Upregulated Apelin Signaling in Pancreatic Cancer Activates Oncogenic Signaling Pathways to Promote Tumor Development.

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated ß-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.

Understanding Peptide Binding in Class A G Protein-Coupled Receptors.

Many physiologic processes are controlled through the activation of G protein-coupled receptors (GPCRs) by regulatory peptides, making peptide GPCRs particularly useful targets for major human diseases such as diabetes and cancer. Peptide GPCRs are also being evaluated as next-generation targets for the development of novel antiparasite agents and insecticides in veterinary medicine and agriculture. Resolution of crystal structures for several peptide GPCRs has advanced our understanding of peptide-receptor interactions and fueled interest in correlating peptide heterogeneity with receptor-binding properties. In this review, the knowledge of recently crystalized peptide-GPCR complexes, previously accumulated peptide structure-activity relationship studies, receptor mutagenesis, and sequence alignment are integrated to better understand peptide binding to the transmembrane cavity of class A GPCRs. Using SAR data, we show that peptide class A GPCRs can be divided into groups with distinct hydrophilic residues. These characteristic residues help explain the preference of a receptor to bind the C-terminal free carboxyl group, the C-terminal amidated group, or the N-terminal ammonium group of peptides.

Real-Time Analysis of Magnetic Hyperthermia Experiments on Living Cells under a Confocal Microscope.

Combining high-frequency alternating magnetic fields (AMF) and magnetic nanoparticles (MNPs) is an efficient way to induce biological responses through several approaches: magnetic hyperthermia, drug release, controls of gene expression and neurons, or activation of chemical reactions. So far, these experiments cannot be analyzed in real-time during the AMF application. A miniaturized electromagnet fitting under a confocal microscope is built, which produces an AMF of frequency and amplitude similar to the ones used in magnetic hyperthermia. AMF application induces massive damages to tumoral cells having incorporated nanoparticles into their lysosomes without affecting the others. Using this setup, real-time analyses of molecular events occurring during AMF application are performed. Lysosome membrane permeabilization and reactive oxygen species production are detected after only 30 min of AMF application, demonstrating they occur at an early stage in the cascade of events leading eventually to cell death. Additionally, lysosomes self-assembling into needle-shaped organization under the influence of AMF is observed in real-time. This experimental approach will permit to get a deeper insight into the physical, molecular, and biological process occurring in several innovative techniques used in nanomedecine based on the combined use of MNPs and high-frequency magnetic fields.

Distinct CCK-2 receptor conformations associated with ß-arrestin-2 recruitment or phospholipase-C activation revealed by a biased antagonist.

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Nearly 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signaling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2R(G)) or recruiting ß-arrestin2 (CCK2R(ß)) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150013X) acted as a high affinity competitive antagonist on CCK2R(G) but was nearly inefficient as inhibitor of CCK2R(ß). Several structural elements on both GV150013X and in CCK2R binding cavity, which hinder binding of GV150013X only to the CCK2R(ß) were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulfur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2R(ß) state. These data establish structural evidence for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.

Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists.

Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of ß-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or ß-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and ß-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for ß-arrestin recruitment. However, this region and ß-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R·ß-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor.

Reduction of stress responses in honey bees by synthetic ligands targeting an allatostatin receptor.

Honey bees are of great economic and ecological importance, but are facing multiple stressors that can jeopardize their pollination efficiency and survival. Therefore, understanding the physiological bases of their stress response may help defining treatments to improve their resilience. We took an original approach to design molecules with this objective. We took advantage of the previous identified neuropeptide allatostatin A (ASTA) and its receptor (ASTA-R) as likely mediators of the honey bee response to a biologically relevant stressor, exposure to an alarm pheromone compound. A first series of ASTA-R ligands were identified through in silico screening using a homology 3D model of the receptor and in vitro binding experiments. One of these (A8) proved also efficient in vivo, as it could counteract two behavioral effects of pheromone exposure, albeit only in the millimolar range. This putative antagonist was used as a template for the chemical synthesis of a second generation of potential ligands. Among these, two compounds showed improved efficiency in vivo (in the micromolar range) as compared to A8 despite no major improvement in their affinity for the receptor in vitro. These new ligands are thus promising candidates for alleviating stress in honey bees.

Magneto-mechanical destruction of cancer-associated fibroblasts using ultra-small iron oxide nanoparticles and low frequency rotating magnetic fields.

The destruction of cells using the mechanical activation of magnetic nanoparticles with low-frequency magnetic fields constitutes a recent and interesting approach in cancer therapy. Here, we showed that superparamagnetic iron oxide nanoparticles as small as 6 nm were able to induce the death of pancreatic cancer-associated fibroblasts, chosen as a model. An exhaustive screening of the amplitude, frequency, and type (alternating vs. rotating) of magnetic field demonstrated that the best efficacy was obtained for a rotating low-amplitude low-frequency magnetic field (1 Hz and 40 mT), reaching a 34% ratio in cell death induction; interestingly, the cell death was not maximized for the largest amplitudes of the magnetic field. State-of-the-art kinetic Monte-Carlo simulations able to calculate the torque undergone by assemblies of magnetic nanoparticles explained these features and were in agreement with cell death experiments. Simulations showed that the force generated by the nanoparticles once internalized inside the lysosome was around 3 pN, which is in principle not large enough to induce direct membrane disruption. Other biological mechanisms were explored to explain cell death: the mechanical activation of magnetic nanoparticles induced lysosome membrane permeabilization and the release of the lysosome content and cell death was mediated through a lysosomal pathway depending on cathepsin-B activity. Finally, we showed that repeated rotating magnetic field exposure halted drastically the cell proliferation. This study established a proof-of-concept that ultra-small nanoparticles can disrupt the tumor microenvironment through mechanical forces generated by mechanical activation of magnetic nanoparticles upon low-frequency rotating magnetic field exposure, opening new opportunities for cancer therapy.

Id3 modulates cellular localization of bHLH Ptf1-p48 protein.

Ptf1-p48 is a pancreas-specific bHLH transcriptional protein, which, in the normal adult pancreas, shows a restricted expression in acinar cells where it is predominantly localized in the nucleus and activates the transcription of exocrine-specific genes. Ptf1-p48 partners with two proteins to form the PTF1 active complex: a bHLH E-protein and suppressor of hairless RBP-J. Cytoplasmic mislocalization of Ptf1-p48 has been reported in pancreatic pathologies, suggesting its contribution in the early steps of pancreatic carcinogenesis. The aim of the our work was to elucidate the mechanisms regulating Ptf1-p48 subcellular localization. We hypothesized a role of Id proteins acting in a dominant-negative fashion by heterodimerizing with bHLH proteins. We reproduced Ptf1-p48 cytoplasmic mislocalization in acinar AR4-2J cells and demonstrated that a proliferative signal elicited by gastrin leads to increases in Id3 protein expression and levels of Id3/E47 and Id3/Ptf1-p48 interactions, and a decrease in the level of E47/Ptf1-p48 interaction. By contrast, Id3 silencing reversed the cytoplasmic mislocalization of Ptf1-p48 induced by gastrin. As E47 is responsible for the nuclear import of the PTF1 complex, disruption of this complex via Id3 interactions with both E47 and Ptf1-p48 appears to induce cytoplasmic mislocalization of Ptf1-p48. We then found that Ptf1-p48 is either absent or mislocalized in the cytoplasm and Id3 is overexpressed in human and murine pancreatic preneoplastic lesions. Our data provide novel insight into the regulation of Ptf1-p48 function and provide evidence that Ptf1-p48 cytoplasmic mislocalization and Id3 overexpression are early events in pancreatic cancer progression.

Identification of determinants of glucose-dependent insulinotropic polypeptide receptor that interact with N-terminal biologically active region of the natural ligand.

Glucose-dependent insulinotropic polypeptide receptor (GIPR), a member of family B of the G-protein coupled receptors, is a potential therapeutic target for which discovery of nonpeptide ligands is highly desirable. Structure-activity relationship studies indicated that the N-terminal part of glucose-dependent insulinotropic polypeptide (GIP) is crucial for biological activity. Here, we aimed at identification of residues in the GIPR involved in functional interaction with N-terminal moiety of GIP. A homology model of the transmembrane core of GIPR was constructed, whereas a three-dimensional model of the complex formed between GIP and the N-terminal extracellular domain of GIPR was taken from the crystal structure. The latter complex was docked to the transmembrane domains of GIPR, allowing in silico identification of putative residues of the agonist binding/activation site. All mutants were expressed at the surface of human embryonic kidney 293 cells as indicated by flow cytometry and confocal microscopy analysis of fluorescent GIP binding. Mutation of residues Arg183, Arg190, Arg300, and Phe357 caused shifts of 76-, 71-, 42-, and 16-fold in the potency to induce cAMP formation, respectively. Further characterization of these mutants, including tests with alanine-substituted GIP analogs, were in agreement with interaction of Glu3 in GIP with Arg183 in GIPR. Furthermore, they strongly supported a binding mode of GIP to GIPR in which the N-terminal moiety of GIP was sited within transmembrane helices (TMH) 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5), and Phe357 (TMH6). These data represent an important step toward understanding activation of GIPR by GIP, which should facilitate the rational design of therapeutic agents.

GIP receptor: Expression in neuroendocrine tumours, internalization, signalling from endosomes and structure-function relationship studies.

GIP is well known as a peptide regulating metabolic functions. In this review paper, we summarize a series of data on GIP receptor (GIPR). First, expression study of GIPR in human neuroendocrine tumours showed a very high incidence (nearly 100%) and a high density in both functional and non functional pancreatic tumours, ileal tumours, bronchial tumours and medullary thyroid carcinomas. Then, data on internalization of GIPR following stimulation by GIP are reported. Rapid and abundant internalization of GIPR also found in tumor pancreatic endocrine cells opens the possibility of tumor imaging and eradication using radiolabeled GIP. Interestingly, internalized GIPR continues to signal in early endosomes stimulating production of cAMP and activation of PKA, thus, supporting the view that GIPR signals from both plasma membrane and vesicles of internalization. At last, we summarize data from studies using in synergy molecular modeling and site-directed mutagenesis, which identified crucial amino acids of transmembrane domains of GIPR involved in GIPR binding site of GIP and/or in its activation and coupling to Gs protein. All together, these last molecular data may help to better understand structure-activity relationship data on GIP and GIPR.

Evidence for a direct and functional interaction between the regulators of G protein signaling-2 and phosphorylated C terminus of cholecystokinin-2 receptor.

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active Galpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q)-dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical, functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

Insights into the binding and activation sites of the receptors for cholecystokinin and gastrin.

CCK receptors represent potential targets in a number of diseases. Knowledge of CCK receptor binding sites is a prerequisite for the understanding of the molecular basis for their ligand recognition, partial agonism, ligand-induced trafficking of signalling. In the current paper, we report studies from our laboratory and others which have provided new data on the molecular basis of the pharmacology and functioning of CCK1 and CCK2 receptors. It has been shown that: 1) homologous regions of the two receptors are involved in the binding site of CCK, however, positioning of CCK slightly differs in agreement with distinct pharmacophores of CCK toward the two receptors and receptor sequence variations; 2) Binding sites of most of non-peptide agonists/ antagonist are buried in the pocket formed by transmembrane helices and overlap that of CCK; Aromatic amino acids within and near the binding site, especially in helix VI, are involved in receptor activation; 4) Like for other members of family A of G-protein coupled receptors, residues of the binding sites as well as of conserved motifs such as E/DRY, NPXXY are crucial for receptor activation.

[Internalized receptor for glucose-dependent insulinotropic peptide stimulates adenylyl cyclase on early endosomes]. / Signalisation endosomale du récepteur du peptide insulinotrope dépendant du glucose (GIP).

G-protein coupled receptors represent the largest family of membrane receptors. G-protein dependent signal of GPCR is classically thought to originate exclusively from the plasma membrane and, until very recently, internalized GPCRs were considered silent. At present, experimental proofs exist showing that GPCR can continue to signal via G proteins after internalization. We demonstrated that, once internalized in early endosomes, Glucose-dependent Insulinotropic Peptide Receptor (GIPR) continues to stimulate production of cAMP and activate PKA. In addition to indirect proofs showing that kinetics of cAMP production and PKA activation depend on internalization and GIPR trafficking, we identified the active form of Gαs on early endosomes containing GIPR and detected a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, relative to endosomes without GIP.

Combined Treatments of Magnetic Intra-Lysosomal Hyperthermia with Doxorubicin Promotes Synergistic Anti-Tumoral Activity.

Doxorubicin is a cytotoxic drug used for the treatment of many cancer types. However, its significant dose-related adverse effects including cardiotoxicity may hamper its efficiency. Moreover, the multidrug resistance that appears during treatments limits anti-cancer therapies. Hyperthermia has been introduced as an adjuvant anti-cancer therapy and presents promising opportunities especially in combination with chemotherapy. However, hyperthermia methods including standard magnetic hyperthermia do not discriminate between the target and the surrounding normal tissues and can lead to side effects. In this context, a Magnetic Intra-Lysosomal Hyperthermia (MILH) approach, which occurs without perceptible temperature rise, has been developed. We previously showed that minute amounts of iron oxide magnetic nanoparticles targeting the gastrin receptor (CCK2R) are internalized by cancer cells through a CCK2R-dependent physiological process, accumulated into their lysosomes and kill cancer cells upon high frequency alternating magnetic field (AMF) application through lysosomal cell death. Here, we show that the combination of MILH with doxorubicin increases the efficiency of the eradication of endocrine tumor cells with synergism. We also demonstrate that these two treatments activate two different cell death pathways that are respectively dependent on Caspase-1 and Caspase-3 activation. These findings will result in the development of new anti-tumoral, intra-lysosomal-thermo/chemotherapy with better curative effects than chemotherapy alone and that are devoid of adverse effects linked to standard hyperthermia approaches.

Targeted Magnetic Intra-Lysosomal Hyperthermia produces lysosomal reactive oxygen species and causes Caspase-1 dependent cell death.

Therapeutic strategies using drugs which cause Lysosomal Cell Death have been proposed for eradication of resistant cancer cells. In this context, nanotherapy based on Magnetic Intra-Lysosomal Hyperthermia (MILH) generated by magnetic nanoparticles (MNPs) that are grafted with ligands of receptors overexpressed in tumors appears to be a very promising therapeutic option. However, mechanisms whereby MILH induces cell death are still elusive. Herein, using Gastrin-grafted MNPs specifically delivered to lysosomes of tumor cells from different cancers, we provide evidences that MILH causes cell death through a non-apoptotic signaling pathway. The mechanism of cell death involves a local temperature elevation at the nanoparticle periphery which enhances the production of reactive oxygen species through the lysosomal Fenton reaction. Subsequently, MILH induces lipid peroxidation, lysosomal membrane permeabilization and leakage of lysosomal enzymes into the cytosol, including Cathepsin-B which activates Caspase-1 but not apoptotic Caspase-3. These data highlight the clear potential of MILH for the eradication of tumors overexpressing receptors.

An ITIM-like motif within the CCK2 receptor sequence required for interaction with SHP-2 and the activation of the AKT pathway.

SHP-2 is a tyrosine phosphatase which functions as a positive regulator downstream of RTKs, activating growth-stimulatory signalling pathways. To date, very few G protein-coupled receptors (GPCRs) have been shown to be connected to SHP-2 and very little is known about the positive role of SHP-2 in GPCR signalling. The CCK2 receptor (CCK2R), a GPCR, is now recognized to mediate mitogenic effects of gastrin on gastrointestinal cells. In the present study, we demonstrate the role of SHP-2 in the activation of the AKT pathway by the CCK2R in COS-7 cells transfected with the CCK2R and in a pancreatic cancer cell line expressing the endogenous receptor. Using surface plasmon resonance analysis, we identified a highly conserved ITIM motif, containing the tyrosine residue 438, located in the C-terminal intracellular tail of the CCK2R which directly interacts with the SHP-2 SH2 domains. The interaction was confirmed by pull down assays and co-immunoprecipitation of the receptor with SHP-2. This interaction was transiently increased following gastrin stimulation of the CCK2R and correlated with the tyrosine phosphorylation of SHP-2. Mutational analysis of the key ITIM residue 438 confirmed that the CCK2R ITIM sequence is required for interaction with SHP-2 and the activation of the AKT pathway.

Involvement of cholecystokinin 2 receptor in food intake regulation: hyperphagia and increased fat deposition in cholecystokinin 2 receptor-deficient mice.

The role of cholecystokinin (CCK) as a satiety factor has been extensively documented. Although most work implies that CCK1 receptor mediates the control of food intake, a contributing role for CCK2 receptor (CCK2R) in the CCK-induced satiety cannot be totally excluded. The hypothesis that CCK2R invalidation disrupts regulatory pathways with impact on feeding behavior was examined in CCK2R(-/-) mice. CCK2R(-/-) mice developed obesity that was associated with hyperphagia. Obesity was related with increased fat deposition resulting from adipocyte hypertrophy. Expression of several adipokines was dysregulated consistently with obesity. Moreover, obesity was associated with disturbed glucose homeostasis as revealed by increased fasting glycemia and insulinemia, impaired glucose tolerance, and hepatic insulin resistance in CCK2R(-/-) mice. In vitro analysis of isolated adipocytes metabolism was consistent with increased storage but preserved insulin sensitivity. Suppression of feeding and concomitant increased expression of hypothalamic proopiomelanocortin after intracerebroventricular injection of gastrin into control mice demonstrates that hypothalamic CCK2 receptors mediate inhibition of food intake. Comparative analysis of hypothalamic mediator gene expression in fed knockout and control mice demonstrated overexpression of ghrelin receptors in CCK2R(-/-) mice, indicating up-regulation of orexigenic pathways. This effect was also observed after body weight normalization, indicating a causative role in the development of hyperphagia and obesity of CCK2R(-/-) mice. Our results give evidence that CCK2 receptor activity plays a contributing regulatory role in the control of food intake.

Signaling pathways associated with colonic mucosa hyperproliferation in mice overexpressing gastrin precursors.

MTI/G-Gly mice and hGAS mice, overexpressing glycine-extended gastrin (G-Gly) and progastrin, respectively, display colonic mucosa hyperplasia, hyperproliferation, and an increased susceptibility to intestinal neoplasia. Here, we have used these transgenic mice to analyze in vivo the modulation of intracellular signaling pathways that may be responsible for the proliferative effects of gastrin precursors. The expression, activation, and localization of signaling and cell-to-cell adhesion molecules were studied using immunofluorescence and Western blot techniques on colonic tissues derived from MTI/G-Gly, hGAS, or wild-type FVB/N mice. These analyses revealed an up-regulation of Src tyrosine kinase and related signaling pathways [phosphatidyl inositol 3'-kinase (PI3K)/Akt, Janus-activated kinase (JAK) 2, signal transducer and activator of transcription (STAT) 3, and extracellular-signal regulated kinases (ERK)] in both MTI/G-Gly and hGAS mice compared with the wild-type control animals as well as an overexpression of transforming growth factor-alpha (TGF-alpha). In contrast, overexpression of the gastrin precursors did not affect the activation status of STAT1 nor the expression and the distribution of adhesion proteins (focal adhesion kinase, cadherins, and catenins). We report for the first time that the transition from a normal colonic epithelium to a hyperproliferative epithelium in MTI/G-Gly and hGAS mice may be a consequence of the up-regulation of Src, PI3K/Akt, JAK2, STAT3, ERKs, and TGF-alpha. Deregulation of cell adhesion, a late event in tumor progression, does not occur in these transgenic models.

Cholecystokinin 1 receptor modulates the MEKK1-induced c-Jun trans-activation: structural requirements of the receptor.

In cells overexpressing active MEKK1 to enhance c-Jun trans-activation, expression of rat cholecystokinin 1 receptor increased the activity of c-Jun while in the same experimental conditions overexpression of mouse cholecystokinin 1 receptor repressed it. This differential trans-activation is specific, since it was not observed for either the other overexpressed kinases (MEK, PKA) or for other transcription factors (ATF2, ELK-1, CREB). This differential behaviour was also detected in a human colon adenocarcinoma cell-line naturally producing high levels of endogenous MEKK1. This differential behaviour between the two receptors on the MEKK1-induced c-Jun trans-activation was independent of the activation state of JNK, of the phosphorylation level of c-Jun and of its ability to bind its specific DNA responsive elements. Two amino acids (Val43 and Phe50 in the mouse cholecystokinin 1 receptor, replaced by Leu43 and Ileu50 in the rat cholecystokinin 1 receptor) localized in the first transmembrane domain were found to play a crucial role in this differential behaviour. MEKK1 probably activates a transcriptional partner of c-Jun whose activity is maintained or increased in the presence of the rat cholecystokinin 1 receptor but repressed in the presence of the mouse cholecystokinin 1 receptor.