP pulses failed to induce cell streaming: the HSB61 cells chemotaxed towards the capillary mostly as single cells, suggesting that spontaneous cAMP relay was still reduced in the mutant.
In this study we have reported the isolation and characterization of a novel RasGEF, named RasGEFM, which is required for proper Dictyostelium development. RasGEFM is peculiar in that it contains 6 poly-proline stretches that represent putative interacting sites for SH3, WW and/or EVH1 containing proteins , and a DEP1 motif.
Disrupting rasGEFM results in cells to be blocked at the aggregation stage, forming rather small, flattened aggregates that fail to develop further. The RasGEFM-null phenotype could be partially rescued by pulsing cells with cAMP. Gene expression studies and functional assays indicate that the mutant phenotype is due to defective developmental expression of cAMP relay and are consistent with a role of RasGEFM in regulating cAMP signalling. The mutant phenotype differs, however, from the phenotype displayed by other mutants that are strictly defective in adenylyl cyclase activation, such as mutants in RasGEFA , PIA [21, 22] CRAC  and RasC . PIA and CRAC are cytosolic proteins that couple the G protein to adenylyl cyclase, and RasGEFA as well as RasC have been shown to also be essential for adenylyl cyclase activation. In contrast to rasGEFM null cells, which form small flat aggregates, all these other mutants fail to aggregate, although they respond to cAMP pulses. Their chemotactic motility in response to cAMP diffusing from a microcapillary is also much less impaired, and guanylyl cyclase activation is rather normal and not enhanced as in the rasGEFM null mutant.
These findings indicate that these mutants display different alterations in signal transduction downstream of the cAMP receptor. The RASGEFM protein, in contrast to RasGEFA, PIA and CRAC, appears to be located downstream of the cAR1 receptor, but upstream of the G protein, thus possibly affecting both G protein dependent and G protein independent pathways.
RasGEFM null cells have defects in both adenylyl cyclase activation and in chemotaxis, characteristics similar to those of rasC null cells. It might have been expected, therefore, that RasGEFM would act upstream of RasC. However, we now have direct evidence that RasGEFA directly activates RasC and is the only GEF responsible for the activation of RasC (Kae et al, unpublished). Consistent with this result, there was no major effect on the cAMP dependent activation of both RasC and RasG in the rasGEFM null cells.
Several attempts have been performed to try to rescue the HSB61 phenotype but so far none of them succeeded, although a RasGEFM GFP-fused protein of the correct size was expressed (data not shown). This observation, combined with the rather peculiar rasGEFM mRNA expression, in which two transcripts are detected and regulated independently, suggests that the failed rescue might be due to the necessity for the RasGEFM protein to be expressed in a regulated way and not under the control of a constitutive promoter, such as the actin 15 promoter. Experiments are in progress in this direction and they will be crucial to confirm that the phenotype is due to direct disruption of the rasGEFM gene and not to additional detrimental effects of the transformation. The possibility of a second random mutation, which independently of rasGEFM disruption could be responsible for the observed phenotype, cannot be excluded but it seems unlikely to us. The GTPγS inhibition of cAMP binding, together with the stimulating effects of GTPγS on adenylyl cyclase, and the rescuing effect of cAMP, clearly locate the mutant defect upstream of the G protein and suggest a single event to be involved.
When rasGEFM null cells are challenged with a cAMP filled micropipette, the chemotactic response is impaired. Apparently the cells sense the gradient but they seem incapable of organizing a leading front. Moreover, several lateral pseudopodia are continuously extended and retracted. As a consequence, the cell fails to polarize, maintains a rather flattened shape and moves "spastically" towards the micropipette. If mutant cells are treated with cAMP pulses for at least 6 to 8 hours, chemotactic orientation and speed are improved, and many cells become elongated and move faster towards the micropipette.
Formation of a leading front, acquisition of polarity and suppression of lateral pseudopodia are processes characterized by the redistribution of cytoskeletal components, with F-actin and numerous actin-binding proteins being enriched at the front and myosin II assembled in filaments at the back of cells . These processes are regulated by the balanced action of several sub-cellular components, including second messengers (e.g. Ca2+, cGMP) and proteins such as PI3K and PTEN . A mechanism by which localized Ras activation mediates leading edge formation through activation of PI3K and other Ras effectors required for chemotaxis has been recently proposed for Dictyostelium cells by Sasaki et al. .
Cyclic AMP elicits in the mutant a biphasic actin polymerization response comparable to the parental strain, but the absolute peak values are strongly reduced. Strikingly, when mutant cells are pulsed for additional 2–4 hours the actin response is comparable to that of the wild type. These results suggest to us that RasGEFM is not directly involved in mediating a putative Ras-induced actin polymerization. They are instead consistent with the notion that a developmentally regulated component, required for proper actin recruitment in response to chemoattractant, is absent or expressed under a threshold level in the mutant and is induced by prolonged cAMP treatment.
Taken together our findings support a role for RasGEF M in developmental acquisition of chemotactic efficiency and aggregation competence. The proposed localization of Ras GEFM downstream the cAMP receptor lead us to suggest that Ras GEFM and its cognate Ras may act as dissociating components between G protein coupled receptors and the G protein or molecular switches acting in a G protein independent way. A physical interaction between β 1 adrenergic receptors and a RasGEF has been recently reported in mammalian cells .
A peculiarity of the RasGEFM null mutant is the elevated cyclic GMP response to cAMP stimulation both at early and late starvation times, which suggests a role for RasGEFM as negative regulator of cAMP receptor-induced guanylyl cyclase activation. Cyclic GMP accumulation has been proposed to be regulated by adaptation  and to inhibit pseudopodia formation at the back of the cell by inducing myosin filament formation in the cell cortex [42–45]. Myosin filament formation at the presumptive leading front would be counteracted by myosin phosphorylation due to myosin heavy chain kinase A (MHCK-A), which is selectively recruited at the leading front . As a result, a high cGMP concentration leads to improved orientation in a chemoattractant gradient, as shown in mutant cells lacking cGMP phosphodiesterase . RasGEFM null cells, challenged with cAMP diffusing from a microcapillary, show, however, reduced orientation and increased lateral pseudopodia, despite a stronger cGMP response to cAMP. It may be argued that cGMP is rapidly hydrolysed in the HSB61 mutant, in contrast to the cGMP phosphodiesterase null cells. However, if cGMP is down-regulated by adaptation, it must be assumed that as long as cells are exposed to a chemoattractant gradient, as occurs when they are stimulated with cAMP diffusing from a microcapillary, adaptation of guanylyl cyclase is switched off , and this should lead in RasGEF M null cells to constantly higher levels of cGMP. Why is then chemotactic orientation reduced in the mutant?
A possible explanation for this discrepancy is offered by the reduced actin response to chemoattractant. We have suggested that a developmentally regulated component, which is required for actin recruitment at the presumptive leading front, is lacking/down-regulated in the mutant. Strongly reduced actin polymerization at the front would result in impaired translocation of myosin heavy chain kinase A (MHCK-A), which has been shown to require F-actin binding . As a consequence, a high cGMP level in the mutant, as in wild type cells or PDE null mutants, would induce myosin filaments all over the cell, consistent with its proposed role as global inhibitor [33, 45], but myosin filaments in the front would not be dissociated by MHCK-A, due to its impaired recruitment. Thus we propose that in the RasGEFM null mutant chemotaxis is inhibited due firstly to altered F-actin polymerization at the presumptive leading front and secondarily to impaired recruitment of MHCK-A.
All the defects observed in the rasGEFM null mutant can be explained by assuming a modulatory role of the RasGEFM close to the cAMP receptor, which regulates guanylyl and adenylyl cyclases. RasGEFM appears to act as a negative regulator of guanylyl cyclase and a positive regulator of adenylyl cyclase. Currently we have no obvious explanation for this opposite activity. It must be kept in mind, however, that in contrast to adenylyl cyclase, which is stimulated by the Gβ subunit of the heterotrimeric G protein, receptor-dependent stimulation of guanylyl cyclase is more complex and less well understood. Several lines of evidence suggest a role for small GTPases in its activation, independently and in addition to the heterotrimeric G protein [48–50]. If RasGEFM and its putative Ras target act as receptor-linked molecular switches, this may lead to differential, albeit opposite, effects on heterotrimeric G-protein-dependent or independent pathways. Identifying the putative RasGEFM regulated Ras may help in understanding these complex transduction pathways.
Cell cultures, growth, and developmental conditions
Wild type strain AX2-214 and rasGEFM null mutant, referred as HSB61, were grown either in liquid nutrient medium at 23°C under shaking at 150 rpm  or on nutrient agar plates with Escherichia coli B/2 . When cultured in liquid medium, HSB61 cells were supplemented with blasticidin (ICN) at a final concentration of 10 μg/ml. For development on solid substratum, cells were grown to a density of 2–3 × 106 cell/ml, washed three times in 0.017 M Na+/K+ Soerensen phosphate buffer, pH 6.0 and, once deposited on non nutrient 1.5% (w/v) agar plates, allowed to develop at 23°C. For development in suspension, cells were incubated at a concentration of 1 × 107 cell/ml in Soerensen phosphate buffer. For cAMP treatment, pulses of 20 nM cAMP were applied every 6 minutes using a Braun perfusor VI equipped with 10 ml-syringe .
Measurement of EDTA-stable contacts
EDTA-stable contacts were measured as described by using the agglutinometer of Beug and Gerisch .
Chemotaxis was studied by using the microcapillary assay . Briefly, cells were seeded onto 35 mm glass base dishes (Iwaki) at a density of approximately 1 × 105/cm2 and local stimulation of chemotaxis was obtained by passive diffusion of cAMP from a microcapillary (Femtotips1, Eppendorf), filled with a 1.0 mM solution of cAMP. The microcapillary was positioned with an automated Zeiss micromanipulator and cells observed either with a 20× or with a Neofluar 100x/1.3 oil immersion objective, equipped with DIC filter. Images were captured at an interval of 0.66 sec. and recorded in a Panasonic videorecorder (AG-TL700) with a ZVS-47DE camera (Zeiss) mounted on a microscope (Zeiss, Axiovert HAL100). The recorded time-lapse movies were transferred to a computer using USB Instant Video package (ADS Technologies).
Actin polymerization assay
Actin polymerization assays were carried out as previously described [36, 55]. Briefly, cells were starved at 2 × 107cells/ml for 4 and 6 hours and pulsed with 1 μm cAMP. At the indicated time points, 100 μl samples were taken and transferred to 1 ml of actin buffer (20 mM K2PO4, 10 mM PIPES, 5 mM EGTA, 2 mM MgCl2, 3.7% formaldehyde, 0.1% Triton X-100, 0.25 μM TRITC-phalloidin, pH6.8). After shaking for 1 hour at room temperature, samples were centrifuged for 10 minutes at 16000 g and the resulting pellet was resuspended in 1 ml methanol. After shaking overnight, the amount of F-actin was determined by measuring the fluorescence with a fluorimeter (Kontron SFM 25). Setting: excitation wavelength 540 nm, emission 570 nm.
Measurement of cAMP-induced Ca2+-influx
Cells were developed in suspension as described above. For accelerating developmental gene expression cells were pulsed with 20 nM cAMP every 6 minutes, overnight in case of HSB61 cells, and for 2 hours in case of wild-type cells. At appropriate time points the cells were washed in nominally Ca2+ free tricine buffer (tricine pH 7.0, supplemented with 5 mM KCl) and resuspended at a cell density of 5 × 107 cells/ml. The cell suspension was then stimulated with cAMP and Ca2+-influx measured with a Ca2+-sensitive electrode (Möller) and a voltmeter (Metrohm). Statistical analysis was performed with "Wilcoxon test". Light scattering measurements of cells in suspension were done has described by Gerisch and Hess . The cell suspension (2 × 107cell/ml) was aerated in a cuvette and extinction was concomitantly monitored at 500 nm in a Zeiss PM6 spectrophotometer.
In vitro stimulation of adenylyl cyclase
Adenylyl cyclase in Dictyostelium lysates was assayed in the presence of 2'-deoxy-cAMP or GTPγS as described by Lilly and Devreotes . Briefly starving cells were treated with 20 nM cAMP pulses every 6 min for different hours. A total of 1 × 108 cells were pelleted and resuspended in 1 ml of Soerensen phosphate buffer. An equal volume of ice cold lysis buffer containing either 4 mM MgCl2 or 4 mM MnCl2, 20 mM Tris pH 8.0 was added. Cells were lysed by passage through a 3-μm pore size Nucleopore membrane in the absence or presence of 30 μM GTPγS or 50 μM 2'-deoxy-cAMP and the lysates incubated on ice for 5 min. A 40 μl aliquot of cell lysate was added to a 40 μl assay mix (20 mM DTT, 1 mM ATP, 2 mM MgCl2 or 2 mM MnCl2 in 10 mM Tris pH 8.0, 0.4 mM IBMX) and incubated at 20°C for 5 min.
The reaction was stopped, by adding 40 μl 0.1 M EDTA pH 8.0 and boiling the sample for 2 min. The total concentration of cAMP in the samples was determined by using the "Biotrak cAMP assay Kit" according to manufacturer's instructions (Amersham Pharmacia Biotech).
cAMP binding assays
Binding of cAMP to cell surface receptors was determined as previously described by Van Haastert . Briefly, wild-type and mutant cells were starved by shaking in Soerensen phosphate buffer, without or with cAMP pulses for 5 or 8 hours respectively, washed and resuspended at a density of 1 × 108 cell/ml.
Aliquots of 80 μl of cells were incubated with a radioactive binding mixture, containing 300 nM of [3H]cAMP (Amersham Pharmacia Biotech), 50 mM dithiothreitol in 90% saturated ammonium sulphate, and a variety of cAMP concentration ranging between 700 to 19700 nM. Specific binding was obtained by subtracting non specific binding determined in the presence of 1 mM cAMP.
After 5 min. incubation at 0°C, cells were collected by centrifugation at 14000 × g for 2 min, the pellet resuspended in 100 μl of 0.1 M acetic acid and dissolved in 1.3 ml scintillation fluid. Scatchard plots of cAMP binding were done with GraphPad software (GraphPad Inc.).
GTP-inhibition of cAMP binding to plasma membranes
The assay was performed as described earlier . Brieflly, GTP-inhibition of cAMP binding was measured in a total volume of 100 μl containing PB (10 mM KH2PO4/Na2HPO4 pH 6.5), 5 nM [3H] cAMP, 10 mM dithiothreitol, GTPγS (300 μM when present) and 70 μl membranes. Samples were incubated 5 min. at 0°C, centrifuged for 2 min. at 14000 × g, the supernatant was aspirated and the pellet dissolved in 100 μl acetic acid. Radioactivity was determined after the addition of 1.3 ml of liquid scintillation.
Cyclic AMP and cGMP determination
For the determination of cyclic nucleotides, cells aliquots were taken at different time points before and after a cAMP pulse, and quenched with 1 vol of 2 N perchloric acid . After centrifugation, neutralization of the supernatant with potassium carbonate, and acetylation, the concentration of cAMP and cGMP in the extract was measured using the 125I radioimmunoassay kit according to manufacturer's instructions (Amersham Pharmacia Biotech).
RBD binding assay
The RBD binding assay was performed as described elsewhere . Briefly, 6 h or 10 h pulsed cells were washed twice and resuspended at 5 × 107 cell/ml in Soerensen phosphate buffer. Cells were stimulated with 200 nM cAMP, aliquots (0.5 ml) were selected at the indicated time points and lysed in an equal volume of ice-cold 2× HK-LB (20 mM sodium phosphate pH 7.2, 2% Triton X-100, 20% glycerol, 300 mM NaCl, 20 mM MgCl2, 2 mM EDTA, 2 mM Na3VO4, 10 mM NaF, with protease inhibitor Roche), and incubated on ice for 5 min. The lysates were cleared by centrifugation for 10 min and protein concentrations were determined using DC Protein Assay (Bio-Rad). A 0.8 mg portion of protein lysates was incubated with 100 μg of GST-Byr2 (RBD) and the mixture was incubated at 4°C for 1 h. Beads were harvested by centrifugation and washed three times in 1× HK-LB. A volume of 40 μl of 1× SDS gel loading buffer was added to the pellet beads and the mixture was boiled for 5 min. Samples were fractionated by SDS-PAGE, blotted, blocked and probed with RasC or RasG antibody. Bands were determined by enhanced chemiluminescence reaction (Amersham Pharmacia Biotech).
Molecular cloning and sequence analysis
The rasGEFM gene was isolated using PCR based method. Through the sequence derived from DNA database screening [13, 14] a pair of primers was designed (5'-ATGATGAATGAAGTTTCTTCAAATTC-3' and 5'-CCATCGATAATTATCTAAATAATGGATTTGA-3') and used for PCR amplification. As template, cDNA isolated with "First strand cDNA synthesis kit" (Amersham Pharmacia Biotech), from RNA of cells developed for 5 hours on solid substrata, was used. The DNA fragment, of approximately 2.7 kb, was then ligated into "pGEM-T easy vector" (PROMEGA) and cloned into DH5α E. coli strain. PCR products were purified from gel with "High Pure PCR Product Purification" kit (Roche) and verified by sequencing.
Construction of the D.d. rasGEFM null strain (HSB61)
To construct the rasGEFM null strain the rasGEFM locus was disrupted via homologous recombination. The blasticidin resistance gene (bsr), used as selectable marker, was excised from pUCBsrΔ Bam  with Hind III and Xba I and subsequently inserted into the JC2a86b07.s1 clone (Dictyostelium genome project) digested with Hind III and Xba I. Subsequently the N-terminal portion (from position 0 to 617 bp) of the gene was ligated into Kpn I site of the above vector, using the "DNA ligation kit" (Amersham Pharmacia Biotech). The vector, carrying the selectable marker was electroporated  into parental strain and transformed cells selected for blasticidin resistence. Resistant cells were cloned, and clones subsequently screened via Southern blot in order to identify Dictyostelium clones in which the RasGEF M locus was disrupted.
Southern and Northern hybridization analysis
Genomic DNA was extracted and purified by CsCl gradient centrifugation as previously described by Nellen, et al. , digested with EcoR I, run onto 0.8% agarose gel, blotted onto Hybond-N membrane (Amersham Pharmacia Biotech), and subjected to Southern assay . The membrane was probed with a 800 bp RasGEF M cDNA specific probe, corresponding to bp. 760–1518 of the cDNA clone, previously radiolabelled by the "Megaprimer™DNA Labelling System" using [α32] dATP (Amersham).
For Northern blots, total RNA was prepared using TRIZOL reagent (GIBCO) according to manifacturer's instructions. RNA was then resuspended in DEPC treated water, quantified, and 15 μg were size separated on 1.2% agarose gel in presence of formaldehyde. Equal loading of samples, was checked by probing membranes with the actin gene. The radiolabelled DNA fragments used as probes were as follow: car1, csA, acaA (fragment from 2.7 kb to 4.2 kb of cDNA clone), RasGEF M. After being hybridised with the first cDNA probe, Northern blots were stripped with 0.1% SDS in boiling water and then re-hybridised with a second probe.
We thank Barbara Peracino for technical help and D. Malchow for helpful suggestions. This work was supported by funds of MIUR (cofin 2002) to S.B. and local funds of the University of Turin to E.B.
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