RESUMEN
The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.
Asunto(s)
Dopamina/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Multimerización de Proteína , Receptores de Dopamina D2/química , Receptores de Ghrelina/química , Transducción de Señal , Dopamina/genética , Dopamina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Humanos , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Ghrelina/genética , Receptores de Ghrelina/metabolismoRESUMEN
How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Receptores de Ghrelina/química , Transferencia de Energía , Conformación ProteicaRESUMEN
The G protein-coupled receptor GHS-R1a mediates ghrelin-induced growth hormone secretion, food intake, and reward-seeking behaviors. GHS-R1a signals through Gq, Gi/o, G13, and arrestin. Biasing GHS-R1a signaling with specific ligands may lead to the development of more selective drugs to treat obesity or addiction with minimal side effects. To delineate ligand selectivity at GHS-R1a signaling, we analyzed in detail the efficacy of a panel of synthetic ligands activating the different pathways associated with GHS-R1a in HEK293T cells. Besides ß-arrestin2 recruitment and ERK1/2 phosphorylation, we monitored activation of a large panel of G protein subtypes using a bioluminescence resonance energy transfer-based assay with G protein-activation biosensors. We first found that unlike full agonists, Gq partial agonists were unable to trigger ß-arrestin2 recruitment and ERK1/2 phosphorylation. Using G protein-activation biosensors, we then demonstrated that ghrelin promoted activation of Gq, Gi1, Gi2, Gi3, Goa, Gob, and G13 but not Gs and G12. Besides, we identified some GHS-R1a ligands that preferentially activated Gq and antagonized ghrelin-mediated Gi/Go activation. Finally, we unambiguously demonstrated that in addition to Gq, GHS-R1a also promoted constitutive activation of G13. Importantly, we identified some ligands that were selective inverse agonists toward Gq but not of G13. This demonstrates that bias at GHS-R1a signaling can occur not only with regard to agonism but also to inverse agonism. Our data, combined with other in vivo studies, may facilitate the design of drugs selectively targeting individual signaling pathways to treat only the therapeutically relevant function.
Asunto(s)
Receptores de Ghrelina/agonistas , Receptores de Ghrelina/antagonistas & inhibidores , Arrestinas/metabolismo , Diseño de Fármacos , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Fosfatos de Inositol/biosíntesis , Cinética , Ligandos , Sistema de Señalización de MAP Quinasas , Receptores de Ghrelina/metabolismo , Transducción de Señal , Relación Estructura-Actividad , beta-ArrestinasRESUMEN
Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.
Asunto(s)
Receptores de Ghrelina/metabolismo , Triazoles/química , Transferencia Resonante de Energía de Fluorescencia , Indoles/química , Indoles/farmacología , Concentración 50 Inhibidora , Ligandos , Receptores de Ghrelina/agonistas , Relación Estructura-Actividad , Sustancia P/química , Triptófano/análogos & derivados , Triptófano/química , Triptófano/farmacologíaRESUMEN
Ghrelin receptor ligands based on a trisubstituted 1,2,4-triazole scaffold were recently synthesized and evaluated for their in vitro affinity for the GHS-R1a receptor and their biological activity. In this study, replacement of the α-aminoisobutyryl (Aib) moiety (a common feature present in numerous growth hormone secretagogues described in the literature) by aromatic and heteroaromatic groups was explored. We found potent antagonists incorporating the picolinic moiety in place of the Aib moiety. In an attempt to increase affinity and activity of our lead compound 2, we explored the modulation of the pyridine ring. Herein we report the design and the structure-activity relationships study of these new ghrelin receptor ligands.
Asunto(s)
Receptores de Ghrelina/antagonistas & inhibidores , Receptores de Ghrelina/metabolismo , Triazoles/síntesis química , Triazoles/metabolismo , Animales , Línea Celular , Humanos , Ratones , Unión Proteica/fisiología , Relación Estructura-Actividad , Triazoles/farmacologíaRESUMEN
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.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Ligandos , Receptores de Ghrelina/antagonistas & inhibidores , Unión Competitiva , Complejos de Coordinación/química , Éteres Corona/química , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Cinética , Receptores de Ghrelina/agonistas , Receptores de Ghrelina/metabolismo , Terbio/químicaRESUMEN
Ghrelin receptor ligands based on trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the replacement of the alpha-aminoisobutyryl moiety by aromatic or heteroaromatic groups. Compounds 5 and 34 acted as potent in vivo antagonists of hexarelin-stimulated food intake. These two compounds did not stimulate growth hormone secretion in rodents and did not antagonize growth hormone secretion induced by hexarelin.
Asunto(s)
Fármacos Antiobesidad/síntesis química , Picolinas/síntesis química , Pirazinas/síntesis química , Receptores de Ghrelina/antagonistas & inhibidores , Triazoles/síntesis química , Animales , Fármacos Antiobesidad/química , Fármacos Antiobesidad/farmacología , Ingestión de Alimentos/efectos de los fármacos , Hormona del Crecimiento/metabolismo , Humanos , Células LLC-PK1 , Oligopéptidos/farmacología , Picolinas/química , Picolinas/farmacología , Pirazinas/química , Pirazinas/farmacología , Ensayo de Unión Radioligante , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Porcinos , Triazoles/química , Triazoles/farmacologíaRESUMEN
A series of ghrelin receptor ligands based on the trisubstituted 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and biological activity. In this study, we explored the significance of the aminoisobutyryl (Aib) moiety, a common feature in numerous growth hormone secretagogues described in the literature. Potent agonist and antagonist ligands of the growth hormone secretagogue receptor type 1a (GHS-R1a) were obtained, i.e., compounds 41 (JMV2894) and 17 (JMV3031). The best compounds were evaluated for their in vivo activity on food intake, after sc injection in rodents. Among the tested compounds, few of them were able to stimulate food intake and some others, i.e., compounds 4 (JMV2959), 17, and 52 (JMV3021), acted as potent in vivo antagonist of hexarelin-stimulated food intake. These compounds did not stimulate growth hormone secretion in rats and furthermore did not antagonize growth hormone secretion induced by hexarelin, revealing that it is possible to modulate food intake without altering growth hormone secretion.
Asunto(s)
Receptores de Ghrelina/agonistas , Receptores de Ghrelina/antagonistas & inhibidores , Triazoles/síntesis química , Animales , Calcio/metabolismo , Línea Celular , Cricetinae , Ingestión de Alimentos/efectos de los fármacos , Hormona del Crecimiento/metabolismo , Humanos , Ligandos , Masculino , Oligopéptidos/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Estereoisomerismo , Relación Estructura-Actividad , Triazoles/química , Triazoles/farmacologíaRESUMEN
A new series of growth hormone secretagogue (GHS) analogues based on the 1,2,4-triazole structure were synthesized and evaluated for their in vitro binding and their ability to stimulate intracellular calcium release to the cloned hGHS-1a ghrelin receptor expressed in LLC PK-1 cells. We have synthesized potent ligands of this receptor, some of them behaving as agonists, partial agonists, or antagonists. Some compounds among the most potent, i.e., agonist 29c (JMV2873), partial agonists including 21b (JMV2810), antagonists 19b (JMV2866) and 19c (JMV2844), were evaluated for their in vivo activity on food intake, after sc injection in rodents. Some compounds were found to stimulate food intake like hexarelin; some others were identified as potent hexarelin antagonists in this assay. Among the tested compounds, 21b was identified as an in vitro ghrelin receptor partial agonist, as well as a potent in vivo antagonist of hexarelin-stimulated food intake in rodents. Compound 21b was without effect on GH release from rat. However, in this series of compounds, it was not possible to find a clear correlation between in vitro and in vivo results.
Asunto(s)
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Triazoles/síntesis química , Animales , Calcio/metabolismo , Línea Celular , Técnicas Químicas Combinatorias , Ingestión de Alimentos/efectos de los fármacos , Hormona del Crecimiento/metabolismo , Humanos , Ligandos , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Ghrelina , Estereoisomerismo , Relación Estructura-Actividad , Triazoles/química , Triazoles/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Quinasa 1 de Quinasa de Quinasa MAP/metabolismo , Receptores de Colecistoquinina/metabolismo , Activación Transcripcional/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Ratones , Fosforilación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Ratas , Receptores de Colecistoquinina/genética , Especificidad de la EspecieRESUMEN
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 (epsilon, delta), but not conventional or atypical PKCs. Further analyses suggest that PKCepsilon 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 PKCepsilon-dependent pathway.
Asunto(s)
Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Hormonas Peptídicas/fisiología , Proteína Quinasa C-epsilon/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células CHO , Cricetinae , Ghrelina , Humanos , Receptores de Ghrelina , Transfección , Fosfolipasas de Tipo C/metabolismo , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Radiolabeling of ligands is still the gold standard in the study of high-affinity receptor-ligand interactions. In an effort toward safer and simpler alternatives to the use of radioisotopes, we developed a quantitative and highly sensitive matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) method that relies on the use of chemically tagged ligands designed to be specifically detectable when present as traces in complex biological mixtures such as cellular lysates. This innovative technology allows easy, sensitive detection and accurate quantification of analytes at the sub-nanomolar level. After statistical validation, we were able to perform pharmacological evaluations of Gâ protein-coupled receptor (V1A-R)-ligand interactions. Both saturation and competitive binding assays were successfully processed.
Asunto(s)
Técnicas de Química Analítica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Animales , Unión Competitiva , Células CHO , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Cricetinae , Cricetulus , Marcaje Isotópico , Ligandos , Péptidos/síntesis química , Péptidos/química , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Using an in-house bioinformatics programme, we identified and synthesized a novel nonapeptide, H-Pro-Pro-Thr-Thr-Thr-Lys-Phe-Ala-Ala-OH. Here, we have studied its biological activity, in vitro and in vivo, and have identified its target in the brain. EXPERIMENTAL APPROACH: The affinity of the peptide was characterized using purified whole brain and striatal membranes from guinea pigs and rats . Its effect on behaviour in rats following intra-striatal injection of the peptide was investigated. A photoaffinity UV cross-linking approach combined with subsequent affinity purification of the ligand covalently bound to its receptor allowed identification of its target. KEY RESULTS: The peptide bound with high affinity to a single class of binding sites, specifically localized in the striatum and substantia nigra of brains from guinea pigs and rats. When injected within the striatum of rats, the peptide stimulated in vitro and in vivo dopamine release and induced dopamine-like motor effects. We purified the target of the peptide, a ~151 kDa protein that was identified by MS/MS as angiotensin converting enzyme (ACE I). Therefore, we decided to name the peptide acein. CONCLUSION AND IMPLICATIONS: The synthetic nonapeptide acein interacted with high affinity with brain membrane-bound ACE. This interaction occurs at a different site from the active site involved in the well-known peptidase activity, without modifying the peptidase activity. Acein, in vitro and in vivo, significantly increased stimulated release of dopamine from the brain. These results suggest a more important role for brain ACE than initially suspected.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Dopamina/metabolismo , Oligopéptidos/farmacología , Peptidil-Dipeptidasa A/metabolismo , Animales , Encéfalo/enzimología , Dominio Catalítico/efectos de los fármacos , Biología Computacional , Cobayas , Masculino , Oligopéptidos/administración & dosificación , Oligopéptidos/síntesis química , Ratas , Ratas Sprague-DawleyRESUMEN
The 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-I134M 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-I134M being the first example of a GHS-R1a mutant with a higher basal activity than the wild type receptor.
Asunto(s)
Receptores de Ghrelina/química , Receptores de Ghrelina/metabolismo , Transducción de Señal , Triptófano/fisiología , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Ghrelina/metabolismo , Humanos , Isoleucina/metabolismo , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , Receptores de la Hormona Gastrointestinal/química , Receptores de Ghrelina/genética , Receptores de Neuropéptido/química , Triptófano/química , Valina/metabolismoRESUMEN
Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser ([octa])-Phe-NH (2) and Gly-Ser-Ser ([octa])-Phe-Leu-NH (2) allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.
Asunto(s)
Receptores de Ghrelina/agonistas , Receptores de Ghrelina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Cristalografía por Rayos X , Ghrelina/química , Ghrelina/genética , Ghrelina/metabolismo , Humanos , Técnicas In Vitro , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estabilidad Proteica , Receptores Adrenérgicos beta 2/química , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Ghrelina/genética , Receptores de Ghrelina/metabolismo , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopsina/química , Homología Estructural de Proteína , TermodinámicaRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Receptores de Colecistoquinina/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adenosina Trifosfato/química , Animales , Benzodiazepinonas/farmacología , Sitios de Unión , Western Blotting , Células COS , Devazepida/farmacología , Relación Dosis-Respuesta a Droga , Genes Reporteros , Antagonistas de Hormonas/farmacología , Imidazoles/farmacología , Immunoblotting , Fosfatos de Inositol/química , Cinética , Ligandos , Sistema de Señalización de MAP Quinasas , Modelos Químicos , Modelos Moleculares , Compuestos de Fenilurea/farmacología , Unión Proteica , Conformación Proteica , Piridinas/farmacología , Ratas , Receptor de Colecistoquinina A/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
It is well known that the amidated C-terminal part of gastrin is crucial for its interaction with the classical seven transmembrane domain receptors CCK-1 or CCK-2. Nevertheless, over the past 10 years, several groups have characterized new binding sites using peptides related to gastrin (particularly glycine-extended forms of gastrin) on various tumoral and nontumoral cell lines. In the present study, we focused on the human astrocytic tumoral cell line U373. Although it has been described that gastrin was able to inhibit the motility of these cells, we were unable to detect any classical CCK/gastrin receptor. On the other hand, by using the radiolabeled C-terminal heptapeptide of gastrin ((125)I-G-7), we evidenced a new binding site that possessed a pharmacological profile different from the classical CCK/gastrin receptors. This new gastrin binding site seemed to be coupled to G proteins and be implicated in c-Fos transcription gene. Moreover, we showed that G-7 was able to induce a strong inhibition of U373 cell migration, a crucial biological effect when we know that astrocytoma cells' migration in brain parenchyma constitutes a major feature of malignancy in astrocytic tumors.