RESUMEN
Activator of G protein signaling 3 (AGS3) is a newly identified protein shown to act at the level of the G protein itself. AGS3 belongs to the GoLoco family of proteins, sharing the 19-aa GoLoco motif that is a Galpha(i/o) binding motif. AGS3 interacts only with members of the Galpha(i/o) subfamily. By surface plasmon resonance, we found that AGS3 binds exclusively to the GDP-bound form of Galpha(i3). In GTPgammaS binding assays, AGS3 behaves as a guanine dissociation inhibitor (GDI), inhibiting the rate of exchange of GDP for GTP by Galpha(i3). AGS3 interacts with both Galpha(i3) and Galpha(o) subunits, but has GDI activity only on Galpha(i3), not on Galpha(o). The fourth GoLoco motif of AGS3 is a major contributor to this activity. AGS3 stabilizes Galpha(i3) in its GDP-bound form, as it inhibits the increase in tryptophan fluorescence of the Galpha(i3)-GDP subunit stimulated by AlF(4)(-). AGS3 is widely expressed as it is detected by immunoblotting in brain, testis, liver, kidney, heart, pancreas, and in PC-12 cells. Several different sizes of the protein are detected. By Northern blotting, AGS3 shows 2.3-kb and 3.5-kb mRNAs in heart and brain, respectively, suggesting tissue-specific alternative splicing. Taken together, our results demonstrate that AGS3 is a GDI. To the best of our knowledge, no other GDI has been described for heterotrimeric G proteins. Inhibition of the Galpha subunit and stimulation of heterotrimeric G protein signaling, presumably by stimulating Gbetagamma, extend the possibilities for modulating signal transduction through heterotrimeric G proteins.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Guanina/metabolismo , Compuestos de Aluminio , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Fraccionamiento Celular , Fluoruros , Guanosina Difosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Datos de Secuencia Molecular , Ratas , Distribución TisularRESUMEN
Mitochondria play a key part in the regulation of apoptosis (cell death). Their intermembrane space contains several proteins that are liberated through the outer membrane in order to participate in the degradation phase of apoptosis. Here we report the identification and cloning of an apoptosis-inducing factor, AIF, which is sufficient to induce apoptosis of isolated nuclei. AIF is a flavoprotein of relative molecular mass 57,000 which shares homology with the bacterial oxidoreductases; it is normally confined to mitochondria but translocates to the nucleus when apoptosis is induced. Recombinant AIF causes chromatin condensation in isolated nuclei and large-scale fragmentation of DNA. It induces purified mitochondria to release the apoptogenic proteins cytochrome c and caspase-9. Microinjection of AIF into the cytoplasm of intact cells induces condensation of chromatin, dissipation of the mitochondrial transmembrane potential, and exposure of phosphatidylserine in the plasma membrane. None of these effects is prevented by the wide-ranging caspase inhibitor known as Z-VAD.fmk. Overexpression of Bcl-2, which controls the opening of mitochondrial permeability transition pores, prevents the release of AIF from the mitochondrion but does not affect its apoptogenic activity. These results indicate that AIF is a mitochondrial effector of apoptotic cell death.
Asunto(s)
Apoptosis/fisiología , Flavoproteínas/fisiología , Proteínas de la Membrana/fisiología , Mitocondrias/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Factor Inductor de la Apoptosis , Línea Celular , Núcleo Celular/fisiología , Sistema Libre de Células , Cromatina/fisiología , Grupo Citocromo c/fisiología , Flavoproteínas/genética , Flavoproteínas/aislamiento & purificación , Células HeLa , Humanos , Membranas Intracelulares/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Ratones , Microinyecciones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Ratas , Proteínas Recombinantes/farmacologíaRESUMEN
Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the alpha subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein gamma subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different Gbeta subunits reveals specific interaction between RGS11 and Gbeta5. The expression of mRNA for RGS11 and Gbeta5 in human tissues overlaps. The Gbeta5/RGS11 heterodimer acts as a GAP on Galphao, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for Galpha proteins and form complexes with specific Gbeta subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Proteínas/química , Proteínas/metabolismo , Retina/metabolismo , Transcripción Genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Caenorhabditis elegans , Bovinos , Secuencia de Consenso , Secuencia Conservada , Proteínas de Unión al GTP/genética , Proteínas Activadoras de GTPasa , Humanos , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Spodoptera , TransfecciónRESUMEN
PTEN is a tumor suppressor with sequence homology to protein tyrosine phosphatases and the cytoskeletal protein tensin. mPTEN-mutant mouse embryos display regions of increased proliferation. In contrast, mPTEN-deficient immortalized mouse embryonic fibroblasts exhibit decreased sensitivity to cell death in response to a number of apoptotic stimuli, accompanied by constitutively elevated activity and phosphorylation of protein kinase B/Akt, a crucial regulator of cell survival. Expression of exogenous PTEN in mutant cells restores both their sensitivity to agonist-induced apoptosis and normal pattern of PKB/Akt phosphorylation. Furthermore, PTEN negatively regulates intracellular levels of phosphatidylinositol (3,4,5) trisphosphate in cells and dephosphorylates it in vitro. Our results show that PTEN may exert its role as a tumor suppressor by negatively regulating the PI3'K/PKB/Akt signaling pathway.
Asunto(s)
Apoptosis , Genes Supresores de Tumor , Monoéster Fosfórico Hidrolasas , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor , Animales , División Celular , Supervivencia Celular , Células Cultivadas , Femenino , Fibroblastos/citología , Ratones , Mutagénesis , Fosfohidrolasa PTEN , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Especificidad por SustratoRESUMEN
Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein, and recent studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Plasticidad Neuronal/fisiología , Neuronas/enzimología , Transducción de Señal/fisiología , Animales , Células COS/química , Células COS/enzimología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Corteza Cerebral/química , Corteza Cerebral/citología , Corteza Cerebral/enzimología , Cocaína/farmacología , Antagonistas de Dopamina/farmacología , Inhibidores de Captación de Dopamina/farmacología , Femenino , GTP Fosfohidrolasas/metabolismo , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Genes Inmediatos-Precoces/fisiología , Haloperidol/farmacología , Hipocampo/química , Hipocampo/citología , Hipocampo/enzimología , Hidrólisis , Metabolismo de los Lípidos , Masculino , Neuronas/química , Neuronas/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas F344 , Ratas Sprague-Dawley , Receptores Muscarínicos/fisiología , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.