Some chemotactic mutants can be progress through development in chimeric populations.

Cell migration in response to morphogen gradients affects morphogenesis. Chemotaxis towards adenosine 3', 5'-monophosphate (cAMP) is essential for the early stage of morphogenesis in the slime mold Dictyostelium discoideum. Here, we show that D. discoideum completes morphogenesis without cAMP-chemotaxis-dependent cell migration. The extracellular cAMP gradient is believed to cause cells to form a slug-shaped multicellular structure and fruiting body. The cAMP receptor, cAR1, was not expressed at the cell surface during these stages, correlating with reduced chemotactic activity. Gß-null cells expressing temperature sensitive Gß are unable to generate extracellular cAMP (Jin et al., 1998) and thus unable to aggregate and exhibit proper morphogenesis under restrictive temperature. However, when mixed with wild type cells ts-Gß expressing gß-null cells normally aggregated and exhibited normal morphogenesis under restrictive temperature. Furthermore, cells migrated after aggregation in a mixture containing wild-type cells. KI-5 cells, which do not show aggregation or morphogenesis, spontaneously migrated to a transplanted wild-type tip and underwent normal morphogenesis and cell differentiation; this was not observed in cells lacking tgrB1and tgrC1 cells adhesion molecules. Thus, cAMP gradient-dependent cell migration may not be required for multicellular pattern formation in late Dictyostelium development.

Sycrp2 Is Essential for Twitching Motility in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Two cAMP receptor proteins (CRPs), Sycrp1 (encoded by sll1371) and Sycrp2 (encoded by sll1924), exist in the cyanobacterium Synechocystis sp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report that sycrp2 disruption results in the loss of motility of Synechocystis sp. PCC 6803, and that the phenotype can be recovered by reintroducing the sycrp2 gene into the genome of sycrp2-disrupted mutants. Electron microscopy showed that the sycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of the pilA9-pilA11 operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased in sycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region of pilA9 Together, these findings indicate that in Synechocystis sp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCE cAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacterium Synechocystis sp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

Unusually Situated Binding Sites for Bacterial Transcription Factors Can Have Hidden Functionality.

A commonly accepted paradigm of molecular biology is that transcription factors control gene expression by binding sites at the 5' end of a gene. However, there is growing evidence that transcription factor targets can occur within genes or between convergent genes. In this work, we have investigated one such target for the cyclic AMP receptor protein (CRP) of enterotoxigenic Escherichia coli. We show that CRP binds between two convergent genes. When bound, CRP regulates transcription of a small open reading frame, which we term aatS, embedded within one of the adjacent genes. Our work demonstrates that non-canonical sites of transcription factor binding can have hidden functionality.

Rice TUTOU1 Encodes a Suppressor of cAMP Receptor-Like Protein That Is Important for Actin Organization and Panicle Development.

Panicle development, a key event in rice (Oryza sativa) reproduction and a critical determinant of grain yield, forms a branched structure containing multiple spikelets. Genetic and environmental factors can perturb panicle development, causing panicles to degenerate and producing characteristic whitish, small spikelets with severely reduced fertility and yield; however, little is known about the molecular basis of the formation of degenerating panicles in rice. Here, we report the identification and characterization of the rice panicle degenerative mutant tutou1 (tut1), which shows severe defects in panicle development. The tut1 also shows a pleiotropic phenotype, characterized by short roots, reduced plant height, and abnormal development of anthers and pollen grains. Molecular genetic studies revealed that TUT1 encodes a suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous (SCAR/WAVE)-like protein. We found that TUT1 contains conserved functional domains found in eukaryotic SCAR/WAVE proteins, and was able to activate Actin-related protein2/3 to promote actin nucleation and polymerization in vitro. Consistently, tut1 mutants show defects in the arrangement of actin filaments in trichome. These results indicate that TUT1 is a functional SCAR/WAVE protein and plays an important role in panicle development.

Revisiting the mechanism of activation of cyclic AMP receptor protein (CRP) by cAMP in Escherichia coli: lessons from a subunit-crosslinked form of CRP.

Cyclic AMP receptor protein (CRP), the global transcription regulator in prokaryotes, is active only as a cAMP-CRP complex. Binding of cAMP changes the conformation of CRP, transforming it from a transcriptionally 'inactive' to an 'active' molecule. These conformers are also characterized by distinct biochemical properties including the ability to form an S-S crosslink between the C178 residues of its two monomeric subunits. We studied a CRP variant (CRP(cl)), in which the subunits are crosslinked. We demonstrate that CRP(cl) can activate transcription even in the absence of cAMP. Implications of these results for the crystallographically-determined structure of cAMP-CRP are discussed.

A novel indirect sequence readout component in the E. coli cyclic AMP receptor protein operator.

The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid nucleobase interactions in the major groove of the two operator half-sites. Crystal structure analyses have revealed that the interaction results in two strong kinks at the TG/CA steps closest to the 6-base-pair spacer (N6). This spacer exhibits high sequence variability among the more than 100 natural binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold or abolished by varying the N6 spacer sequences. Furthermore, on the basis of sequence analysis and uranyl (UO2(2+)) probing data, we propose that the underlying mechanism relies on N6 deformability.

How do amoebae swim and crawl?

The surface behaviour of swimming amoebae was followed in cells bearing a cAR1-paGFP (cyclic AMP receptor fused to a photoactivatable-GFP) construct. Sensitized amoebae were placed in a buoyant medium where they could swim toward a chemoattractant cAMP source. paGFP, activated at the cell's front, remained fairly stationary in the cell's frame as the cell advanced; the label was not swept rearwards. Similar experiments with chemotaxing cells attached to a substratum gave the same result. Furthermore, if the region around a lateral projection near a crawling cell's front is marked, the projection and the labelled cAR1 behave differently. The label spreads by diffusion but otherwise remains stationary in the cell's frame; the lateral projection moves rearwards on the cell (remaining stationary with respect to the substrate), so that it ends up outside the labelled region. Furthermore, as cAR1-GFP cells move, they occasionally do so in a remarkably straight line; this suggests they do not need to snake to move on a substratum. Previously, we suggested that the surface membrane of a moving amoeba flows from front to rear as part of a polarised membrane trafficking cycle. This could explain how swimming amoebae are able to exert a force against the medium. Our present results indicate that, in amoebae, the suggested surface flow does not exist: this implies that they swim by shape changes.

Alkaline pH Is a signal for optimal production and secretion of the heat labile toxin, LT in enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) cause secretory diarrhea in children and travelers to endemic areas. ETEC spreads through the fecal-oral route. After ingestion, ETEC passes through the stomach and duodenum before it colonizes the lower part of the small intestine, exposing bacteria to a wide range of pH and environmental conditions. This study aimed to determine the impact of external pH and activity of the Cyclic AMP receptor protein (CRP) on the regulation of production and secretion of heat labile (LT) enterotoxin. ETEC strain E2863wt and its isogenic mutant E2863ΔCRP were grown in LBK media buffered to pH 5, 7 and 9. GM1 ELISA, cDNA and cAMP analyses were carried out on bacterial pellet and supernatant samples derived from 3 and 5 hours growth and from overnight cultures. We confirm that CRP is a repressor of LT transcription and production as has been shown before but we show for the first time that CRP is a positive regulator of LT secretion both in vitro and in vivo. LT secretion increased at neutral to alkaline pH compared to acidic pH 5 where secretion was completely inhibited. At pH 9 secretion of LT was optimal resulting in 600 percent increase of secreted LT compared to unbuffered LBK media. This effect was not due to membrane leakage since the bacteria were viable at pH 9. The results indicate that the transition to the alkaline duodenum and/or exposure to high pH close to the epithelium as well as activation of the global transcription factor CRP are signals that induce secretion of the LT toxin in ETEC.

Cyclic AMP receptor protein regulates pheromone-mediated bioluminescence at multiple levels in Vibrio fischeri ES114.

Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45-50, 1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170:4040-4046, 1988; P. V. Dunlap, J. Bacteriol. 171:1199-1202, 1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87-91, 1983). Consistent with this model, we found that bioluminescence in V. fischeri ES114 is modulated by glucose and stimulated by cAMP. In addition, a Δcrp mutant was ∼100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking crp were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only luxR but also the alternative pheromone synthase gene ainS. We found that His-tagged V. fischeri CRP could bind sequences upstream of both luxR and ainS, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the ainS or luxR system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in transgenic Escherichia coli, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the luxI promoter was enhanced by CRP. Second, the effect of CRP on the ainS promoter depended on whether the V. fischeri regulatory gene litR was also introduced. These results suggest an integral role for CRP in pheromone signaling that goes beyond sensing cell density.

The NMRA/NMRAL1 homologue PadA modulates the expression of extracellular cAMP relay genes during aggregation in Dictyostelium discoideum.

NMRA-like proteins belong to a class of conserved transcriptional regulators that function as direct sensors of the metabolic state of the cell and link basic metabolism to changes in gene expression. PadA was the first NMRA-like protein described in Dictyostelium discoideum and was shown to be necessary for prestalk cell differentiation and correct development. We describe and characterize padA(-) mutant phenotype during the onset of development, which results in the formation of abnormally small territories and impairment of cAMP responses. Transcriptional analysis shows that cAMP-induced gene expression is downregulated in padA(-), particularly the genes that establish the extracellular cAMP relay. The mutant phenotype can be rescued with the constitutive expression of one of these genes, carA, encoding the cAMP receptor. Transcriptional analysis of padA(-)/A15::carA showed that carA maximum mRNA levels were not reached during aggregation. Our data support a regulatory role for PadA on the regulation of extracellular cAMP relay genes during aggregation and suggest that PadA is required to achieve carA full induction.

Transcriptional modulation of enterotoxigenic Escherichia coli virulence genes in response to epithelial cell interactions.

Enterotoxigenic Escherichia coli (ETEC) strains are a leading cause of morbidity and mortality due to diarrheal illness in developing countries. There is currently no effective vaccine against these important pathogens. Because genes modulated by pathogen-host interactions potentially encode putative vaccine targets, we investigated changes in gene expression and surface morphology of ETEC upon interaction with intestinal epithelial cells in vitro. Pan-genome microarrays, quantitative reverse transcriptase PCR (qRT-PCR), and transcriptional reporter fusions of selected promoters were used to study changes in ETEC transcriptomes. Flow cytometry, immunofluorescence microscopy, and scanning electron microscopy were used to investigate alterations in surface antigen expression and morphology following pathogen-host interactions. Following host cell contact, genes for motility, adhesion, toxin production, immunodominant peptides, and key regulatory molecules, including cyclic AMP (cAMP) receptor protein (CRP) and c-di-GMP, were substantially modulated. These changes were accompanied by visible changes in both ETEC architecture and the expression of surface antigens, including a novel highly conserved adhesin molecule, EaeH. The studies reported here suggest that pathogen-host interactions are finely orchestrated by ETEC and are characterized by coordinated responses involving the sequential deployment of multiple virulence molecules. Elucidation of the molecular details of these interactions could highlight novel strategies for development of vaccines for these important pathogens.

Seventeen Sxy-dependent cyclic AMP receptor protein site-regulated genes are needed for natural transformation in Haemophilus influenzae.

Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. In Haemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized.

Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance.

One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1-MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1-MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h(-1) while that of wild type was 0.20 h(-1). When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h(-1)) became twice that of the wild type (0.09 h(-1)). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering.

The TonB3 system in the human pathogen Vibrio vulnificus is under the control of the global regulators Lrp and cyclic AMP receptor protein.

TonB systems transduce the proton motive force of the cytoplasmic membrane to energize substrate transport through a specific TonB-dependent transporter across the outer membrane. Vibrio vulnificus, an opportunistic marine pathogen that can cause a fatal septicemic disease in humans and eels, possesses three TonB systems. While the TonB1 and TonB2 systems are iron regulated, the TonB3 system is induced when the bacterium grows in human serum. In this work we have determined the essential roles of the leucine-responsive protein (Lrp) and cyclic AMP (cAMP) receptor protein (CRP) in the transcriptional activation of this system. Whereas Lrp shows at least four very distinctive DNA binding regions spread out from position -59 to -509, cAMP-CRP binds exclusively in a region centered at position -122.5 from the start point of the transcription. Our results suggest that both proteins bind simultaneously to the region closer to the RNA polymerase binding site. Importantly, we report that the TonB3 system is induced not only by serum but also during growth in minimal medium with glycerol as the sole carbon source and low concentrations of Casamino Acids. In addition to catabolite repression by glucose, l-leucine acts by inhibiting the binding of Lrp to the promoter region, hence preventing transcription of the TonB3 operon. Thus, this TonB system is under the direct control of two global regulators that can integrate different environmental signals (i.e., glucose starvation and the transition between "feast" and "famine"). These results shed light on new mechanisms of regulation for a TonB system that could be widespread in other organisms.

Actin cross-linking proteins cortexillin I and II are required for cAMP signaling during Dictyostelium chemotaxis and development.

Starvation induces Dictyostelium amoebae to secrete cAMP, toward which other amoebae stream, forming multicellular mounds that differentiate and develop into fruiting bodies containing spores. We find that the double deletion of cortexillin (ctx) I and II alters the actin cytoskeleton and substantially inhibits all molecular responses to extracellular cAMP. Synthesis of cAMP receptor and adenylyl cyclase A (ACA) is inhibited, and activation of ACA, RasC, and RasG, phosphorylation of extracellular signal regulated kinase 2, activation of TORC2, and stimulation of actin polymerization and myosin assembly are greatly reduced. As a consequence, cell streaming and development are completely blocked. Expression of ACA-yellow fluorescent protein in the ctxI/ctxII-null cells significantly rescues the wild-type phenotype, indicating that the primary chemotaxis and development defect is the inhibition of ACA synthesis and cAMP production. These results demonstrate the critical importance of a properly organized actin cytoskeleton for cAMP-signaling pathways, chemotaxis, and development in Dictyostelium.

Phenotype, virulence and immunogenicity of Edwardsiella ictaluri cyclic adenosine 3',5'-monophosphate receptor protein (Crp) mutants in catfish host.

Edwardsiella ictaluri is an Enterobacteriaceae that causes lethal enteric septicemia in catfish. Being a mucosal facultative intracellular pathogen, this bacterium is an excellent candidate to develop immersion-oral live attenuated vaccines for the catfish aquaculture industry. Deletion of the cyclic 3',5'-adenosine monophosphate (cAMP) receptor protein (crp) gene in several Enterobacteriaceae has been utilized in live attenuated vaccines for mammals and birds. Here we characterize the crp gene and report the effect of a crp deletion in E. ictaluri. The E. ictaluri crp gene and encoded protein are similar to other Enterobacteriaceae family members, complementing Salmonella enterica Δcrp mutants in a cAMP-dependent fashion. The E. ictaluri Δcrp-10 in-frame deletion mutant demonstrated growth defects, loss of maltose utilization, and lack of flagella synthesis. We found that the E. ictaluri Δcrp-10 mutant was attenuated, colonized lymphoid tissues, and conferred immune protection against E. ictaluri infection to zebrafish (Danio rerio) and catfish (Ictalurus punctatus). Evaluation of the IgM titers indicated that bath immunization with the E. ictaluri Δcrp-10 mutant triggered systemic and skin immune responses in catfish. We propose that deletion of the crp gene in E. ictaluri is an effective strategy to develop immersion live attenuated antibiotic-sensitive vaccines for the catfish aquaculture industry.

Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP.

Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an "on/off" signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. Coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria . Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of the bioluminescent signal from aequorin. The "on/off" bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as In Vivo cyclic AMP detection and/or imaging.

Disrupting microtubule network immobilizes amoeboid chemotactic receptor in the plasma membrane.

Signaling cascades are initiated in the plasma membrane via activation of one molecule by another. The interaction depends on the mutual availability of the molecules to each other and this is determined by their localization and lateral diffusion in the cell membrane. The cytoskeleton plays a very important role in this process by enhancing or restricting the possibility of the signaling partners to meet in the plasma membrane. In this study we explored the mode of diffusion of the cAMP receptor, cAR1, in the plasma membrane of Dictyostelium discoideum cells and how this is regulated by the cytoskeleton. Single-particle tracking of fluorescently labeled cAR1 using Total Internal Reflection Microscopy showed that 70% of the cAR1 molecules were mobile. These receptors showed directed motion and we demonstrate that this is not because of tracking along the actin cytoskeleton. Instead, destabilization of the microtubules abolished cAR1 mobility in the plasma membrane and this was confirmed by Fluorescence Recovery after Photobleaching. As a result of microtubule stabilization, one of the first downstream signaling events, the jump of the PH domain of CRAC, was decreased. These results suggest a role for microtubules in cAR1 dynamics and in the ability of cAR1 molecules to interact with their signaling partners.

The tib adherence locus of enterotoxigenic Escherichia coli is regulated by cyclic AMP receptor protein.

Enterotoxigenic Escherichia coli (ETEC) is a Gram-negative enteric pathogen that causes profuse watery diarrhea through the elaboration of heat-labile and/or heat-stable toxins. Virulence is also dependent upon the expression of adhesive pili and afimbrial adhesins that allow the pathogen to adhere to the intestinal epithelium or mucosa. Both types of enterotoxins are regulated at the level of transcription by cyclic AMP (cAMP) receptor protein (CRP). To further our understanding of virulence gene regulation, an in silico approach was used to identify putative CRP binding sites in the genome of H10407 (O78:H11), an ETEC strain that was originally isolated from the stool of a Bangledeshi patient with cholera-like symptoms circa 1971. One of the predicted binding sites was located within an intergenic region upstream of tibDBCA. TibA is an autotransporter and afimbrial adhesin that is glycosylated by TibC. Expression of the TibA glycoprotein was abolished in an H10407 crp mutant and restored when crp was provided in trans. TibA-dependent aggregation was also abolished in a cyaA::kan strain and restored by addition of exogenous cAMP to the growth medium. DNase I footprinting confirmed that the predicted site upstream of tibDBCA is bound by CRP. Point mutations within the CRP binding site were found to abolish or significantly impair CRP-dependent activation of the tibDB promoter. Thus, these studies demonstrate that CRP positively regulates the expression of the glycosylated afimbrial adhesin TibA through occupancy of a binding site within tibDBp.

Chemoattractant-mediated Rap1 activation requires GPCR/G proteins.

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through the G-protein coupled receptors cAR1/cAR3 and Gα2 proteins in a way independent of the cGMP signaling pathway.