RESUMEN
OBJECTIVES: Concern has recently been expressed with regard to the physiologic effects of primary intramedullary femoral nailing in seriously injured patients. "Damage control orthopaedics" techniques have been proposed, which comprise principally the use of primary external fixation. The aim of this study was to compare the physiologic effects of external femoral fixation with those of intramedullary stabilization over the first 24 hours after femoral fracture using an established large animal (ovine) trauma model. METHODS: Under terminal anesthesia, bilateral high-energy femoral fractures and hypovolemic shock were produced using a pneumatic actuator. Twenty-four sheep were randomized into 4 groups and monitored for 24 hours. Group 1--control, group 2--trauma only, group 3--trauma and external fixation, and group 4--trauma and reamed intramedullary nailing. Outcome measures included the following: pulmonary embolic load (transesophageal echocardiography), metabolic base excess, plasma coagulation markers, and polymorphonuclear cell counts obtained from bronchoalveolar lavage samples. RESULTS: The total embolic load was significantly higher (P < 0.001) in the intramedullary nailing group. All trauma groups had a significant increase (P < 0.05) in prothrombin times with a fall in antithrombin III and fibrinogen levels. However, the type of fracture stabilization used did not significantly affect any of the other outcome measurements. CONCLUSIONS: A higher pulmonary embolic load can be expected during early intramedullary femoral fracture stabilization compared with primary external fixation. However, the degree of stimulation to systemic coagulation and pulmonary inflammation by each type of surgery was comparable.
Asunto(s)
Clavos Ortopédicos , Fijadores Externos/efectos adversos , Fracturas del Fémur/cirugía , Fijación Intramedular de Fracturas/efectos adversos , Embolia Pulmonar/etiología , Choque/etiología , Animales , Modelos Animales de Enfermedad , Ecocardiografía , Fracturas del Fémur/fisiopatología , Fijación Intramedular de Fracturas/instrumentación , Fijación Intramedular de Fracturas/métodos , Hemodinámica/fisiología , Masculino , Embolia Pulmonar/diagnóstico por imagen , Embolia Pulmonar/fisiopatología , Ovinos , Choque/fisiopatologíaRESUMEN
BACKGROUND AND PURPOSE: GPR119 is a G protein-coupled receptor that is preferentially expressed in islet cells and mediates insulin secretion. Oleoyl-lysophosphatidylcholine and oleoylethanolamide (OEA) act as endogenous ligands for this receptor, whereas PSN375963 and PSN632408 are two recently reported synthetic agonists. In this study, we explored mechanisms underlying GPR119-induced insulin secretion. In addition, we assessed the potential utility of the synthetic agonists as tools for exploring GPR119 biology. EXPERIMENTAL APPROACH: We examined natural and synthetic GPR119 agonist activity at GPR119 in MIN6c4 and RINm5f insulinoma cells. We evaluated insulin secretion, intracellular calcium [Ca(2+)](i), ion channel involvement and levels of cAMP. KEY RESULTS: We report that increases in insulin secretion induced by OEA were associated with increased cAMP and a potentiation of glucose-stimulated increases in [Ca(2+)](i). We also demonstrate that ATP-sensitive K(+) and voltage-dependent calcium channels were required for GPR119-mediated increases in glucose-stimulated insulin secretion. In contrast to OEA, the synthetic GPR119 agonist PSN375963 and PSN632408 have divergent effects on insulin secretion, cAMP and intracellular calcium in MIN6c4 cells. CONCLUSIONS AND IMPLICATIONS: The endogenous ligand OEA signals through GPR119 in a manner similar to glucagon-like peptide-1 (GLP-1) and its receptor with respect to insulin secretion, [Ca(2+)](i) and cAMP. In addition, PSN375963 and PSN632408 substantially differ from OEA and from one another. These studies suggest that the commercially available synthetic agonists, although they do activate GPR119, may also activate GPR119-independent pathways and are thus unsuitable as GPR119-specific pharmacological tools.
Asunto(s)
Insulina/metabolismo , Insulinoma/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Ácidos Heterocíclicos/farmacología , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Endocannabinoides , Glucosa/metabolismo , Secreción de Insulina , Canales KATP/metabolismo , Lisofosfatidilcolinas/metabolismo , Ratones , Ácidos Oléicos/metabolismo , Oxadiazoles/farmacología , Piridonas/farmacología , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
A series of potent and selective inhibitors of h-MCH-R1 has been developed based on the piperidine glycineamide compounds I and II. These structurally more rigid tetrahydroisoquinolines (III and IV) showed better pharmacokinetics. The highly potent compounds 12d and 12g displayed excellent rat pk.
Asunto(s)
Receptores de Somatostatina/antagonistas & inhibidores , Tetrahidroisoquinolinas/síntesis química , Tetrahidroisoquinolinas/farmacología , Animales , Bencimidazoles/química , Humanos , Estructura Molecular , Ratas , Receptores de Somatostatina/metabolismo , Relación Estructura-Actividad , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/farmacocinéticaRESUMEN
Melanin-concentrating hormone (MCH) is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G protein-coupled receptor family. Recently an orphan receptor, SLC-1, has been identified as an MCH receptor (MCH-R1). Herein we identify and characterize a novel receptor for human MCH (MCH-R2). The receptor is composed of 340 amino acids encoded by a 1023-base pair cDNA and is 35% homologous to SLC-1. (125)I-MCH specifically bound to Chinese hamster ovary cells stably expressing MCH-R2. MCH stimulated dose-dependent increases in intracellular free Ca(2+) and inositol phosphate production in these cells but did not affect cAMP production. The pharmacological profile for mammalian MCH, [Phe(13),Tyr(19)]MCH, and salmon MCH at MCH-R2 differed compared with MCH-R1 as assessed by intracellular signaling and radioligand binding assays. The EC(50) in signaling assays and the IC(50) in radioligand binding assays of salmon MCH was an order of magnitude higher than mammalian MCH at MCH-R2. By comparison, the EC(50) and IC(50) values of salmon MCH and mammalian MCH at MCH-R1 were relatively similar. Blot hybridization revealed exclusive expression of MCH-R2 mRNA in several distinct brain regions, particularly in the cortical area, suggesting the involvement of MCH-R2 in the central regulation of MCH-mediated functions.
Asunto(s)
Receptores de la Hormona Hipofisaria/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células CHO , Calcio/metabolismo , Cricetinae , Fosfatos de Inositol/metabolismo , Datos de Secuencia Molecular , ARN Mensajero/análisis , Receptores de la Hormona Hipofisaria/genética , Receptores de la Hormona Hipofisaria/metabolismoRESUMEN
Protein tyrosine kinases (PTKs) are ubiquitous enzymes that are integrally involved in the regulation of transformation mechanisms, normal and pathological growth, cell cycle regulation, immune responses, and a variety of intracellular signaling mechanisms. This rapidly growing family of enzymes is generally divided into two groups: receptor PTKs (with more than twelve distinct families) and nonreceptor PTKs (with more than nine distinct families). PTKs mediate the enzymatic transfer of the gamma phosphate of ATP to the phenolic groups on tyrosine residues to generate phosphate monoesters. In this unit, several assays are provided to measure the ability of PTKs to transphosphorylate protein and peptide substrates, and to autophosphorylate. Phosphorylation of exogenous substrates or autophosphorylation is detected using a ³²P- or ³³P-phosphorylated protein. Alternatively, antibodies recognizing phosphorylated tyrosine residues can be used to quantify PTK activity. In some cases, antibodies are available for context-specific phosphotyrosine residues, thereby enabling the detection of PTK-specific substrate phosphorylation.
Asunto(s)
Pruebas de Enzimas/métodos , Proteínas Tirosina Quinasas/metabolismo , Células Cultivadas , Activación Enzimática/fisiología , FosforilaciónRESUMEN
The receptor for melanin-concentrating hormone (MCH) was recently identified as the orphan G protein-coupled receptor SLC-1. In this study, a CHO cell line expressing the MCH receptor (Kd = 1.3 nM; binding capacity, 3.6 pmol/mg protein) is used to assess the ability of the MCH receptor to couple to Gi, Go, and Gq proteins. The results demonstrate that MCH inhibits forskolin-stimulated cAMP production in a pertussis toxin- (PTX)-sensitive manner in CHO-MCHR cells (EC50 = 100 pM), indicating that the MCH receptor couples to one or more members of the Gi subfamily of G proteins. In addition, MCH stimulates increases in phosphoinositide metabolism (EC50 = 50 nM) and in intracellular free Ca2+ levels (EC50 = 10 nM). MCH-stimulated inositol phosphate production and increases in intracellular free Ca2+ are partially inhibited (60% and 40%, respectively) by PTX pretreatment, demonstrating that there are at least two components of each of these signaling pathways. One component is PTX sensitive and therefore mediated through a Gi/Go protein. A distinct G protein-coupled (probably Gq type) mediates the PTX-insensitive component. To distinguish Gi vs. Go coupling, MCH-stimulated mitogen-activated protein (MAP) kinase activity was examined. Gi and Go use separate signaling pathways to mediate MAP kinase activation in CHOcells. Protein kinase C (PKC) activity is essential in the Go-dependent MAP kinase signaling pathway, but is not required in the GC-dependent MAP kinase signaling pathway. MCH stimulated MAP kinase activity is decreased (50%), but not abolished, by inhibition of PKC activity or depletion of cellular PKC, indicating that MCH-stimulated MAP kinase activity is mediated through both Gi- and Go-dependent signaling mechanisms. The results of this study are the first to clearly demonstrate that the MCH receptor couples to multiple G proteins to mediate several diverse intracellular signaling pathways.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Receptores de la Hormona Hipofisaria/fisiología , Transducción de Señal , Adenilil Ciclasas/metabolismo , Animales , Células CHO , Membrana Celular/metabolismo , Colforsina/farmacología , Cricetinae , AMP Cíclico/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Expresión Génica , Proteínas de Unión al GTP Heterotriméricas/fisiología , Humanos , Hormonas Hipotalámicas/metabolismo , Hormonas Hipotalámicas/farmacología , Fosfatos de Inositol/biosíntesis , Lisofosfolípidos/farmacología , Melaninas/metabolismo , Melaninas/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Toxina del Pertussis , Hormonas Hipofisarias/metabolismo , Hormonas Hipofisarias/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Receptores de la Hormona Hipofisaria/genética , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The recent identification of the nociceptin receptor-nociceptin system and the description of its role in nociceptive processing has produced numerous investigative studies. A fundamental part of this research is to understand the cellular signaling events (i.e. the building blocks) upon which the pharmacology of this intriguing system is based. As anticipated, nociceptin receptor activation inhibits the formation of cAMP formation via a pertussis toxin-sensitive G-protein. This indicates that nociceptin receptor couples to the G(i)/G(o) class of G-protein(s). However, there is now growing evidence for nociceptin activation of additional signaling pathways, including MAP kinase and phospholipase C/[Ca(2+)](i). These signaling events are discussed in this review.
Asunto(s)
Péptidos Opioides/metabolismo , Receptores Opioides/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Células CHO , Células COS , Calcio/metabolismo , Cricetinae , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Concentración 50 Inhibidora , Canales Iónicos , Cinética , Sistema de Señalización de MAP Quinasas , Modelos Biológicos , Péptidos Opioides/química , Receptores Opioides/química , Transducción de Señal , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismo , Vasodilatadores/química , Vasodilatadores/metabolismo , Receptor de Nociceptina , NociceptinaRESUMEN
The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity (K(D) = 0.47 nM) and saturable binding with 125I-galanin (Bmax = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin approximately = M40 approximately = M15 approximately = M35 approximately = C7 approximately = galanin(2-29) approximately = galanin(1-16) >> galanin(10-29) approximately = galanin(3-29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.
Asunto(s)
Clonación Molecular , ADN Complementario/genética , Genoma , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Isomerismo , Ratones , Datos de Secuencia Molecular , Ratas , Receptor de Galanina Tipo 2 , Receptores de Galanina , Homología de Secuencia de AminoácidoRESUMEN
The recently identified 17-amino acid peptide nociceptin (orphanin FQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) receptor. A physiologic role for nociceptin (OFQ) activation of the ORL-1 receptor (OFQR) may be to modulate opioid-induced analgesia. The molecular mechanism by which nociceptin (OFQ) and ORL-1 (OFQR) modify opioid-stimulated effects, however, is unclear. Both ORL-1 (OFQR) and opioid receptors mediate pertussis toxin (PTX)-sensitive signal transduction, indicating these receptors are capable of coupling to Gi/Go proteins. This study determines that nociceptin stimulates an intracellular signaling pathway, leading to activation of mitogen-activated protein (MAP) kinase in CHO cells expressing ORL-1 receptor (OFQR). Nociceptin (OFQ)-stimulated MAP kinase activation was inhibited by PTX or by expression of the carboxyl terminus of beta-adrenergic receptor kinase (betaARKct), which specifically blocks Gbetagamma-mediated signaling. Expression of the proline-rich domain of SOS (SOS-PRO), which inhibits SOS interaction with p21ras, also attenuated nociceptin (OFQ)-stimulated MAP kinase activation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY294002 reduced nociceptin (OFQ)-stimulated MAP kinase activation, whereas inhibition of protein kinase C (PKC) activity by bisindolylmaleimide I or cellular depletion of PKC had no effect. In a similar manner, in cells expressing mu-opioid receptor, [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO; a mu-opioid receptor-selective agonist) stimulated PTX-sensitive MAP kinase activation that was inhibited by wortmannin, LY294002, betaARKct expression, or SOS-PRO expression but not affected by inhibition of PKC activity. These results indicate that both ORL-1 (OFQR) and mu-opioid receptors mediate MAP kinase activation via a signaling pathway using the betagamma-subunit of Gi, a PI-3K, and SOS, independent of PKC activity. In cells expressing both ORL-1 (OFQR) and mu-opioid receptors, pretreatment with nociceptin decreased subsequent nociceptin (OFQ)- or DAMGO-stimulated MAP kinase activation. In contrast, pretreatment of cells with DAMGO decreased subsequent DAMGO-stimulated MAP kinase but had no effect on subsequent nociceptin (OFQ)-stimulated MAP kinase activation. These results demonstrate that nociceptin (OFQ) activation of ORL-1 (OFQR) can modulate mu-opioid receptor signaling in a cellular system.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/fisiología , Receptores Opioides mu/fisiología , Receptores Opioides/fisiología , Transducción de Señal/fisiología , Animales , Células CHO , Cricetinae , Activación Enzimática/fisiología , Humanos , Péptidos Opioides/farmacología , Receptores Opioides/agonistas , Receptores Opioides/efectos de los fármacos , Receptores Opioides mu/agonistas , Receptores Opioides mu/efectos de los fármacos , Receptor de Nociceptina , NociceptinaRESUMEN
The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Líquido Intracelular/metabolismo , Receptores de la Hormona Gastrointestinal/fisiología , Transducción de Señal , Animales , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Galanina/metabolismo , Galanina/farmacología , Líquido Intracelular/fisiología , Ratas , Receptores de Galanina , Receptores de la Hormona Gastrointestinal/biosíntesis , Receptores de la Hormona Gastrointestinal/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Porcinos , TransfecciónAsunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Receptores de Catecolaminas/fisiología , Animales , Células CHO , Células COS , Cricetinae , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Modelos Biológicos , Receptores de Catecolaminas/biosíntesis , Proteínas Recombinantes/biosíntesis , Transfección , Factores de Virulencia de Bordetella/farmacología , Proteínas ras/metabolismoRESUMEN
G-protein-coupled receptors that mediate cellular responses to a variety of humoral, endothelial-, or platelet-derived substances are able to stimulate MAP kinase activity. In transfected model systems, G-protein-coupled receptors that couple to pertussis toxin-insensitive G proteins of the Gq/11 family mediate this activation predominantly via a PKC-dependent mechanism. In contrast, activation of MAP kinase by receptors that couple to pertussis toxin-sensitive Gi proteins is PKC-independent and requires downstream activation of the low-molecular-weight G protein, Ras. This pathway can be inhibited by coexpression of peptides that sequester Gbetagamma subunits, and is mimicked by overexpression of Gbetagamma subunits. This Ras-dependent MAP kinase activation requires tyrosine phosphorylation of "docking proteins," including the shc adapter protein, and depends upon recruitment of Grb2/Sos1 complexes to the plasma membrane, thus resembling the pathway of MAP kinase activation employed by the receptor tyrosine kinases. Other molecules, including PI-3-kinases and phosphotyrosine phosphatases, probably also contribute to Gbetagamma-subunit-mediated assembly of a mitogenic signaling complex. Identification of the G-protein-coupled, receptor-regulated tyrosine kinase(s), and the means by which the mitogenic signaling complex is assembled at the plasma membrane, remain subjects of further study.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Fosfoproteínas , Receptores de Superficie Celular/metabolismo , Animales , Proteínas Sanguíneas/metabolismo , Activación Enzimática , Humanos , Fosforilación , Transducción de Señal , Tirosina/metabolismo , Proteínas ras/metabolismoRESUMEN
Opioid receptor-like 1 (ORL1) receptor, a member of the superfamily of G-protein-coupled receptors has significant primary sequence homology to the mu-, delta- and kappa-opioid receptors. The ORL1 receptor is selectively activated by the recently discovered peptide nociceptin. To probe the functional homology amongst these receptors, a Chinese hamster ovary (CHO) cell line expressing the human ORL1 receptor has been characterized. Nociceptin inhibited forskolin-stimulated increases in intracellular cAMP with an IC50 of 70 pM. Stimulation by nociceptin caused a 2-fold increase in the rate of [35S]GTPgammaS binding to membranes derived from CHO cells expressing the ORL1 receptor. Following incubation with nociceptin mitogen-activated protein kinase activity was increased by 2-fold in cells expressing the ORL1 receptor. In non-transfected CHO cells, nociceptin had no effect on cAMP accumulation, the rate of [35S]GTPgammaS binding or mitogen-activated protein kinase activity. Human ORL1 receptors expressed in CHO cells selectively bound [125I][Tyr14]nociceptin with a Kd of 2.1 pM and a Bmax of 2.6 pmol/mg protein. Similar to opioid receptors, nociceptin binding to the ORL1 receptor was altered by Na+, GTPgammaS and dithiothreitol. Na+ increased the Kd of nociceptin binding to the ORL1 receptor. GTPgammaS decreased the apparent Bmax of [125I][Tyr14]nociceptin binding but had no effect on the Kd of the remaining sites. Pretreatment with dithiothreitol inhibited nociceptin binding to the ORL1 receptor. Nociceptin binding was insensitive to low nanomolar concentrations of opioid receptor-selective agonists and antagonists. However, high micromolar levels of opioid receptor-selective agents inhibited the binding. Morphine, naloxone, naltrindole, nor-Binaltorphimine and CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) inhibited nociceptin binding to ORL1 receptor with Ki values of 36, 24, 0.4, 8 and 28 microM, respectively. These results imply that ORL1 is a G-protein-coupled receptor with functional as well as structural homology to opioid receptors. In addition, opioid receptor ligands may serve as starting templates for the development of ORL1 specific ligands.
Asunto(s)
Péptidos Opioides/farmacología , Receptores Opioides/agonistas , Animales , Células CHO/efectos de los fármacos , Células CHO/metabolismo , Cricetinae , AMP Cíclico/metabolismo , Ditiotreitol/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Receptor de Nociceptina , NociceptinaAsunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Proteínas de Unión al GTP/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Receptores de Superficie Celular , Transducción de Señal , Animales , Humanos , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiologíaRESUMEN
Several G protein-coupled receptors that interact with pertussis toxin-sensitive heterotrimeric G proteins mediate Ras-dependent activation of mitogen-activated protein (MAP) kinases. The mechanism involves Gbetagamma subunit-mediated increases in tyrosine phosphorylation of the Shc adapter protein, Shc*Grb2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity. We have investigated the role of the ubiquitous nonreceptor tyrosine kinase c-Src in activation of the MAP kinase pathway via endogenous G protein-coupled lysophosphatidic acid (LPA) receptors or by transient expression of Gbetagamma subunits in COS-7 cells. In vitro kinase assays of Shc immunoprecipitates following LPA stimulation demonstrated rapid, transient recruitment of tyrosine kinase activity into Shc immune complexes. Recruitment of tyrosine kinase activity was pertussis toxin-sensitive and mimicked by cellular expression of Gbetagamma subunits. Immunoblots for coprecipitated proteins in Shc immunoprecipitates revealed a transient association of Shc and c-Src following LPA stimulation, which coincided with increases in Shc-associated tyrosine kinase activity and Shc tyrosine phosphorylation. LPA stimulation or expression of Gbetagamma subunits resulted in c-Src activation, as assessed by increased c-Src autophosphorylation. Overexpression of wild-type or constitutively active mutant c-Src, but not kinase inactive mutant c-Src, lead to increased tyrosine kinase activity in Shc immunoprecipitates, increased Shc tyrosine phosphorylation, and Shc.Grb2 complex formation. MAP kinase activation resulting from LPA receptor stimulation, expression of Gbetagamma subunits, or expression of c-Src was sensitive to dominant negatives of mSos, Ras, and Raf. Coexpression of Csk, which inactivates Src family kinases by phosphorylating the regulatory C-terminal tyrosine residue, inhibited LPA stimulation of Shc tyrosine phosphorylation, Shc.Grb2 complex formation, and MAP kinase activation. These data suggest that Gbetagamma subunit-mediated formation of Shc.c-Src complexes and c-Src kinase activation are early events in Ras-dependent activation of MAP kinase via pertussis toxin-sensitive G protein-coupled receptors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Animales , Proteína Tirosina Quinasa CSK , Proteínas Portadoras/metabolismo , Línea Celular , Activación Enzimática , Humanos , Fosforilación , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Proteínas ras/metabolismo , Familia-src QuinasasRESUMEN
The beta gamma-subunit of Gi mediates mitogen-activated protein (MAP) kinase activation through a signaling pathway involving Shc tyrosine phosphorylation, subsequent formation of a multiprotein complex including Shc, Grb2, and Sos, and sequential activation of Ras, Raf, and MEK. The mechanism by which G beta gamma mediates tyrosine phosphorylation of Shc, however, is unclear. This study assesses the role of phosphatidylinositol 3-kinase (PI-3K) in G beta gamma-mediated MAP kinase activation. We show that Gi-coupled receptor- and G beta gamma-stimulated MAP kinase activation is attenuated by the PI-3K inhibitors wortmannin and LY294002 or by over expression of a dominant negative mutant of the p85 subunit of PI-3K. Wortmannin and LY294002 also inhibit Gi-coupled receptor-stimulated Ras activation. The PI-3K inhibitors do not affect MAP kinase activation stimulated by over-expression of Sos, a constitutively active mutant of Ras, or a constitutively active mutant of MEK. These results demonstrate that PI-3K activity is required in the G beta gamma-mediated MAP kinase signaling pathway at a point upstream of Sos and Ras activation.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Androstadienos/farmacología , Animales , Células CHO , Línea Celular , Cromonas/farmacología , Cricetinae , Inhibidores Enzimáticos/farmacología , Morfolinas/farmacología , Mutación , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , WortmaninaRESUMEN
Mitogen-activated protein kinase (MAPK) is activated in response to both receptor tyrosine kinases and G-protein-coupled receptors. Recently, Gi-coupled receptors, such as the alpha 2A adrenergic receptor, were shown to mediate Ras-dependent MAPK activation via a pathway requiring G-protein beta gamma subunits (G beta gamma) and many of the same intermediates involved in receptor tyrosine kinase signaling. In contrast, Gq-coupled receptors, such as the M1 muscarinic acetylcholine receptor (M1AChR), activate MAPK via a pathway that is Ras-independent but requires the activity of protein kinase C (PKC). Here we show that, in Chinese hamster ovary cells, the M1AChR and platelet-activating factor receptor (PAFR) mediate MAPK activation via the alpha-subunit of the G(o) protein. G(o)-mediated MAPK activation was sensitive to treatment with pertussis toxin but insensitive to inhibition by a G beta gamma-sequestering peptide (beta ARK1ct). M1AChR and PAFR catalyzed G(o) alpha-subunit GTP exchange, and MAPK activation could be partially rescued by a pertussis toxin-insensitive mutant of G(o) alpha but not by similar mutants of Gi. G(o)-mediated MAPK activation was insensitive to inhibition by a dominant negative mutant of Ras (N17Ras) but was completely blocked by cellular depletion of PKC. Thus, M1AChR and PAFR, which have previously been shown to couple to Gq, are also coupled to G(o) to activate a novel PKC-dependent mitogenic signaling pathway.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Toxina del Pertussis , Proteína Quinasa C/metabolismo , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Factores de Virulencia de Bordetella/farmacología , Animales , Células CHO , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Activación Enzimática , Sustancias Macromoleculares , Modelos Biológicos , Mutagénesis , Glicoproteínas de Membrana Plaquetaria/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Muscarínicos/fisiología , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección , Proteínas ras/metabolismoRESUMEN
The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Receptores Adrenérgicos alfa 2/fisiología , Androstadienos/farmacología , Animales , Benzoquinonas , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Chlorocebus aethiops , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/biosíntesis , Genisteína , Humanos , Isoflavonas/farmacología , Riñón , Cinética , Lactamas Macrocíclicas , Sustancias Macromoleculares , Toxina del Pertussis , Fosfatos/metabolismo , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinonas/farmacología , Receptores Adrenérgicos alfa 2/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Rifabutina/análogos & derivados , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Acetato de Tetradecanoilforbol/farmacología , Transfección , Factores de Virulencia de Bordetella/farmacología , WortmaninaRESUMEN
Mitogen-activated protein (MAP) kinases mediate the phosphorylation and activation of nuclear transcription factors that regulate cell growth. MAP kinase activation may result from stimulation of either tyrosine-kinase (RTK) receptors, which possess intrinsic tyrosine kinase activity, or G-protein-coupled receptors (GPCR). RTK-mediated mitogenic signalling involves a series of SH2- and SH3-dependent protein-protein interactions between tyrosine-phosphorylated receptor, Shc, Grb2 and Sos, resulting in Ras-dependent MAP kinase activation. The beta gamma subunits of heterotrimeric G proteins (G beta gamma) also mediate Ras-dependent MAP kinase activation by an as-yet unknown mechanism. Here we demonstrate that activation of MAP kinase by Gi-coupled receptors is preceded by the G beta gamma-mediated tyrosine phosphorylation of Shc, leading to an increased functional association between Shc, Grb2 and Sos. Moreover, disruption of the Shc-Grb2-Sos complex blocks G beta gamma-mediated MAP kinase activation, indicating that G beta gamma does not mediate MAP kinase activation by a direct interaction with Sos. These results indicate that G beta gamma-mediated MAP kinase activation is initiated by a tyrosine phosphorylation event and proceeds by a pathway common to both GPCRs and RTKs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Línea Celular , Activación Enzimática , Factor de Crecimiento Epidérmico/metabolismo , Proteína Adaptadora GRB2 , Proteínas de la Membrana/metabolismo , Fosforilación , Proteínas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteínas Son Of Sevenless , Tirosina/metabolismoRESUMEN
Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.