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

Etude pharmacologique de la signalisation intracellulaire d'un récepteur couplé aux protéines G : le récepteur de la cholécystokinine de type I

Le récepteur de la cholécystokinine de type 1 (R-CCK1) est un récepteur à sept domaines trans-membranaires couplé aux protéines G dont la structure primaire varie légèrement entre les espèces. Nous avons pu montrer que les R-CCK1 murin et humain, contrairement au R-CCK1 de rat, induisent une inhibition de l'activation transcriptionnelle de c-Jun, en présence d'une sur-expression de MEKK1. A l'aide d'expériences complémentaires, il a pu être déterminé que cet effet serait indépendant de l'état de phosphorylation de la SAPK/JNK et de c-Jun, et de l'activité kinase de la SAPK/JNK, principale kinase connue jusqu'à présent comme activant c-Jun. Par ailleurs, les expériences menées sur des mutants du R-CCK1 murin tendent à prouver que la troisième boucle intracellulaire ne serait pas impliquée dans cette différence de signalisation intracellulaire, et que, par contre, le premier domaine trans-membranaires et l'extrémité C-terminale des R-CCK1 pourraient jouer un rôle dans ce phénomène.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Clonage et caractérisation pharmacologique du récepteur de la cholécystokinine de type 1 de souris (par Laure Di Malta)

MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

JMV641: a potent bombesin receptor antagonist that inhibits Swiss 3T3 cell proliferation

International audienc

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