Cholecystokinin B Receptor from Human Jurkat Lymphoblastic T Cells Is Involved in Activator Protein-1-Responsive Gene Activation

SUMMARY The aim of this study was to analyze the role of cholecystokinin (CCK B ) receptor in human lymphoblastic Jurkat T cells. We investigated the trophic effect resulting from activation of such a receptor by using the reporter gene strategy. For this purpose, we transiently transfected Jurkat T cells with the reporter plasmid p[(TRE)3-tk-Luc] and found that CCK-8 was able to dose-dependently induce luciferase expression related to activator protein-1 (AP-1) activation with a maximal response identical to that obtained with compounds known to activate AP-1 complex (quantitatively, the same level of induction was obtained with 1 nM 12-O-tetradecanoylphorbol-13-acetate, 100 M diacylglycerol, or 4 nM epidermal growth factor). The involvement of the CCK B receptor in such a stimulation was demonstrated by the inhibiting effect of the selective CCK B receptor antagonist 158. This effect was confirmed in COS-7 cells transfected with the cDNA of CCK B receptor cloned from Jurkat T cells. To better understand the AP-1-dependent luciferase expression in Jurkat T cells, we tested two specific inhibitors of serine/threonine phosphatases-1 and -2A: okadaic acid and calyculin A. These compounds strongly increased the phorbol-12-myristate-13-acetate response, whereas we have not observed a contribution of phosphatase inhibitors on a CCK-8-induced luciferase activity. To confirm that CCK B receptors are involved in AP-1 response, we investigated the CCK-8 effect on interleukin-2 expression, a natural endogenous gene regulated by several factors, including AP-1. In Jurkat T cells activated by phorbol-12-myristate-13-acetate and phytohemagglutinin, CCK-8 induced IL-2 expression. This induction was abolished by PD-135,158. Our results indicate that CCK-8 exerts a trophic effect in Jurkat T cells through stimulation of CCK B receptors by modulation of expression of AP-1-regulated genes. Several studies have shown that various gastrointestinal peptides may be involved in the control of proliferation of various tissues and neoplastic cells (1). For example, CCK was shown to increase growth of tumors in nude mice bearing transplanted pancreatic cancer tissues (2). CCK is also known to increase the number of animals developing nitrosamine-induced pancreatic cancers (3), and CCK was shown to increase the rate of growth of cultured pancreatic cancer cells (2). Similar observations were described for bombesin/ gastrin-releasing peptide in human glioblastoma in vitro and in vivo in small-cell lung carcinoma, prostatic, mammary, and pancreatic cancer cell lines (1). In addition, gastrointestinal peptides can function as autocrine growth factors in neoplastic tissues as shown for bombesin/gastrin-releasing peptide in small-cell lung carcinoma cells, for gastrin an

Interactions croisées entre inhibiteurs de MAP Kinases et analogues de la cholécystokinine sur leur cible respective : le récepteur de type 1 et P38 MAP Kinase (étude pharmacologique et moléculaire)

La cholécystokinine est une hormone peptidique majeure du tractus gastro-intestinal et du système nerveux central. Elle est impliquée dans de nombreuses fonctions biologiques au travers de sa liaison sur le récepteur CCKl, parmi lesquelles la sécrétion d'enzymes digestives et la régulation de la satiété. Elle est également responsable de l'activation des différentes voies de transduction du signal telle que la voie p38 MAP kinase, au niveau des acini pancréatiques de rat. P38 MAP kinase est une enzyme clé des réponses inflammatoires et de nombreux composés ont été développés pour inhiber son activité. Parmi ces composés, deux sont couramment utilisés, le SB202190 et le SB203580. Cependant, des études récentes ont montré que le composé SB203580 inhibait significativement d'autres protéines à activité kinase, suggérant le contrôle de la spécificité de l'effet observé lors de son utilisation. Nous avons observé que ces deux inhibiteurs de p38 MAP kinase diminuaient fortement la liaison de la 125I-BH-CCK8s sur son récepteur CCKI. Il est apparu que cette inhibition était dose-dépendante et que l'affinité apparente de ces deux composés pour le récepteur CCKI était du même ordre de grandeur que les concentrations classiquement utilisées pour inhiber l'activité .de p38 MAP kinase. Nous avons également montré que le SB202190 et le SB203580 interagissait directement sur le récepteur CCKl, via un site distinct du site de liaison de la CCK8s. Après dosage des seconds messagers et de la sécrétion d'amylase, les deux inhibiteurs de p38 MAP kinase se sont avérés être des antagonistes du récepteur CCKI. Nous avons ensuite montré qu'ils étaient spécifiques du récepteur CCKI et qu'ils étaient sélectifs de ce récepteur. Après alignement de séquences et mutagénèse dirigée, nous avons mis en évidence que les boucles intracellulaires i2 et i3 du récepteur CCKI jouaient un rôle prépondérant dans l'interaction avec le SB202190 et le SB203580. De manière réciproque, nous avons établi que des analogues de la CCK, dérivés de benzodiazépines, étaient capables d'inhiber directement et spécifiquement l'activité de p38 MAP kinase, via une interaction avec le site de liaison de l'ATP de cette dernière. Enfin, nous avons montré que des analogues peptidiques de la CCK8s et de la CCK4, modifiés sur leur extrémité C-terminale par des groupements aromatiques, induisaient une inhibition de l'activité de p38 MAP kinase.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Trisubstituted 1,2,4-triazoles as ligands for the ghrelin receptor: On the significance of the orientation and substitution at position 3

270KS Times Cited:0 Cited References Count:14International audienceThe synthesis and structure-activity relationships concerning 3,4,5-trisubstituted 1,2,4-triazoles as ghrelin receptor ligands are described. The importance of the starting aminoacid material as well as its configuration was explored and the (D) Trp residue was found to lead to the best agonist or antagonist compounds. (C) 2007 Elsevier Ltd. All rights reserved

Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme

International audienceHumanACE is a central component of the renin–angiotensin systemand amajor therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE

Involvement of tryptophan W276 and of two surrounding amino acid residues in the high constitutive activity of the ghrelin receptor GHS-R1a

642NO Times Cited:0 Cited References Count:30The human ghrelin receptor (GHS-R1a) is known to display a high level of signaling in the absence of ligand. The Trp276, located in the fully conserved CWXP motif of G protein-coupled receptors, is believed to function as a rotameric switch in these receptors. A comparative modelling of GHS-R1a with the motilin receptor, the most related G protein-coupled receptor to GHS-R1a known to date, but characterized by a very low ligand-independent signaling level, revealed that only two surrounding residues of Trp276, that are Val131 and Ile134, were different from these receptors. We mutated them at once in GHS-R1a to create a "motilin receptor-like" environment of Trp276 in order to study the consequences on GHS-R1a activation. We studied the pharmacological properties of the W276A, V131L-1134M GHS-R1a mutants. Basal as well as maximal ghrelin-induced signaling was assessed both by inositol-phosphate accumulation and SRE pathways. As compared to the wild type receptor, the SRE-luciferase assay displayed a markedly impaired basal activity for W276A whereas that of V131L-I134M was, strikingly, two fold increased. Nevertheless, the efficacy of ghrelin to bind or to stimulate mutant receptors remained unchanged. It is concluded that Trp276, Val131 and Ile134 have a significant impact on constitutive signaling of GHS-R1a, V131L-1134M being the first example of a GHS-R1a mutant with a higher basal activity than the wild type receptor. (C) 2010 Elsevier B.V. All rights reserved