Yersinia enterocolitica outer protein T (YopT)

Pathogenic Yersinia strains evade the innate immune responses of the host by producing effector proteins (Yersinia outer proteins (Yops)), which are directly injected into mammalian cells by a type III secretion system (TTSS). One of these effector proteins (YopT) disrupts the actin cytoskeleton of the host cell. YopT is a cysteine protease which cleaves Rho proteins directly upstream of the post-translationally modified cysteine. Thereby, it releases the GTPases from the membrane leading to their inactivation. Besides a biochemical characterisation of the molecular mechanism and substrate specificity also delivery into host cells with chaperone binding and guidance to the injection apparatus and the patho-physiological role of YopT have been studied and are summarised in this review.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.     

Rho GTPases Are Involved in the Regulation of NF-κB by Genotoxic Stress

A common cellular response to genotoxic agents and inflammatory cytokines is the activation of NF-κB. Here, we addressed the question of whether small GTPases of the Rho family are involved in the stimulation of NF-κB signaling by genotoxic agents or TNFα in HeLa cells. Inhibition of isoprenylation of Rho proteins by use of the HMG-CoA reductase inhibitor lovastatin attenuated UV-, doxorubicin-, and TNFα-induced degradation of IκBα as well as drug-stimulated DNA binding activity of NF-κB. Furthermore, NF-κB-regulated gene expression stimulated by either UV irradiation or treatment with TNFα was abrogated by lovastatin pretreatment. This indicates that isoprenylated regulatory proteins participate in the regulation of NF-κB by DNA-damaging agents as well as by TNFα. Specific blockage of Rho signaling by Clostridium difficile toxin B attenuated UV- and doxorubicin-induced activation of NF-κB, but did not affect stimulation of NF-κB by TNFα. Obviously, signaling to NF-κB by genotoxic and nongenotoxic stimuli occurs via different molecular mechanisms, either involving Rho GTPases or not. Based on the data, we suggest Rho GTPases to be essentially required for genotoxic stress-induced signaling to NF-κB.     

A stretch of polybasic residues mediates Cdc42 GTPase-activating protein (CdGAP) binding to phosphatidylinositol 3,4,5-trisphosphate and regulates its GAP activity

The Rho family of small GTPases are membrane-associated molecular switches involved in the control of a wide range of cellular activities, including cell migration, adhesion, and proliferation. Cdc42 GTPase-activating protein (CdGAP) is a phosphoprotein showing GAP activity toward Rac1 and Cdc42. CdGAP activity is regulated in an adhesion-dependent manner and more recently, we have identified CdGAP as a novel molecular target in signaling and an essential component in the synergistic interaction between TGFβ and Neu/ErbB-2 signaling pathways in breast cancer cells. In this study, we identified a small polybasic region (PBR) preceding the RhoGAP domain that mediates specific binding to negatively charged phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). In vitro reconstitution of membrane vesicles loaded with prenylated Rac1 demonstrates that the PBR is required for full activation of CdGAP in the presence of PI(3,4,5)P3. In fibroblast cells, the expression of CdGAP protein mutants lacking an intact PBR shows a significant reduced ability of the protein mutants to induce cell rounding or to mediate negative effects on cell spreading. Furthermore, an intact PBR is required for CdGAP to inactivate Rac1 signaling into cells, whereas it is not essential in an in vitro context. Altogether, these studies reveal that specific interaction between negatively charged phospholipid PI(3,4,5)P3 and the stretch of polybasic residues preceding the RhoGAP domain regulates CdGAP activity in vivo and is required for its cellular functions.     

The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences

Many small GTPases in the Ras and Rho families have a C-terminal polybasic region (PBR) comprised of multiple lysines or arginines. The PBR controls diverse functions of these small GTPases, including their ability to associate with membranes, interact with specific proteins, and localize in subcellular compartments. Different signaling pathways mediated by Ras and Rho family members may converge when the small GTPases are directed by their PBRs to shared binding sites in specific proteins or at cell membranes. The PBR promotes the interactions of small GTPases with SmgGDS, which is a nucleocytoplasmic shuttling protein that stimulates guanine nucleotide exchange by small GTPases. The PBR of Rac1 was recently found to have a functional nuclear localization signal (NLS) sequence, which enhances the nuclear accumulation of protein complexes containing SmgGDS and Rac1. Sequence analysis demonstrates that canonical NLS sequences (K-K/R-x-K/R) are present in the PBRs of additional Ras and Rho family members, and are evolutionarily conserved across several phyla. These findings suggest that the PBR regulates the nucleocytoplasmic shuttling of some Ras and Rho family members when they are in protein complexes that are too large to diffuse through nuclear pores. These diverse functions of the PBR indicate its critical role in signaling by Ras and Rho family GTPases.     

The Yersinia enterocolitica type 3 secretion system (T3SS) as toolbox for studying the cell biological effects of bacterial Rho GTPase modulating T3SS effector proteins

The bacterial effector proteins IpgB(1) and IpgB(2) of Shigella and Map of Escherichia coli activate the Rho GTPases Rac1, RhoA and Cdc42, respectively, whereas YopE and YopT of Yersinia inhibit these Rho family GTPases. We established a Yersinia toolbox which allows to study the cellular effects of these effectors in different combinations in the context of Yersinia type 3 secretion system (Ysc)-T3SS-mediated injection into HeLa cells. For this purpose hybrid proteins were constructed by fusion of YopE with the effector protein of interest. As expected, injected hybrid proteins induced membrane ruffles and Yersinia uptake for IpgB(1) , stress fibres for IpgB(2) and microspikes for Map. By co-infection experiments we could demonstrate (i) IpgB(2) -mediated and ROCK-dependent inhibition of IpgB(1) -mediated Rac1 effects, (ii) YopT-mediated suppression of IpgB(1) -induced Yersinia invasion and (iii) failure of YopE-mediated suppression of IpgB(1) -induced Yersinia invasion, presumably due to preferential inhibition of RhoG by YopE GAP function. By infecting polarized MDCK cells we could demonstrate that Map or IpgB(1) but not IpgB(2) affects cell monolayer integrity. In summary, the Yersinia toolbox is suitable to study cellular effects of effector proteins of diverse bacterial species separately or in combination in the context of bacterial T3SS-mediated injection.     

Effect of natural polymorphism on structure and function of the Yersinia pestis outer membrane porin F (OmpF protein): a computational study

The Yersinia pestis outer membrane porin F (OmpF) is a transmembrane protein located in the outer membrane of this Gram-negative bacterium which is the causative agent of plague, where it plays a significant role in controlling the selective permeability of the membrane. The amino acid sequences of OmpF proteins from 48 Y. pestis strains representing all currently available phylogenetic groups of this Gram-negative bacterium were recently deduced. Comparison of these amino acid sequences revealed that the OmpF can be present in four isoforms, the pestis-pestis type, and the pestis-microtus types I, II, and III. OmpF of the most recent pestis-pestis type has an alanine residue at the position 148, where all the pestis-microtus types have threonine there (T148A polymorphism). The variability of different pestis-microtus types is caused by an additional polymorphism at the 193rd position, where the OmpFs of the pestis-microtus type II and type III have isoleucine-glycine (IG⁺₁₉₃) or isoleucine-glycine-isoleucine-glycine (IGIG⁺₁₉₃) insertions, respectively (IG⁺₁₉₃ and IGIG⁺₁₉₃ polymorphism). To investigate potential effects of these sequence polymorphisms on the structural properties of the OmpF protein, we conducted multi-level computational analysis of its isoforms. Analysis of the I-TASSER-generated 3D-models revealed that the Yersinia OmpF is very similar to other non-specific enterobacterial porins. The T148A polymorphism affected a loop located in the external vestibule of the OmpF channel, whereas IG⁺₁₉₃ and IGIG⁺₁₉₃ polymorphisms affected one of its β-strands. Our analysis also suggested that polymorphism has moderate effect on the predicted local intrinsic disorder predisposition of OmpF, but might have some functional implementations.     

Identification of a negative regulatory region for the exchange activity and characterization of T332I mutant of Rho guanine nucleotide exchange factor 10 (ARHGEF10)

The T332I mutation in Rho guanine nucleotide exchange factor 10 (ARHGEF10) was previously found in persons with slowed nerve conduction velocities and thin myelination of peripheral nerves. However, the molecular and cellular basis of the T332I mutant is not understood. Here, we show that ARHGEF10 has a negative regulatory region in the N terminus, in which residue 332 is located, and the T332I mutant is constitutively active. An N-terminal truncated ARHGEF10 mutant, ARHGEF10 ΔN (lacking amino acids 1-332), induced cell contraction that was inhibited by a Rho kinase inhibitor Y27632 and had higher GEF activity for RhoA than the wild type. The T332I mutant also showed the phenotype similar to the N-terminal truncated mutant. These data suggest that the ARHGEF10 T332I mutation-associated phenotype observed in the peripheral nerves is due to activated GEF activity of the ARHGEF10 T332I mutant.     

Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica

Post-translational modification of proteins is an efficient way cells use to control the activity of structural proteins, gene expression regulatory proteins, and enzymes. In eukaryotes, the Sir2-dependent system of protein acetylation/deacetylation controls a number of processes that affect cell longevity. Sir2 proteins have NAD⁺-dependent protein deacetylase activity and are found in all forms of life. Although the identity of the acetyltransferases that partner with Sir2 enzymes is known in eukaryotes, the identity of the prokaryotic acetyltransferases is not. We report the identification of the gene of Salmonella enterica serovar Typhimurium LT2 encoding the major protein acetyltransferase (Pat) enzyme that, in concert with the CobB sirtuin of this bacterium, regulates the activity of the central metabolic enzyme acetyl-coenzyme A synthetase (Acs). The Pat enzyme uses acetyl-CoA as substrate to modify residue Lys609 of Acs. The Pat/CobB system of S. enterica should serve as the paradigm to further investigate the contributions of this system to the physiology of prokaryotes.     

Activation of Rac1 by phosphatidylinositol 3-kinase in vivo: role in activation of mitogen-activated protein kinase (MAPK) pathways and retinoic acid-induced neuronal differentiation of SH-SY5Y cells

Rho GTPases such as RhoA, Rac1 and Cdc42 are crucial players in the regulation of signal transduction pathways required for neuronal differentiation. Using an in vitro cell culture model of neuroblastoma SH-SY5Y cells, we demonstrated previously that RhoA is an in vivo substrate of tissue transglutaminase (TGase) and retinoic acid (RA) promoted activation of RhoA by transamidation. Although activation of RhoA promoted cytoskeletal rearrangement in SH-SY5Y cells, it was not involved in induction of neurite outgrowth. Here, we demonstrate that RA promotes activation of Rac1 in SH-SY5Y cells in a transamidation-independent manner. RA-induced activation of Rac1 is mediated by phosphatidylinositol 3-kinase (PI3K), probably because of phosphorylation of the p85 regulatory subunit by Src kinases. Over-expression of constitutively active PI3K or Rac1-V12 induces neurite outgrowth, activation of mitogen activated protein kinases (MAPKs), and expression of neuronal markers. The PI3K inhibitor LY294002, or over-expression of dominant negative Rac1-N17, blocks RA-induced neurite outgrowth, activation of MAPKs, and expression of neuronal markers, suggesting that activation of PI3K/Rac1 signaling represents a potential mechanism for regulation of neuronal differentiation in SH-SY5Y cells.     

Involvement of Rho family G protein in the cell signaling for sperm incorporation during fertilization of mouse eggs: inhibition by Clostridium difficile toxin B

Sperm-egg interaction was investigated in mouse eggs freed from the zona pellucida and injected with Clostridium difficile toxin B, the inhibitor of Rho family small G proteins. Toxin B reduced in a dose-dependent manner the percentage of eggs associated with sperm fusion on the surface or sperm nucleus decondensation in the ooplasm, examined by injection of a DNA-staining dye into the egg and transfer of the dye to the fused sperm head after recording intracellular Ca(2+) responses for 100 min postinsemination. The mean number of decondensed sperm nuclei per egg was remarkably decreased by approximately 1 microg/ml toxin B in the ooplasm. This was because spermatozoa were arrested at the fusion state without developing to sperm incorporation and tended to lose cytoplasmic continuity to the egg. The fusion-arrested spermatozoa caused transient small Ca(2+) oscillations in most of eggs, while an injected spermatozoon produced repetitive large Ca(2+) spikes unaffected by toxin B. A decrease in the rate of fused spermatozoa and decondensed sperm nuclei was also caused by 20-40 microM cytochalasin D, the inhibitor of actin polymerization. Immunostaining of Rho proteins showed that Rac1 and RhoB are present in the cortical ooplasm, but Cdc42 is absent. Actin filaments in the cortex appeared to be reduced in toxin B-injected eggs. This study suggests that Rho protein(s) regulating actin-based cytoskeletal reorganization is involved in the process leading to sperm incorporation.     

Solution structure of a bacterial immunoglobulin‐like domain of the outer membrane protein (LigB) from Leptospira

Leptospiral immunoglobulin‐like (Lig) proteins are surface proteins expressed in pathogenic strains of Leptospira. LigB, an outer membrane protein containing tandem repeats of bacterial Ig‐like (Big) domains and a no‐repeat tail, has been identified as a virulence factor involved in adhesion of pathogenic Leptospira interrogans to host cells. A Big domain of LigB, LigBCen2R, was reported previously to bind the GBD domain of fibronectin, suggesting its important role in leptospiral infections. In this study, we determined the solution structure of LigBCen2R by nuclear magnetic resonance (NMR) spectroscopy. LigBCen2R adopts a canonical immunoglobulin‐like fold which is comprised of a beta‐sandwich of ten strands in three sheets. We indicated that LigBCen2R is able to bind to Ca²⁺ with a high affinity by isothermal titration calorimetry assay. NMR perturbation experiment identified a number of residues responsible for Ca²⁺ binding. Structural comparison of it with other Big domains demonstrates that they share a similar fold pattern, but vary in some structural characters. Since Lig proteins play a vital role in the infection to host cells, our study will contribute a structural basis to understand the interactions between Leptospira and host cells. Proteins 2015; 83:195–200. © 2014 Wiley Periodicals, Inc.     

Post-translational modification of RNase R is regulated by stress-dependent reduction in the acetylating enzyme Pka (YfiQ)

RNase R is a processive exoribonuclease that plays an important role in degradation of structured RNAs in Escherichia coli. RNase R is unstable in exponential phase cells; however, under certain stress conditions, RNase R levels increase dramatically due to its stabilization. Binding of tmRNA and SmpB to the C-terminal region of RNase R is required for its instability, and this binding is regulated by acetylation of a single residue, Lys544, in exponential phase cells. RNase R is not acetylated in stationary phase. We show here that only exponential phase RNase R is acetylated because the modifying enzyme, protein lysine acetyltransferase, Pka (YfiQ), is absent from late exponential and stationary phase cells. As a consequence, newly synthesized RNase R remains unmodified. Together with the turnover of preexisting acetylated RNase R, no modified RNase R remains in stationary phase. We find that RNase R in cold-shocked cells also lacks the acetyl modification due to the absence of Pka. These data indicate that RNase R stability depends on Pka, which itself is regulated under stress conditions.     

Cloning,expression and characterization of attachment‐invasion locus protein (Ail) of Yersinia enterocolitica and its utilization in rapid detection by immunoassays

Aims: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment‐invasion locus (Ail) protein. Methods and Results: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l^?1 final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. Conclusion: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. Significance and Impact of the Study: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.     

Homophilic interactions of the amyloid precursor protein (APP) ectodomain are regulated by the loop region and affect beta-secretase cleavage of APP

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of alpha-secretory APP increased, soluble beta-cleaved APP levels decreased. Because Abeta40 and Abeta42 decreased to similar levels as soluble beta-cleaved APP, we conclude either that beta-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.     

The phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs)

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.     

Metal-catalyzed oxidation and cleavage of octopus glutathione transferase by the CU(II)-ascorbate system

Glutathione transferase (GST) from octopus hepatopancreas was rapidly inactivated by micromolar concentration of Cu(II) in the presence of ascorbate at neutral pH and 0°C. Omitting the metal ion or ascorbate, or replacing the Cu(II) with Fe(II) did not result in any inactivation. Glutathione or the conjugation product of glutathione and 1-chloro-2,4-dinitrobenzene offered complete protection of the enzyme from Cu(II)-induced inactivation. 1-Chloro-2,4-dinitrobenzene, however, did not provide any protection. The inactivation was time and Cu(II) concentration dependent. The dependence of inactivation rate on Cu(II) concentration displayed saturation kinetics, which suggests that the inactivation occurs in two steps with Cu(II) binding with the enzyme first (K_dCu = 260 μM), then the locally generated free radicals modify the essential amino acid residues in the active center, which results in enzyme inactivation. The Cu(II)-ascorbate system is, thus, an affinity reagent for the octopus GST. The enzyme inactivation was demonstrated to be followed by protein cleavage. Native octopus GST has a subunit M_r of 24,000. The inactivated enzyme was cleaved at the C-terminal domain (domain II) of the enzyme molecule and resulted in the formation of peptide fragment of M_r 15,300, which has the identical N-terminal amino acid sequence as the native enzyme. The other half of the peptide with M_r approximately 7700 was visible in the gels only after silver staining, which also revealed a minor cleavage site, also located at the domain II, to produce peptide fragments of M_r approximately 11,300 and 8300. The oxygen carrier molecule in the cephalopods' blood is the copper-containing hemocyanin, which during turnover will release Cu(II). Our results indicate that Cu(II) catalyzes a site-specific oxidation of the essential amino acid residues at the C-terminus of GST causing enzyme inactivation. The modified-enzyme is then affinity cleaved at the putative metal binding site. The ability of octopus GST to bind with free Cu(II) may have important biological implications to enable cephalopods to avoid copper-induced cellular toxicity.     

Evidence for the involvement of the RNA-dependent protein kinase (PKR) in the induction of human cytotoxic T lymphocytes against a synthetic peptide of HIV-1 by regulatory RNA

The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms . After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation.To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation of WEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHI-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation.