RESUMO
The protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin. We investigated the expression of PAR-2 and the role of trypsin in cell proliferation in human colon cancer cell lines. A total of 10 cell lines were tested for expression of PAR-2 mRNA by Northern blot and RT-PCR. PAR-2 protein was detected by immunofluorescence. Trypsin and the peptide agonist SLIGKV (AP2) were tested for their ability to induce calcium mobilization and to promote cell proliferation on serum-deprived cells. PAR-2 mRNA was detected by Northern blot analysis in 6 out of 10 cell lines [HT-29, Cl.19A, Caco-2, SW480, HCT-8 and T84]. Other cell lines expressed low levels of transcripts, which were detected only by RT-PCR. Further results were obtained with HT-29 cells: (1) PAR-2 protein is expressed at the cell surface; (2) an increase in intracellular calcium concentration was observed upon trypsin (1-100 nM) or AP2 (10-100 microM) challenges; (3) cells grown in serum-deprived media supplemented with trypsin (0.1-1 nM) or AP2 (1-300 microM) exhibited important mitogenic responses (3-fold increase of cell number). Proliferative effects of trypsin or AP2 were also observed in other cell lines expressing PAR-2. These data show that subnanomolar concentrations of trypsin, acting at PAR-2, promoted the proliferation of human colon cancer cells. The results of this study indicate that trypsin could be considered as a growth factor and unravel a new mechanism whereby serine proteases control colon tumours.
Assuntos
Neoplasias do Colo/metabolismo , Proteínas de Ligação a DNA/farmacologia , Proteínas de Neoplasias/metabolismo , Receptores de Trombina/metabolismo , Tripsina/farmacologia , Northern Blotting , Células CACO-2 , Cálcio/análise , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Técnica Indireta de Fluorescência para Anticorpo , Células HT29 , Humanos , RNA Mensageiro/análise , Receptor PAR-2 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
A peptide YY (PYY)-preferring receptor [PYY > neuropeptide Y (NPY)] was previously characterized in rat small intestinal crypt cells, where it mediates inhibition of fluid secretion. Here, we investigated the possible status of this receptor as a peripheral Y(2) receptor in rats. Typical Y(2) agonists (PYY(3-36), NPY(3-36), NPY(13-36), C2-NPY) and very short PYY analogs (N-alpha-Ac-PYY(22-36) and N-alpha-Ac-PYY(25-36)) acting at the intestinal PYY receptor were tested for their ability to inhibit the binding of (125)I-PYY to membranes of rat intestinal crypt cells and of CHO cells stably transfected with the rat hippocampal Y(2) receptor cDNA. Similar PYY preference was observed and all analogs exhibited comparable high affinity in both binding assays. The same held true for the specific Y(2) antagonist BIIE0246 with a K(i) value of 6.5 and 9.0 nM, respectively. BIIE0246 completely abolished the inhibition of cAMP production by PYY in crypt cells and transfected CHO cells. Moreover, the antagonist 1) considerably reversed the PYY-induced reduction of short-circuit current in rat jejunum mucosa in Ussing chamber and 2) completely abolished the antisecretory action of PYY on vasoactive intestinal peptide (VIP)-induced fluid secretion in rat jejunum in vivo. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that Y(2) receptor transcripts were present in intestinal crypt cells (3 x 10(2) molecules/100 ng RNA(T)) with no expression in villus cells, in complete agreement with the exclusive binding of PYY in crypt cells. Finally, a full-length Y(2) receptor was cloned by RT-PCR from rat intestinal crypt cells and also from human small intestine. We conclude that the so-called PYY-preferring receptor mediating inhibition of intestinal secretion is a peripheral Y(2) receptor.
Assuntos
Arginina/análogos & derivados , Jejuno/fisiologia , Peptídeo YY/metabolismo , Receptores dos Hormônios Gastrointestinais/genética , Sequência de Aminoácidos , Animais , Arginina/farmacologia , Sequência de Bases , Benzazepinas/farmacologia , Células CHO , Cricetinae , AMP Cíclico/metabolismo , DNA Complementar/análise , Hipocampo/fisiologia , Jejuno/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Peptídeo YY/farmacologia , Ratos , Ratos Wistar , Receptores dos Hormônios Gastrointestinais/antagonistas & inibidores , Receptores dos Hormônios Gastrointestinais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Intestinos/química , Receptores de Neuropeptídeo Y/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Animais , Clonagem Molecular , Colo/química , Humanos , Hipotálamo/química , Intestino Delgado/química , Ratos , Análise de Sequência de DNA , Distribuição TecidualAssuntos
Cisteína , Receptores de Peptídeo Intestinal Vasoativo/química , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Sequência de Aminoácidos , Animais , Células COS , AMP Cíclico/metabolismo , DNA Complementar , Humanos , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Estrutura Secundária de Proteína , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
Vasoactive intestinal peptide (VIP)1 receptors in rats and humans recognize peptide histidine isoleucineamide (PHI) with high and low affinity, respectively. We took advantage of this phenotypic difference to identify the domain responsible for the selective recognition of PHI by rat and human receptors which display >80% sequence identity. After transfection of human and rat receptors in COS cells, the ratio of IC50 for PHI/IC50 for VIP (referred to as P/V) in inhibiting 125I-VIP binding was shown to be >1,000 and <40, respectively. Construction of eight rat/human receptor chimerae by overlap polymerase chain reaction and determination of their P/V ratios demonstrated that the critical domain for PHI recognition is present within a sequence comprising part of the first extracellular loop and third transmembrane domain. This domain contains three different amino acids numbered according to human and rat sequences, respectively, e.g. Gln207 (human) versus His208 (rat), Gly211 versus Ala212 and Met219 versus Val220. Site-directed mutagenesis introducing individual, double, or triple mutations in a chimeric construct revealed that all three amino acids were involved in the recognition of PHI. Triple mutations were then introduced in the wild-type receptors i.e. Q207H, G211A, M219V human VIP1 receptor and H208Q, A212G, V220M rat VIP1 receptor, resulting in a complete change in their phenotype from human to rat and from rat to human, respectively. The results demonstrate that three nonadjacent amino acids are responsible for the selective recognition of PHI by human and rat VIP1 receptors.
Assuntos
Peptídeo PHI/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , DNA Complementar , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Ratos , Receptores de Peptídeo Intestinal Vasoativo/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Galanin receptors have been characterized in normal human hypothalamus using 125I-galanin binding assays. Competition experiments of porcine 125I-galanin binding to human hypothalamic membranes with native human, porcine and rat galanin (10(-11) M to 10(-8) M) gave comparable results with IC50 close to 0.1 nM. Scatchard analysis indicated one type of high affinity binding sites (Kd = 0.11 nM) with a capacity of 460 fmol/mg protein. Galanin-(1-15) and galanin-(2-29) inhibited tracer binding (IC50 = 1.5 nM), galanin-(3-29) and galanin-(10-29) being inactive. The galanin receptor antagonist, galantide, 10(-14) M to 10(-8) M, also strongly displaced binding of 125I-galanin to the human receptor (IC50 close to 0.15 nM). Guanine nucleotides (from 10(-8) M to 10(-4) M) decreased tracer binding to human membranes by increasing the dissociation of the galanin-receptor complexes. Structural analysis by covalent labelling indicated that the human galanin receptor behaves as a monomeric protein with a molecular mass of 54,000 daltons.