RESUMEN
Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-ß peptide (Aß) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aß-targeted treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Cognición , Terapia Genética/métodos , Interleucina-10/genética , Neurogénesis/genética , Presenilina-1/genética , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/sangre , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Técnicas de Cocultivo , Dependovirus/genética , Hipocampo/metabolismo , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Mutación , Células-Madre Neurales , Neuronas/metabolismoRESUMEN
Thermal injury causes a hypermetabolic state associated with increased levels of catabolic hormones, but the molecular bases for the metabolic abnormalities are poorly understood. We investigated the lipolytic responses after beta(3)-adrenoceptor (beta(3)-AR) agonists and evaluated the associated changes in beta-AR and its downstream signaling molecules in adipocytes isolated from rats with thermal injury. Maximal lipolytic responses to a specific beta(3)-AR agonist, BRL-37344, were significantly attenuated at post burn days (PBD) 3 and 7. Despite significant reduction of the cell surface beta(3)-AR number and its mRNA at PBD 3 and 7, BRL-37344 and forskolin-stimulated cAMP levels were not decreased. Glycerol production in response to dibutyryl cAMP, a direct stimulant of hormone-sensitive lipase (HSL) via protein kinase A (PKA), was significantly attenuated. Although immunoblot analysis indicated no differences in the expression and activity of PKA or in the expression of HSL, HSL activity showed significant reductions. Finally, beta(3)-AR-induced insulin secretion was indeed attenuated in vivo. These studies indicate that the beta(3)-AR system is desensitized after burns, both in the adipocytes and in beta(3)-AR-induced secretion of insulin. Furthermore, these data suggest a complex and unique mechanism underlying the altered signaling of lipolysis at the level of HSL in animals after burns.
Asunto(s)
Quemaduras/metabolismo , Lipólisis/fisiología , Receptores Adrenérgicos beta/fisiología , Adipocitos/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Etanolaminas/farmacología , Insulina/sangre , Cinética , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Transducción de Señal/fisiología , Esterol Esterasa/metabolismoRESUMEN
Caveolins-1 and -2 are normally co-expressed, and they form a hetero-oligomeric complex in many cell types. These caveolin hetero-oligomers are thought to represent the assembly units that drive caveolae formation in vivo. However, the functional significance of the interaction between caveolins-1 and -2 remains unknown. Here, we show that caveolin-1 co-expression is required for the transport of caveolin-2 from the Golgi complex to the plasma membrane. We identified a human erythroleukemic cell line, K562, that expresses caveolin-2 but fails to express detectable levels of caveolin-1. This allowed us to stringently assess the effects of recombinant caveolin-1 expression on the behavior of endogenous caveolin-2. We show that expression of caveolin-1 in K562 cells is sufficient to reconstitute the de novo formation of caveolae in these cells. In addition, recombinant expression of caveolin-1 allows caveolin-2 to form high molecular mass oligomers that are targeted to caveolae-enriched membrane fractions. In striking contrast, in the absence of caveolin-1 expression, caveolin-2 forms low molecular mass oligomers that are retained at the level of the Golgi complex. Interestingly, we also show that expression of caveolin-1 in K562 cells dramatically up-regulates the expression of endogenous caveolin-2. Northern blot analysis reveals that caveolin-2 mRNA levels remain constant under these conditions, suggesting that the expression of caveolin-1 stabilizes the caveolin-2 protein. Conversely, transient expression of caveolin-2 in CHO cells is sufficient to up-regulate endogenous caveolin-1 expression. Thus, the formation of a hetero-oligomeric complex between caveolins-1 and -2 stabilizes the caveolin-2 protein product and allows caveolin-2 to be transported from the Golgi complex to the plasma membrane.
Asunto(s)
Caveolinas , Membrana Celular/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Transporte Biológico , Northern Blotting , Caveolina 1 , Caveolina 2 , Humanos , Células K562 , ARN Mensajero/análisisRESUMEN
Here, we investigate the involvement of caveolins in the pathophysiology of Alzheimer's disease (AD). We show dramatic upregulation of caveolin-3 immunoreactivity in astroglial cells surrounding senile plaques in brain tissue sections from authentic AD patients and an established transgenic mouse model of AD. In addition, we find that caveolin-3 physically interacts and biochemically colocalizes with amyloid precursor protein (APP) both in vivo and in vitro. Interestingly, recombinant overexpression of caveolin-3 in cultured cells stimulated beta-secretase-mediated processing of APP. Immunoreactivities of APP and presenilins were concomitantly increased in caveolin-3-positive astrocytes. Because the presenilins also form a physical complex with caveolin-3, caveolin-3 may provide a common platform for APP and the presenilins to associate in astrocytes. In AD, augmented expression of caveolin-3 and presenilins in reactive astrocytes may alter APP processing, leading to the overproduction of its toxic amyloid metabolites.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/química , Caveolinas , Endopeptidasas/fisiología , Proteínas de la Membrana/metabolismo , Anciano , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/fisiología , Animales , Ácido Aspártico Endopeptidasas , Astrocitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Caveolina 3 , Endopeptidasas/metabolismo , Humanos , Proteínas de la Membrana/fisiología , Ratones , Ratones Transgénicos , Presenilina-1 , Presenilina-2 , Proteínas RecombinantesRESUMEN
Caveolins 1, 2 and 3 are the principal protein components of caveolae organelles. It has been proposed that caveolae play a vital role in a number of essential cellular functions including signal transduction, lipid metabolism, cellular growth control and apoptotic cell death. Thus, a major focus of caveolae-related research has been the identification of novel caveolins, caveolae-associated proteins and caveolin-interacting proteins. However, virtually nothing is known about the expression of caveolins in brain tissue. Here, we report the purification and characterization of caveolins from brain tissue under non-denaturing conditions. As a final step in the purification, we employed immuno-affinity chromatography using rabbit polyclonal anti-caveolin IgG and specific elution at alkaline pH. The final purified brain caveolin fractions contained three bands with molecular masses of 52 kDa, 24 kDa and 22 kDa as visualized by silver staining. Sequencing by ion trap mass spectrometry directly identified the major 24-kDa component of this hetero-oligomeric complex as caveolin 1. Further immunocyto- and histochemical analyses demonstrated that caveolin 1 was primarily expressed in brain endothelial cells. Caveolins 2 and 3 were also detected in purified caveolin fractions and brain cells. The cellular distribution of caveolin 2 was similar to that of caveolin 1. In striking contrast, caveolin 3 was predominantly expressed in brain astroglial cells. This finding was surprising as our previous studies have suggested that the expression of caveolin 3 is confined to striated (cardiac and skeletal) and smooth muscle cells. Electron-microscopic analysis revealed that astrocytes possess numerous caveolar invaginations of the plasma membrane. Our results provide the first biochemical and histochemical evidence that caveolins 1, 2 and 3 are expressed in brain endothelial and astroglial cells.
Asunto(s)
Astrocitos/metabolismo , Química Encefálica/fisiología , Caveolinas , Endotelio Vascular/metabolismo , Proteínas de la Membrana/análisis , Secuencia de Aminoácidos , Animales , Encéfalo/citología , Bovinos , Caveolina 1 , Caveolina 2 , Caveolina 3 , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Immunoblotting , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Membranas/química , Datos de Secuencia Molecular , Pruebas de Precipitina , Ratas , Ratas Sprague-DawleyRESUMEN
The p42/44 mitogen-activated protein (MAP)-kinase cascade is a well-established signal transduction pathway that is initiated at the cell surface and terminates within the nucleus. More specifically, receptor tyrosine kinases can indirectly activate Raf, which in turn leads to activation of MEK and ERK and ultimately phosphorylation of Elk, a nuclear transcription factor. Recent reports have suggested that some members of p42/44 MAP kinase cascade can be sequestered within plasmalemmal caveolae in vivo. For example, morphological studies have directly shown that ERK-1/2 is concentrated in plasma membrane caveolae in vivo using immunoelectron microscopy. In addition, constitutive activation of the p42/44 MAP kinase cascade is sufficient to reversibly down-regulate caveolin-1 mRNA and protein expression. However, the functional relationship between the p42/44 MAP kinase cascade and caveolins remains unknown. Here, we examine the in vivo role of caveolins in regulating signaling along the MAP kinase cascade. We find that co-expression with caveolin 1 dramatically inhibits signaling from EGF-R, Raf, MEK-1 and ERK-2 to the nucleus. Using a variety of caveolin-1 deletion mutants, we mapped this in vivo inhibitory activity to caveolin-1 residues 32-95. Peptides derived from this region of caveolin 1 also inhibit the in vitro kinase activity of purified MEK-1 and ERK-2. Thus, we show here that caveolin-1 expression can inhibit signal transduction from the p42/44 MAP kinase cascade both in vitro and in vivo. Taken together with previous data, our results also suggest that a novel form of reciprocal negative regulation exists between p42/44 MAP kinase activation and caveolin-1 protein expression, i.e. up-regulation of caveolin-1 protein expression down-modulates p42/44 MAP kinase activity (this report) and up-regulation of p42/44 MAP kinase activity down-regulates caveolin-1 mRNA and protein expression.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Caveolinas , Proteínas de Unión al ADN , Proteínas de la Membrana/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos , Transducción de Señal/fisiología , Factores de Transcripción , Animales , Células CHO , Caveolina 1 , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Cricetinae , Receptores ErbB/biosíntesis , Receptores ErbB/fisiología , Cinética , MAP Quinasa Quinasa 1 , Proteínas de la Membrana/química , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Conejos , Proteínas Recombinantes/metabolismo , Transfección , Proteína Elk-1 con Dominio etsRESUMEN
Caveolae are plasma membrane invaginations where key signaling elements are concentrated. In this report, both biochemical and histochemical analyses demonstrate that the amyloid precursor protein (APP), a source of Abeta amyloid peptide, is enriched within caveolae. Caveolin-1, a principal component of caveolae, is physically associated with APP, and the cytoplasmic domain of APP directly participates in this binding. The characteristic C-terminal fragment that results from APP processing by alpha-secretase, an as yet unidentified enzyme that cleaves APP within the Abeta amyloid sequence, was also localized within these caveolae-enriched fractions. Further analysis by cell surface biotinylation revealed that this cleavage event occurs at the cell surface. Importantly, alpha-secretase processing was significantly promoted by recombinant overexpression of caveolin in intact cells, resulting in increased secretion of the soluble extracellular domain of APP. Conversely, caveolin depletion using antisense oligonucletotides prevented this cleavage event. Our current results indicate that caveolae and caveolins may play a pivotal role in the alpha-secretase-mediated proteolysis of APP in vivo.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Caveolinas , Endopeptidasas/metabolismo , Procesamiento Proteico-Postraduccional , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Caveolina 1 , Línea Celular , Membrana Celular/enzimología , Membrana Celular/metabolismo , Humanos , Hidrólisis , Proteínas de la Membrana/metabolismoRESUMEN
Burn injury is associated with insulin resistance. The molecular basis of this resistance was investigated by examining insulin receptor signaling in rats after thermal injury. The impaired insulin-stimulated transport of [3H]2-deoxyglucose into soleus muscle strips confirmed the insulin resistance following burns. In vivo insulin-stimulated phosphoinositide 3-kinase activity, pivotal in translocation of GLUT4, was decreased in burns when assessed by its insulin receptor substrate-1 (IRS-1)-associated activity. Insulin-induced tyrosine kinase activity of insulin receptor (IR) and tyrosine phosphorylation of IRS-1 were also attenuated. Immunoprecipitated IR, however, appeared to have normal insulin-responsive kinase activity. Finally, immunoprecipitated IRS-1 was tested for its effect on partially purified recombinant IR and was found to inhibit its kinase activity. This inhibitory effect of IRS-1 was abolished by prior treatment of IRS-1 with alkaline phosphatase, indicating that burn injury-related hyperphosphorylation of IRS-1 is similar to that observed in TNFalpha-induced inhibition of IR signaling. All of these changes were observed in the absence of quantitative changes in IR, IRS-1, and phosphoinositide 3-kinase. Alterations in postreceptor insulin signaling, therefore, may be responsible for the insulin resistance after thermal injury.
Asunto(s)
Quemaduras/fisiopatología , Glucosa/metabolismo , Resistencia a la Insulina , Insulina/farmacología , Proteínas Musculares , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Receptor de Insulina/metabolismo , Fosfatasa Alcalina , Animales , Ayuno , Transportador de Glucosa de Tipo 4 , Proteínas Sustrato del Receptor de Insulina , Cinética , Masculino , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/farmacología , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotirosina/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Insulina/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Valores de ReferenciaRESUMEN
In familial Alzheimer's disease (FAD), three missense mutations, V642I, V642F and V642G, that co-segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin-sensitive apoptosis in an FAD trait-linked manner by activating the G protein Go, which consists of G alpha(o) and G betagamma subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated G alpha(o) or G alpha(i) induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy-terminal amino acids 495-689 of the beta-adrenergic receptor kinase-1, which blocks the specific functions of G betagamma. Co-transfection of G beta2gamma2 cDNAs, but not that of other G beta(x)gamma(z) (x = 1-3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl-2. These data implicate G betagamma as a cell death mediator for the FAD-associated mutant of APP.
Asunto(s)
Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Apoptosis , Proteínas de Unión al GTP/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Southern Blotting , Células COS , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Fragmentación del ADN , ADN Complementario , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/genética , Genes bcl-2 , Humanos , Inmunohistoquímica , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Toxina del Pertussis , Transfección , Factores de Virulencia de Bordetella/farmacología , Quinasas de Receptores Adrenérgicos betaRESUMEN
In familial Alzheimer's disease (FAD), mutations to I, F, and G have been discovered at V642 in the neuron-specific version of the amyloid precursor protein APP695. It has been found that expression of each FAD mutant suppresses the transcriptional activity of the cAMP response element CRE in a G alpha(o)-dependent manner in a COS cell clone NK1 [Ikezu et al. (1996) EMBO J. 15, 2468-2475]. Here we show that adenylyl cyclase (AC) inhibition is probably not the prerequisite for this pathway. First, expression of each FAD mutant in NK1 cells had no effect on AC activity stimulated by cholera toxin and by mutationally activated G alpha(s), although the same expression completely repressed the stimulated CRE. Second, a transfected activating mutant of G alpha(o) inhibited CRE without detectable suppression of AC, whereas similarly transfected activating G alpha(i2) inhibited both AC and CRE. Third, FAD mutant-induced inhibition occurred for CRE activity stimulated by dibutyryl cAMP. These data suggest that CRE suppression by FAD mutants of APP could occur independently of AC.
Asunto(s)
Adenilil Ciclasas/metabolismo , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , AMP Cíclico/farmacología , Mutación , Secuencias Reguladoras de Ácidos Nucleicos , Bucladesina/farmacología , Línea Celular , Toxina del Cólera/farmacología , Proteínas de Unión al GTP/fisiología , Humanos , TransfecciónRESUMEN
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes. In this regard, caveolin co-purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS. Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions. For example, this domain interacts with G-protein alpha subunits and Src-like kinases and can functionally suppress their activity. This caveolinderived protein domain has been termed the caveolin-scaffolding domain. However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules. Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries. These caveolin-selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases. A known caveolin-interacting protein, Gi2alpha, was used as a ligand to further investigate the nature of this interaction. Gi2alpha and other G-protein alpha subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from Gi2alpha interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of Gi2alpha. This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding domain. In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands. Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions. These results serve to further demonstrate the specificity of this interaction. The implications of our current findings are discussed regarding other caveolin- and caveolae-associated proteins.
Asunto(s)
Caveolinas , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Caveolina 1 , Sistema Libre de Células , Citoplasma/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Guanosina Trifosfato/metabolismo , Ligandos , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
In familial Alzheimer's disease (FAD), missense point mutations V642I/F/G, which co-segregate with the disease phenotype, have been discovered in amyloid precursor APP695. Here, we report that three FAD mutants (FAD-APPs) negatively regulated the transcriptional activity of cAMP response element (CRE) by a G(o)-dependent mechanism, but expression of wildtype APP695 had no effect on CRE. Experiments with various Galpha(s) chimeras demonstrated that Phe-APP coupled selectively to the C-terminus of Galpha(0). Again, wild-type APP695 had no effect on its C-terminus. These data indicate that FAD-APPs are gain-of-function mutants of APP695 that negatively regulate the CRE activity through G(o). This negative transactivation of CRE is the first biochemically analyzed signal evoked by the three FAD-APPs, but not by wild-type APP695, in a whole-cell system. We discuss the significance of constitutive CRE suppression by FAD-APPs, which is potentially relevant to synaptic malplasticity or memory disorders.
Asunto(s)
Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , AMP Cíclico/fisiología , Secuencias Reguladoras de Ácidos Nucleicos , Sistemas de Mensajero Secundario/genética , Activación Transcripcional , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Regulación de la Expresión Génica , Humanos , Memoria/fisiología , Datos de Secuencia Molecular , Plasticidad Neuronal/fisiología , Mutación Puntual , Proteínas Recombinantes de Fusión/biosíntesis , Fase de Descanso del Ciclo CelularRESUMEN
We examined the signaling function of the IGF-II/mannose 6-phosphate receptor (IGF-IIR) by transfecting IGF-IIR cDNAs into COS cells, where adenylyl cyclase (AC) was inhibited by transfection of constitutively activated G alpha i cDNA (G alpha i2Q205L). In cells transfected with IGF-IIR cDNA, IGF-II decreased cAMP accumulation promoted by cholera toxin or forskolin. This effect of IGF-II was not observed in untransfected cells or in cells transfected with IGF-IIRs lacking Arg2410-Lys2423. Thus, IGF-IIR, through its cytoplasmic domain, mediates the Gi-linked action of IGF-II in living cells. We also found that IGF-IIR truncated with C-terminal 28 residues after Ser2424 caused G beta gamma-dominant response of AC in response to IGF-II by activating Gi. Comparison with the G alpha i-dominant response of AC by intact IGF-IIR suggests that the C-terminal 28-residue region inactivates G beta gamma. This study not only provides further evidence that IGF-IIR has IGF-II-dependent signaling function to interact with heteromeric G proteins with distinct roles by different cytoplasmic domains, it also suggests that IGF-IIR can separate and sequestrate the G alpha and G beta gamma signals following Gi activation.
Asunto(s)
Proteínas de Unión al GTP/química , Receptor IGF Tipo 2/química , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Datos de Secuencia Molecular , Receptor IGF Tipo 2/fisiologíaRESUMEN
G alpha i2 is a tissue-specific proto-oncogene product, whose activated mutant gip2 induces transformation through less defined downstream pathways. We found that c-fos promoter is a target of gip2 in multiple kinds of cells. Serum response element was shown to be the positive enhancer element that mediates gip2-induced c-fos expression. We further demonstrated that gip2 stimulates the negative silencer activity of the retinoblastoma (Rb) control element (RCE) and inhibits the c-fos promoter activity through RCE located in the c-fos promoter region. The effect of gip2 on RCE was shown to be mediated by the Rb gene product (pRb). Furthermore, gip2 augmented underphosphorylated active form of pRb by promoting pRb expression and by affecting the phosphorylation state of pRb. gip2 therefore propagates both positive and negative signals to the c-fos promoter through two different elements, and pRb mediates the negative signal of gip2. We conclude that gip2 has bifunctional roles in transformation which pRb critically regulates. Given that Rat-1 cells, which gip2 can transform, lack the sensitivity to the gip2/pRb-mediated negative pathway, this study provides a novel insight into oncogenesis by gip2 and its tissue specificity.
Asunto(s)
Transformación Celular Neoplásica , Elementos de Facilitación Genéticos , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP/genética , Regulación Neoplásica de la Expresión Génica , Genes fos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/química , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Proteínas de Unión al GTP/metabolismo , Genes de Retinoblastoma , Genes myc , Técnicas In Vitro , Datos de Secuencia Molecular , Mutación , Proteínas Oncogénicas/metabolismo , ARN Mensajero/genética , Proteína de Retinoblastoma/fisiología , Transducción de Señal , Transcripción Genética , TransfecciónRESUMEN
The 14-residue peptide (peptide 14) corresponding to Arg2410-Lys2423 of the insulin-like growth factor II receptor (IGF-IIR) can activate the adenylate cyclase-inhibitor guanine nucleotide-binding protein Gi, and the 15-residue beta III-2 peptide Arg259-Lys273 of the beta 2-adrenergic receptor (beta 2AR) can activate the stimulatory protein Gs. In phospholipid vesicles, IGF-IIR and beta 2AR activate Gi and Gs in response to IGF-II and isoproterenol, respectively. We constructed a chimeric IGF-II receptor (beta III-2/IGF-IIR) by converting its native peptide 14 sequence to the beta III-2 sequence. In cells expressing beta III-2/IGF-IIR, membrane adenylate cyclase activity markedly increased without IGF-II and was further promoted by IGF-II. This was verified by measuring chloramphenicol acetyltransferase (CAT) activity in beta III-2/IGF-IIR cells with cotransfection of a cAMP response element-CAT construct. This study shows not only the conversion of G-protein specificity of a receptor from Gi to Gs but also the simulation of G protein-coupled receptor signals by using a short receptor region and intact cells. These findings indicate that the G protein-activation signals are interchangeable, self-determined structural motifs that function in the setting of either a single-spanning or multiple-spanning receptor.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptor IGF Tipo 2/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Adenilil Ciclasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/enzimología , Membrana Celular/metabolismo , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/farmacología , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Estructura Secundaria de Proteína , Receptor IGF Tipo 2/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The human alpha 2-adrenergic receptor contains the sequence KASRWRGRQNREKRFTF (amino acids 356-372) at the C-terminal end of its third intracellular loop. This sequence satisfies the structural criteria for G protein-activating sequences [(1992) J. Biol. Chem. 267, 8342-8346] except that the C-terminal sequence is B-B-X-X-Phe instead of B-B-X-B or B-B-X-X-B (B: basic residue, X: non-basic residue). Nevertheless, the synthetic peptide corresponding to this sequence (peptide alpha 2-F) was found to activate Gi and Go strongly with a saturated effect at 1-3 microM. Furthermore, the substitution of the C-terminal Phe of peptide alpha 2-F with Arg, Trp, and Tyr (but not Ala or Asp) did not appreciably affect the Gi-activating potency. It is suggested that the C-terminal basic residue of the B-B-X-X-B motif in Gi-activating sequences can be replaced by an aromatic residue.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Secuencia Conservada , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato) , Modelos Biológicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/genética , Fenilalanina , Unión Proteica , Receptores Adrenérgicos alfa/genéticaRESUMEN
We developed a novel method to quantitatively measure GTP gamma S binding to specific G proteins in crude membranes using G-protein antibodies. The basic strategy was that the materials were initially incubated with [35S]GTP gamma S at 37 degrees C. After 4 degrees C incubation in the wells of an ELISA plate precoated with G-protein antibodies, the radioactivity of each well was counted. This method, using an anti-Gi antiserum and an anti-Gs antiserum, quantitatively and specifically detected the binding of GTP gamma S to purified Gi2 and Gs. In S49 cell membranes, GTP gamma S binding to immunoreactive Gs was observed in a time-dependent manner that obeyed first-order kinetics, and the rate constant was stimulated approximately twofold in response to isoproterenol. The effect of isoproterenol was not observed in unc mutant membranes. The present method thus makes it possible to quantitatively measure GTP gamma S binding to specific G proteins in cell membranes.