RESUMEN
The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.
Asunto(s)
Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/química , Transducción de Señal , Alelos , Animales , Proteínas Bacterianas/metabolismo , Células COS , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster , Eliminación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Hedgehog , Homocigoto , Peroxidasa de Rábano Silvestre/metabolismo , Ligandos , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/metabolismo , Microscopía , Microscopía Confocal , Microscopía Electrónica de Transmisión , Mutagénesis , Proteínas Mutantes Quiméricas , Mutación , Fenotipo , Fosforilación , Mutación Puntual , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular , Proteínas Recombinantes de Fusión/metabolismo , Receptor Smoothened , Factores de TranscripciónRESUMEN
Hedgehog (Hh) signalling plays a central role in many developmental processes in both vertebrates and invertebrates [1]. The multipass membrane-spanning proteins Patched (Ptc) [2-4] and Smoothened (Smo) [5-7] have been proposed to act as subunits of a putative Hh receptor complex. According to this view, Smo functions as the transducing subunit, the activity of which is blocked by a direct interaction with the ligand-binding subunit, Ptc [8]. Activation of the intracellular signalling pathway occurs when Hh binds to Ptc [8-11], an event assumed to release Smo from Ptc-mediated inhibition. Evidence for a physical interaction between Smo and Ptc is so far limited to studies of the vertebrate versions of these proteins when overexpressed in tissue culture systems [8,12]. To test this model, we have overexpressed the Drosophila Smo protein in vivo and found that increasing the levels of Smo protein per se was not sufficient for activation of the pathway. Immunohistochemical staining of wild-type and transgenic embryos revealed distinct patterns of Smo distribution, depending on which region of the protein was detected by the antibody. Our findings suggest that Smo is modified to yield a non-functional form and this modification is promoted by Ptc in a non-stoichiometric manner.
Asunto(s)
Proteínas de Drosophila , Embrión no Mamífero/fisiología , Proteínas de Insectos/fisiología , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Animales , Drosophila/embriología , Embrión no Mamífero/citología , Proteínas Hedgehog , Morfogénesis , Receptores de Superficie Celular/fisiología , Proteínas Recombinantes/análisis , Transducción de Señal/fisiología , Receptor Smoothened , Transfección , beta-Galactosidasa/análisisRESUMEN
Proteolytically activated receptors define a new subclass among the G-protein coupled receptors. Proteinase activated receptor-2 (PAR-2), the second member to be identified of this growing receptor subclass, can be activated by trypsin and trypsin-like serine proteases such as mast cell tryptase. PAR-2 is expressed in endothelial cells. Here we have studied if activation of PAR-2 changes the coagulation properties of cultured human umbilical vein endothelial cells. We show that activation of PAR-2 induces rapid and transient formation of tissue factor mRNA with a maximum level 1 hour after receptor stimulation. The increased mRNA level was accompanied by an increased tissue factor activity at the endothelial cell surface, shortening coagulation time in a standard clotting assay. The level of tissue factor activity after PAR-2 activation was comparable with the effects of thrombin receptor (PAR-1) activation although neither of the two protease receptors were as strong inducers of tissue factor as tumor necrosis factor-alpha.
Asunto(s)
Coagulación Sanguínea , Endotelio Vascular/fisiología , Receptores de Trombina/fisiología , Células Cultivadas , Endotelio Vascular/patología , Humanos , ARN Mensajero/biosíntesis , Receptor PAR-2RESUMEN
Proteinase-activated receptor 2 (PAR-2) is a G protein-coupled receptor related to the thrombin receptor. PAR-2 can be activated by trypsin and by synthetic peptides corresponding to the new amino terminus generated by activating proteolytic cleavage. We show in this report that intravenous injection of PAR-2 agonist peptides has dramatic effects on arterial blood pressure in anesthetized rats. The peptide SLIGRLETQPPI, at 150 nmol/kg, transiently decreased the mean arterial pressure from 104 to 60 mm Hg. The hypotensive response was dose-dependent, and was not secondary to effects on central vasoregulatory systems, heart rate, or the kidneys. A nitric oxide synthase inhibitor attenuated the hypotensive response induced by the PAR-2 agonist peptide. Further experiments in vitro, on preparations of rat femoral artery and vein, showed that PAR-2 agonist peptide elicited a dose-dependent relaxation of both types of vessel. Removal of the endothelium abolished the agonist peptide-induced relaxation. Our results demonstrate that activation of PAR-2 can modulate vascular tone, and that this response was an effect mediated at least partly by nitric oxide. The effect on blood vessels further suggests that the physiological activator of this proteolytically activated receptor is an enzyme present and active in the blood, possibly after a vascular injury.
Asunto(s)
Péptidos/farmacología , Receptores de Superficie Celular/fisiología , Secuencia de Aminoácidos , Animales , Antihipertensivos , Presión Sanguínea/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/antagonistas & inhibidores , Fragmentos de Péptidos/química , Ratas , Ratas Sprague-Dawley , Receptor PAR-2 , Receptores de Superficie Celular/agonistas , Vasodilatación/efectos de los fármacosRESUMEN
Recently, a second member of the protease-activated receptor (PAR) family, named PAR-2, has been identified. Similar to the thrombin receptor, PAR-2 appears to be activated by proteolytic-mediated exposure of a "tethered ligand" sequence and can also be activated by the corresponding synthetic peptides. Similarities in the amino acid sequence of the receptors' tethered ligand sequences suggest that their respective agonist peptides might not be absolutely specific for their particular receptors. To test this, the receptor specificity of each agonist has been determined by measuring the responses of Xenopus oocytes expressing the thrombin receptor or PAR-2 to agonist peptides or enzymes. Thrombin receptors responded to thrombin, the human thrombin receptor-activating peptide SFLLRNP-NH2 (TRAP) (EC50 = 0.1 microM), and Xenopus TRAP, TFRIFD-NH2 (EC50 = 1 microM), but did not show any increase in calcium efflux over control levels with trypsin (50 nM) or PAR-2 agonist peptides (100 microM). Human and murine PAR-2 receptors responded comparably to human and murine PAR-2 agonist peptides (SLIGKVD and SLIGRL, respectively) (EC50 = 0.5-2.0 microM) and trypsin, but not to thrombin. PAR-2 was also found to be responsive to TRAP (EC50 = 1 microM) but was unresponsive to Xenopus TRAP (50 microM). Responses to additional peptide agonist analogs suggest that an amino-terminal serine is critical for PAR-2 agonist activity.
Asunto(s)
Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Receptor PAR-2 , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/genética , Tripsina/farmacología , Xenopus laevisRESUMEN
The proteinase-activated receptor 2 (PAR-2) belongs to the family of seven transmembrane region receptors, and, like the related thrombin receptor, it is activated by specific proteolytic cleavage of its extracellular amino terminus. It is not known which proteinase is the physiological activator of the PAR-2, but candidates can be found among the enzymes involved in the inflammatory cascade systems. Here, we have studied the effects of various mediators on the expression of the PAR-2 and the thrombin receptor in cultured human umbilical vein endothelial cells. Stimulation with the cytokines tumor necrosis factor alpha or interleukin-1 alpha as well as bacterial lipopolysaccharide elevated the expression of PAR-2 in a dose-dependent manner. The time course of induction after cytokine stimulation was similar to those published for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. After 20 h of stimulation, PAR-2 mRNA and protein levels were increased to 5-10-fold basal values, and, in the continued presence of tumor necrosis factor alpha, PAR-2 mRNA expression was found to remain elevated for up to 4 days. In contrast, the thrombin receptor gene was not induced by any of these inflammatory mediators. The responses to phorbol ester treatment also differed between the two genes. Thrombin receptor mRNA levels decreased steadily up to 20 h, whereas PAR-2 mRNA levels first rose to about 3-fold basal values at 4 h before decreasing again. Cell surface protein levels of both receptors were decreased after 20 h of phorbol ester stimulation. Elevating intracellular cAMP levels by treatment with forskolin resulted in decreased expression of both receptors, and inhibition of cAMP degradation appeared to blunt the cytokine-induced increase in PAR-2 expression. The induction of the PAR-2 by cytokine treatment supports the concept of PAR-2 involvement in the acute inflammatory response.
Asunto(s)
Endotelio Vascular/metabolismo , Interleucina-1/farmacología , Receptores de Superficie Celular/biosíntesis , Receptores de Trombina/biosíntesis , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Northern Blotting , Moléculas de Adhesión Celular/farmacología , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Colforsina/farmacología , Sondas de ADN , Regulación hacia Abajo/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Humanos , Inflamación , Cinética , Lipopolisacáridos/farmacología , ARN Mensajero/biosíntesis , Receptor PAR-2 , Proteínas Recombinantes/biosíntesis , Acetato de Tetradecanoilforbol/farmacología , Transfección , Venas UmbilicalesRESUMEN
We previously reported the molecular cloning of a mouse guanosine-nucleotide-binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northern-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.
Asunto(s)
Receptores de Superficie Celular/genética , Receptores de Trombina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , Biblioteca de Genes , Humanos , Ratones , Datos de Secuencia Molecular , Plásmidos , Receptor PAR-2 , Receptores de Trombina/aislamiento & purificación , Receptores de Trombina/metabolismo , Alineación de SecuenciaRESUMEN
We have reported the cloning from mouse genomic DNA of a fragment encoding a G-protein-coupled receptor related to the receptor for the blood clotting enzyme thrombin. Like the thrombin receptor this receptor is activated by proteolytic cleavage of its extracellular amino terminus. Because the physiological agonist at the receptor was unknown, we provisionally named it proteinase-activated receptor 2 (PAR-2). Here we present a PAR-2 cDNA of 2729 nucleotides that differs from the published genomic sequence at the 5' end, including a part of the protein coding region. The differences do not affect the peptide sequence of the activating proteinase cleavage site proper, but may include amino acid residues important for enzyme-substrate recognition. Analysis of the PAR-2 gene structure showed that the cDNA 5' end is derived from a separate exon located about 10 kilobases away from the 3' exon. Results from a primer extension experiment indicate that transcription starts at a unique site around nucleotide -203 respective to the translation initiation ATG. Chinese hamster ovary cells transfected with either the PAR-2 cDNA or a construct made from the published PAR-2 genomic sequence responded with intracellular calcium mobilization to stimulation with 1 nM trypsin, 10 microM PAR-2-activating peptide (SLIGRL), or 1 microM thrombin receptor-activating peptide (SFLLRN). Untransfected cells responded only to stimulation with thrombin receptor activating peptide. Only transcripts corresponding to the PAR-2 cDNA could be detected in three mouse tissues examined.
Asunto(s)
Ratones/genética , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Calcio/metabolismo , Clonación Molecular , Cricetinae , Cartilla de ADN , ADN Complementario , Proteínas de Unión al GTP/metabolismo , Mucosa Gástrica/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptor PAR-2 , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Transcripción Genética , TransfecciónRESUMEN
A DNA sequence encoding a G-protein-coupled receptor was isolated from a mouse genomic library. The predicted protein is similar in structure to the thrombin receptor and has a similar activation mechanism. When expressed in Xenopus laevis oocytes, the receptor was activated by low concentrations of trypsin (EC 3.4.21.4) and by a peptide (SLIGRL) derived from the receptor sequence, but was not activated by thrombin (EC 3.4.21.5). Trypsin failed to activate a mutant receptor in which the presumed cleavage site Arg-34-Ser-35 was changed to an Arg-Pro sequence. The agonist peptide (SLIGRL) activated equally well mutant and wild-type receptors. Northern blot analysis demonstrated receptor transcripts in highly vascularized tissues such as kidney, small intestine, and stomach. Because this, to our knowledge, is the second example, besides the thrombin receptor, of a proteolytically activated seven-transmembrane G-protein-coupled receptor, we have provisionally named it proteinase activated receptor 2.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/metabolismo , Trombina/metabolismo , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Cartilla de ADN , Femenino , Biblioteca Genómica , Humanos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligopéptidos/farmacología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Reacción en Cadena de la Polimerasa , Receptor PAR-2 , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/fisiología , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Trombina/farmacología , Tripsina/farmacología , Xenopus laevisRESUMEN
BACKGROUND AND PURPOSE: Serotonin released from platelets has been suggested as one substance causing the vasospasm following subarachnoid hemorrhage. We studied whether such serotonin is able to constrict pial vessels. METHODS: We studied the uptake of serotonin in pial perivascular nerves by immunohistochemistry. We measured the contractile response in rat basilar artery after in vitro incubation with serotonin and during electrical field stimulation of perivascular nerves following experimental subarachnoid hemorrhage. RESULTS: After incubation with serotonin, electrical field stimulation caused a tetrodotoxin- and ketanserin-blockable contractile response. We observed no such response in vessels from rats treated with 6-hydroxydopamine or after blockade of serotonin uptake. After subarachnoid hemorrhage, a pronounced network of serotonin-immunoreactive nerve fibers was demonstrated in the vessel wall. In vessels from control rats, no serotonin fibers were seen, and in vessels from 6-hydroxydopamine-treated animals with subarachnoid hemorrhage only a few such fibers were seen. Electrical field stimulation of the basilar artery from rats tested 2 or 16 hours (but not 10 minutes or 24 hours) after subarachnoid hemorrhage showed contractile responses that were prevented by tetrodotoxin, ketanserin, and prior 6-hydroxydopamine treatment. CONCLUSIONS: Our study demonstrates a capacity of the perivascular sympathetic nerves to take up serotonin both in vitro and during the early phase of subarachnoid hemorrhage. Such uptake may help to remove excess serotonin from the subarachnoid space. Only if serotonin is subsequently released upon nerve activation may minor smooth muscle contraction develop.