Jean-Claude Galleyrand

Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCɛ pathway

1 The growth hormone secretagogue receptor 1a (GHSR‐1a) is a G‐protein coupled receptor, involved in the biological actions of ghrelin by triggering inositol phosphates and calcium intracellular second messengers. It has also been reported that ghrelin could activate the 44‐ and 42‐kDa extracellular signal‐regulated protein kinases (ERK1/2) in different cell lines, but it is not clear whether this regulation is GHSR‐1a dependent or not. 2 To provide direct evidence for the coupling of GHSR‐1a to ERK1/2 activation, this pathway has been studied in a heterologous expression system. 3 Thus, in Chinese hamster ovary (CHO) cells we showed that ghrelin induced, via the human GHSR‐1a, a transient and dose‐dependent activation of ERK1/2 leading to activation of the transcriptional factor Elk1. 4 We then investigated the precise mechanisms involved in GHSR‐1a‐mediated ERK1/2 activation using various specific inhibitors and dominant‐negative mutants and found that internalization of GHSR‐1a was not necessary. Our results also indicate that phospholipase C (PLC) was involved in GHSR‐1a‐mediated ERK1/2 activation, however, pathways like tyrosine kinases, including Src, and phosphoinositide 3‐kinases were not found to be involved. GHSR‐1a‐mediated ERK1/2 activation was abolished both by a general protein kinase C (PKC) inhibitor, Gö6983, and by PKC depletion using overnight pretreatment with phorbol ester. Moreover, the calcium chelator, BAPTA‐AM, and the inhibitor of conventional PKCs, Gö6976, had no effect on the GHSR‐1a‐mediated ERK1/2 activation, suggesting the involvement of novel PKC isoforms (ɛ, δ), but not conventional or atypical PKCs. Further analyses suggest that PKCɛ is required for the activation of ERK1/2. 5 Taken together, these data suggest that ghrelin, through GHSR‐1a, activates the Elk1 transcriptional factor and ERK1/2 by a PLC‐ and PKCɛ‐dependent pathway.

Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a.

The growth hormone secretagogue receptor type 1a (GHS-R1a) belongs to class A G-protein-coupled receptors (GPCR). This receptor mediates pleiotropic effects of ghrelin and represents a promising target for dysfunctions of growth hormone secretion and energy homeostasis including obesity. Identification of new compounds which bind GHS-R1a is traditionally achieved using radioactive binding assays. Here we propose a new fluorescence-based assay, called Tag-lite binding assay, based on a fluorescence resonance energy transfer (FRET) process between a terbium cryptate covalently attached to a SNAP-tag fused GHS-R1a (SNAP-GHS-R1a) and a high-affinity red fluorescent ghrelin ligand. The long fluorescence lifetime of the terbium cryptate allows a time-resolved detection of the FRET signal. The assay was made compatible with high-throughput screening by using prelabeled cells in suspension under a 384-well plate format. K(i) values for a panel of 14 compounds displaying agonist, antagonist, or inverse agonist properties were determined using both the radioactive and the Tag-lite binding assays performed on the same batches of GHS-R1a-expressing cells. Compound potencies obtained in the two assays were nicely correlated. This study is the first description of a sensitive and reliable nonradioactive binding assay for GHS-R1a in a format amenable to high-throughput screening.

A synthetic glycine-extended bombesin analogue interacts with the GRP/bombesin receptor.

alpha-amidation of a peptide (which takes place from a glycine-extended precursor) is required to produce biologically active amidated hormones, such as gastrin-releasing peptide (GRP)/Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH(2) (bombesin). It was shown that glycine-extended gastrin mediates mitogenic effects on various cell lines by interacting with a specific receptor, different from the classical CCK(1) or CCK(2) receptors. On the basis of this observation, we have extended the concept of obtaining active glycine-extended forms of others amidated peptides to produce new active analogues. In this study, we have tested the biological behaviour of a synthetic analogue of the glycine-extended bombesin (para-hydroxy-phenyl-propionyl-Gln-Trp-Ala-Val-Gly-His-Leu-Met-Gly-OH or JMV-1458) on various in vitro models. We showed that compound JMV-1458 was able to inhibit specific (3-[125I]iodotyrosyl(15)) GRP ([125I]GRP) binding in rat pancreatic acini and in Swiss 3T3 cells with K(i) values of approximately 10(-8) M. In isolated rat pancreatic acini, we found that JMV-1458 induced inositol phosphates production and amylase secretion in a dose-dependent manner. In Swiss 3T3 cells, the glycine-extended bombesin analogue dose-dependently produced [3H]thymidine incorporation. By using potent GRP/bombesin receptor antagonists, we showed that this synthetic glycine-extended bombesin analogue induces its biological activities via the classical GRP/bombesin receptor.

Cross-interactions of Two p38 Mitogen-activated Protein (MAP) Kinase Inhibitors and Two Cholecystokinin (CCK) Receptor Antagonists with the CCK1 Receptor and P38 MAP Kinase

Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

Gastrin13 and the C-terminal octapeptide of cholecystokinin are differently coupled to G-proteins in guinea-pig brain membranes

In the course of our study concerning gastrin and cholecystokinin (CCK) receptors, we synthesized and characterized a labelled gastrin ligand, [125I]BH[Leu15]gastrin-(5-17) (3-(3-[125I]iodo-4-hydroxyphenyl)propionyl[Leu15]gastrin-(5-17)). On isolated canine fundic mucosal cells and human Jurkat lymphoblastic cell line, known to express CCKB/gastrin receptors, the binding experiments performed indicate that [125I]BH[Leu15]gastrin-(5-17) provides a convenient biologically active ligand for cholecystokinin/gastrin receptor studies. We showed in this study that, on guinea-pig brain membranes known to possess CCKB and CCKA receptors, [125I]BH[Leu15]gastrin-(5-17) binds to a single class of high-affinity binding sites in a saturable and specific manner. [125I]BH[Leu15]gastrin-(5-17) interacts with guinea-pig brain membranes with a maximal binding capacity that is about three-fold lower than that of [125I]BHCCK8 (CCK8, the C-terminal octapeptide of cholecystokinin). The apparent affinities of CCK analogues and selective CCK receptor antagonists L-365,260 and MK-329 for the sites labelled by both probes were in accordance with a CCKB-like profile. Association-dissociation kinetics of [125I]BH[Leu15]gastrin-(5-17) and [125I]BHCCK8 were performed and compared. They showed that [125I]BHCCK8 equilibrated more slowly than [125I]BH[Leu15]gastrin-(5-17). The effects of pH, monovalent and divalent cations on binding of both probes were investigated. The results obtained did not indicate strong differences between [125I]BH[Leu15]gastrin-(5-17) and [125I]BHCCK8 binding. Binding experiments in the presence of stable analogues of GTP showed a different behavior between [125I]BH[Leu15]gastrin-(5-17) and [125I]BHCCK8. GTP gamma S strongly decreased [125I]BH[Leu15]gastrin-(5-17) binding whereas it weakly affected [125I]BHCCK8 binding.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of gastrin and/or cholecystokinin antagonists on the proliferation of three human astrocytic tumor cell lines.

We have investigated the potential role of gastrin in the regulation of cell growth in human astrocytic tumors. To this end we have used synthetic analogs of gastrin and cholecystokinin (CCK) which behave as gastrin and/or CCK antagonists, e.g. compounds JMV-97, JMV-209 and JMV-179. Their effects on astrocytic tumor cell proliferation was investigated by the use of the colorimetric MTT assay. The in vitro biological models used in the present study included the SW1088, U87 and U373 astrocytic tumor cell lines. The results demonstrated marked influence of gastrin and CCK antagonists in the regulation of astrocytic tumor growth. This suggests that gastrin and/or CCK antagonists might be tested in experimental glioblastoma.

L-365,260 inhibits in vitro acid secretion by interacting with a PKA pathway

The aim of this study was to analyse the antisecretory mechanism of L‐365,260 in vitro in isolated rabbit gastric glands. We showed that compound L‐365,260, described as a non‐peptide specific competitive CCK‐B receptor antagonist, was able to dose‐dependently inhibit [14C]‐aminopyrine accumulation induced by histamine (10−4 M), carbachol (5×10−5 M), 3‐isobutyl‐1‐methyl‐xanthine (IBMX) (5×10−6 M) and forskolin (5×10−7 M) with similar IC50 values respectively of 1.1±0.6×10−7 M, 1.9±1.2×10−7 M, 4.2±2.0×10−7 M and 4.0±2.8×10−7 M. We showed that L‐365,260 acted beyond receptor activation and production of intracellular second messengers and that it had no action on the H+/K+‐ATPase. We found that L‐365,260 inhibited cyclic AMP‐induced [14C]‐aminopyrine accumulation in digitonin‐permeabilized rabbit gastric glands, suggesting that this compound acted, at least in part, as an inhibitor of the cyclic AMP‐dependent protein kinase (PKA) pathway.