RESUMEN
SignificanceThe modulation of growth hormone secretagogue receptor-1a (GHSR1a) signaling is a promising strategy for treating brain conditions of metabolism, aging, and addiction. GHSR1a activation results in pleiotropic physiological outcomes through distinct and pharmacologically separable G protein- and ß-arrestin (ßarr)-dependent signaling pathways. Thus, pathway-selective modulation can enable improved pharmacotherapeutics that can promote therapeutic efficacy while mitigating side effects. Here, we describe the discovery of a brain-penetrant small molecule, N8279 (NCATS-SM8864), that biases GHSR1a conformations toward Gαq activation and reduces aberrant dopaminergic behavior in mice. N8279 represents a promising chemical scaffold to advance the development of better treatments for GHSR1a-related brain disorders involving the pathological dysregulation of dopamine.
Asunto(s)
Encéfalo/metabolismo , Dopamina/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Receptores de Ghrelina/metabolismo , Animales , Dopamina/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Masculino , Ratones , Ratones Noqueados , Receptores de Ghrelina/genéticaRESUMEN
Diffusion of the complex consisting of low density lipoprotein (LDL) bound to its receptor on the surface of human fibroblasts has been measured with the help of an intensely fluorescent, biologically active LDL derivative, dioctadecylindocarbocyanine LDL (dil(3)-LDL). Fluorescence photobleaching recovering and direct video observations of the Brownian motion of individual LDL-receptor complexes yielded diffusion coefficients for the slow diffusion on cell surfaces and fast diffusion on membrane blebs, respectively. At 10 degrees C, less that 20 percent of the LDL-receptor complex was measurably diffusible either on normal human fibroblasts GM-3348 or on LDL-receptor- internalization-defective J.D. cells GM-2408A. At 21 degrees and 28 degrees C, the diffusion fractions of approximately 75 and 60 percent, respectively, on both cell lines. The lipid analog nitrobenzoxadiazolephosphatidylcholine (NBD-PC) diffused in the GM-2408A cell membrane at 1.5x10(-8) cm(2)/sec at 22 degrees C. On blebs induced in GM-2408A cell membranes, the dil(3)-LDL receptor complex diffusion coefficient increased to approximately 10(-9) cm(2)/s, thus approaching the maximum theoretical predictions for a large protein in the viscous lipid bilayer. Cytoskeletal staining of blebs with NBD- phallacidin, a fluorescent probe specific for F-actin, indicated that loss of the bulk of the F-actin cytoskeleton accompanied the release of the natural constraints on later diffusion observed on blebs. This work shows that the internalization defect of J.D. is not due to immobilization of the LDL-receptor complex since its diffusibility is sufficient to sustain even the internalization rates observed in the native fibroblasts. Nevertheless, as with many other cell membrane receptors, the diffusion coefficient of the LDL-receptor complex is at least two orders of magnitude slower on native membrane than the viscous limit approached on cell membrane blebs where it is released from lateral constraints. However, LDL-receptor diffusion may not limit LDL internalization in normal human fibroblasts.
Asunto(s)
Membrana Celular/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de Superficie Celular/metabolismo , Actinas/análisis , Línea Celular , Difusión , Fibroblastos , Colorantes Fluorescentes , Humanos , Receptores de LDL , TemperaturaRESUMEN
The visible wavelength excited fluorophore 3,3'-dioctadecylindocarbocyanine iodide (Dil[3]) was incorporated into human low density lipoprotein (LDL) to form the highly fluorescent LDL derivative dil(3)-LDL. Dil(3)-LDL binds to normal human fibroblasts and to human fibroblasts defective in LDL receptor internalization but does not bind to LDL receptor-negative human fibroblasts at 4 degrees C or 37 degrees C. It is internalized rapidly at 37 degrees C by normal fibroblasts and depresses the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in a manner similar to that of LDL. It is prevented from binding to the LDL receptor by an excess of unlabeled LDL or by heparin sulfate. Identical distributions of dil(3)-LDL are observed on cells by either indirect immunofluorescence with fluorescein-labeled antibody or directly by dil(3) fluorescence. Upwards of 45 molecules of dil(3) are incorporated per molecule of LDL without affecting binding to the receptor. This labeling renders individual molecules visible by their fluorescence and enables the derivative to be used in dynamic studies of LDL-receptor motion on living fibroblasts by standard fluorescence techniques at low LDL receptor density. Observations with this derivative indicate that the LDL-receptor complex is immobilized on the surface of human fibroblasts but, when free of this linkage, undergoes a Brownian motion consistent with theory.
Asunto(s)
Carbocianinas , Colorantes Fluorescentes , Lipoproteínas LDL/metabolismo , Quinolinas , Receptores de Superficie Celular/metabolismo , Carbocianinas/farmacología , Línea Celular , Fibroblastos , Colorantes Fluorescentes/farmacología , Heparina/farmacología , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Microscopía Fluorescente , Quinolinas/farmacología , Receptores de LDL , TemperaturaRESUMEN
Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait.
Asunto(s)
Actinas/análisis , Transformación Celular Neoplásica , Transformación Celular Viral , Citoesqueleto/análisis , Animales , Virus del Sarcoma Aviar , Línea Celular , Embrión de Pollo , Interfase , Riñón , Virus del Sarcoma Murino de Kirsten , Ratones , Mitosis , Ratas , Virus 40 de los SimiosRESUMEN
The blue light-excited fluorescent phallotoxin derivative nitrobenzoxadiazole phallacidin (NBD-Ph) was used to stain entire tissue culture monolayers of live L6 mouse cells and other mammalian cell lines without the aid of permeabilization treatment. Although cells tend to exclude the fluorescent toxin, reducing the internal concentration by approximately 1,000 times, some of it enters the cells, probably by pinocytosis, and stains actin structures at low intracellular NBD-Ph concentrations (approximately 5-15 nM), where cell toxicity was negligible or at least not detectable by phase-contrast microscopy. Protracted treatments with NBD-Ph did induce pharmacological responses similar to those of phalloidin. The dissociation constant for NBD-Ph with F-actin in fixed and extracted L6 cells was determined, from staining intensity measurements at various NBD-Ph concentrations, to be 1.5-2.5 x 10(-8) M.
Asunto(s)
Actinas/metabolismo , Amanitinas , Citoesqueleto/ultraestructura , Oxadiazoles , Animales , Compartimento Celular , Células Cultivadas , Ratones , Músculos/ultraestructura , Coloración y EtiquetadoRESUMEN
The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.
Asunto(s)
Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/metabolismo , Receptores de LDL/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Electricidad , Técnica del Anticuerpo Fluorescente , Humanos , Lipoproteínas LDL/metabolismo , Fluidez de la MembranaRESUMEN
Various investigations have suggested that cytoplasmic streaming in characean algae is driven by interaction between subcortical actin bundles and endoplasmic myosin. To further test this hypothesis, we have perfused cytotoxic actin-binding drugs and fluorescent actin labels into the cytoplasm of streaming Chara cells. Confirming earlier work, we find that cytochalasin B (CB) reversibly inhibits streaming. In direct contrast to earlier investigators, who have found phalloidin to be a potent inhibitor of movement in amoeba, slime mold, and fibroblastic cells, we find that phalloidin does not inhibit streaming in Chara but does modify the inhibitory effect of CB. Use of two fluorescent actin probes, fluorescein, isothiocyanate-heavy meromyosin (FITC-HMM) and nitrobenzoxadiazole-phallacidin (NBD-Ph), has permitted visualization of the effects of CB and phalloidin on the actin bundles. FITC-HMM labeling in perfused but nonstreaming cells has revealed a previously unobserved alteration of the actin bundles by CB. Phalloidin alone does not perceptibly alter the actin bundles but does block the alteration by CB if applied as a pretreatment, NBD-Ph perfused into the cytoplasm of streaming cells stains actin bundles without inhibiting streaming. NBD-Ph staining of actin bundles is not initially observed in cells inhibited by CB but does appear simultaneously with the recovery of streaming as CB leaks from the cells. The observations reported here are consistent with the established effects of phallotoxins and CB on actin in vitro and support the hypothesis that streaming is generated by actin-myosin interactions.
Asunto(s)
Citocalasina B/farmacología , Corriente Citoplasmática/efectos de los fármacos , Oligopéptidos/farmacología , Faloidina/farmacología , Plantas/efectos de los fármacos , Actinas/análisis , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Plantas/ultraestructuraRESUMEN
EDG-1 is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for sphingosine-1-phosphate (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates sphingosine kinase and increases intracellular SPP, was dependent on expression of EDG-1. Deletion of edg-1 or inhibition of sphingosine kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-1, as measured by translocation of beta-arrestin and phosphorylation of EDG-1. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.
Asunto(s)
Quimiotaxis , Proteínas Inmediatas-Precoces/metabolismo , Lisofosfolípidos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Animales , Arrestinas/metabolismo , Becaplermina , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Eliminación de Gen , Humanos , Proteínas Inmediatas-Precoces/genética , Ratones , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Receptor Cross-Talk , Receptores Lisofosfolípidos , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Esfingosina/farmacología , Transfección , beta-ArrestinasRESUMEN
beta-Arrestins are proteins that bind phosphorylated heterotrimeric GTP-binding protein (G protein)-coupled receptors (GPCRs) and contribute to the desensitization of GPCRs by uncoupling the signal transduction process. Resensitization of GPCR responsiveness involves agonist-mediated receptor sequestration. Overexpression of beta-arrestins in human embryonic kidney cells rescued the sequestration of beta 2-adrenergic receptor (beta 2AR) mutants defective in their ability to sequester, an effect enhanced by simultaneous overexpression of beta-adrenergic receptor kinase 1. Wild-type beta 2AR sequestration was inhibited by the overexpression of two beta-arrestin mutants. These findings suggest that beta-arrestins play an integral role in GPCR internalization and thus serve a dual role in the regulation of GPCR function.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Antígenos/fisiología , Arrestinas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas del Ojo/fisiología , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Antígenos/genética , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/genética , ADN Complementario , Proteínas del Ojo/genética , Humanos , Isoproterenol/farmacología , Mutación , Fosforilación , Mutación Puntual , Receptores Adrenérgicos beta 2/genética , Transfección , Quinasas de Receptores Adrenérgicos beta , beta-ArrestinasRESUMEN
Direct measurements by fluorescence correlation spectroscopy of lateral diffusion coefficients of fluorescent lipid analogs in lipid bilaryer membranes indicate self-diffusion coefficients D greater than 10(-7) square centimeters per second for various lipid systems above their reported transition temperatures. Cholesterol in egg lecithin at mole ratio of 1 : 2 reduces D by about twofold, while retained hydrocarbon solvent can increase it by two- to threefold.
Asunto(s)
Lípidos de la Membrana/fisiología , Colesterol/fisiología , Difusión , Glicéridos , Técnicas In Vitro , Fosfatidilcolinas/fisiología , Espectrometría de Fluorescencia , Relación Estructura-Actividad , TemperaturaRESUMEN
BACKGROUND AND PURPOSE: M2-type pyruvate kinase (M2PK) was found to interact directly with the 'ITAM' region of the gamma chain of the high-affinity IgE receptor (FcvarepsilonRI). Our hypothesis was that mast cell degranulation might require the FcvarepsilonRI-mediated inhibition of M2PK activity. EXPERIMENTAL APPROACH: In rat basophilic leukaemia (RBL-2H3) cells, the effects of directly inhibiting M2PK or preventing the FcvarepsilonRI-mediated inhibition of M2PK (disinhibition) on degranulation was measured by hexosaminidase release. Effects of blocking the FcvarepsilonRI-mediated inhibition of M2PK was also assessed in vivo in a mouse model of allergen-induced airway hyper-responsiveness. KEY RESULTS: Activation of FcvarepsilonRI in RBL-2H3 cells caused the rapid phosphorylation of tyrosine residues in M2PK, associated with a decrease in M2PK enzymatic activity. There was an inverse correlation between M2PK activity and mast cell degranulation. FcvarepsilonRI-mediated inhibition of M2PK involved Src kinase, phosphatidylinositol 3-kinase, PKC and calcium. Direct inhibition of M2PK potentiated FcvarepsilonRI-mediated degranulation and prevention of the FcvarepsilonRI-mediated inhibition of M2PK attenuated mast cell degranulation. Transfection of RBL-2H3 cells with M1PK which prevents FcvarepsilonRI-induced inhibition of M2PK, markedly reduced their degranulation and exogenous M1PK (i.p.) inhibited ovalbumin-induced airway hyper-responsiveness in vivo. CONCLUSIONS AND IMPLICATIONS: We have identified a new control point and a novel biochemical pathway in the process of mast cell degranulation. Our study suggests that the FcvarepsilonRI-mediated inhibition of M2PK is a crucial step in responses to allergens. Moreover, the manipulation of glycolytic processes and intermediates could provide novel strategies for the treatment of allergic diseases.
Asunto(s)
Degranulación de la Célula , Glucólisis , Mastocitos/enzimología , Piruvato Quinasa/metabolismo , Receptores de IgE/metabolismo , Animales , Hiperreactividad Bronquial/enzimología , Hiperreactividad Bronquial/inmunología , Hiperreactividad Bronquial/prevención & control , Calcio/metabolismo , Degranulación de la Célula/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Femenino , Hexosaminidasas/metabolismo , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ovalbúmina , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Piruvato Quinasa/genética , Ratas , Receptores de IgE/genética , Transducción de Señal , Transfección , Familia-src Quinasas/metabolismoRESUMEN
Many measurements of lateral diffusion of proteins and lipids on cell membranes and lipid model membranes have become available through application of fluorescence photobleaching recovery methods. A puzzling aspect of these results is slow diffusion and partial immobilization of protein molecules on the cell surface. Observed protein diffusion coefficients on vertebrate structural tissue cells are consistently D less than or equal to 10(-10) cm2/s, while lipid analogues diffuse with D approx. 10(-8) cm2/s. Substantial fractions of the cell membrane proteins are not diffusible. In a pure viscous membrane, theoretical fluid dynamics has suggested only small differences between lipid and protein diffusion coefficients. Measurements of protein diffusion in model membranes recently showed D less than or equal to 10(-9) cm2/s, as expected. Recent experiments on cell membranes show that uncoupling of the membrane from the cytoskeleton by formation of blebs releases the membrane protein molecules so that diffusion is enhanced to D greater than or equal to 10(-9) cm2/s and the non-diffusible fraction is eliminated.
Asunto(s)
Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Actinas , Células Cultivadas , Difusión , Luz , Membrana Dobles de Lípidos , Unión Proteica , Receptores de Droga , Espectrometría de Fluorescencia , Factores de Tiempo , ViscosidadRESUMEN
Beta-arrestin proteins play a dual role in regulating G protein-coupled receptor (GPCR) responsiveness by contributing to both receptor desensitization and internalization. Recently, beta-arrestins were also shown to be critical determinants for beta2-adrenergic receptor (beta2AR) resensitization. This was demonstrated by overexpressing wild-type beta-arrestins to rescue the resensitization-defect of a beta2AR (Y326A) mutant (gain of function) and overexpressing a dominant-negative beta-arrestin inhibitor of beta2AR sequestration to impair beta2AR dephosphorylation and resensitization (loss of function). Moreover, the ability of the beta2AR to resensitize in different cell types was shown to be dependent upon beta-arrestin expression levels. To further study the mechanisms underlying beta-arrestin function, green fluorescent protein was coupled to beta-arrestin2 (beta arr2GFP), thus allowing the real-time visualization of the agonist-dependent trafficking of beta-arrestin in living cells. Beta arr2GFP translocation from the cytoplasm to the plasma membrane proceeded with a time course, sensitivity and specificity that was indistinguishable from the most sensitive second messenger readout systems. Beta arr2GFP translocation was GRK-dependent and was demonstrated for 16 different ligand-activated GPCRs. Because beta-arrestin binding is a common divergent step in GPCR signalling, this assay represents a universal methodology for screening orphan receptors, GRK inhibitors and novel GPCR ligands. Moreover, beta arr2GFP provides a valuable new tool to dissect the biological function and regulation of beta-arrestin proteins.
Asunto(s)
Arrestinas/fisiología , Proteínas de Unión al GTP/fisiología , Receptores de Superficie Celular/fisiología , Animales , Endocitosis/fisiología , Sensibilidad y Especificidad , beta-ArrestinasAsunto(s)
Proteínas de Unión al GTP/metabolismo , Proteínas Luminiscentes/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Arrestinas/química , División Celular , Proteínas Fluorescentes Verdes , Humanos , Ligandos , Fosforilación , Transporte de Proteínas , Receptores de Superficie Celular/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , beta-ArrestinasAsunto(s)
Arrestinas/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos beta 2/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células COS , Línea Celular , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Secuencia Conservada , Endocitosis , Humanos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Recombinantes/metabolismo , Transfección , Quinasas de Receptores Adrenérgicos beta , beta-ArrestinasRESUMEN
Desensitization of G-protein coupled receptors following agonist occupancy is accompanied by two temporally distinguishable cellular trafficking phenomena of the receptors referred to as sequestration and down regulation. For the beta2-adrenergic receptor, sequestration occurs within minutes of agonist binding and results in a reversible internalization and loss of cell surface receptor binding. With longer occupancy, greater than 1 hour, down regulation results in a variable loss of the complement of cellular receptors. Here we compare the two methods that have been used to monitor these receptor changes, competition of whole cell hydrophobic ligand binding (125I-pindolol) with a hydrophilic ligand (CGP-12177) and flow cytometry quantification of immunologically tagged beta2-adrenergic receptor. While both methods give reliable results, we show that because of a 1:500 partitioning of the hydrophilic ligand into cells, slightly different conditions should be used to assess basally or agonist stimulated sequestered receptor levels. Using a sequestration defective beta2-adrenergic receptor mutant we demonstrate that even though sequestration and down regulation behave as independent processes, sequestration can significantly affect the rate at which receptors are lost by the down regulatory process by removing receptors from the pool of down regulating receptors. A mathematical model expressing these relationships is provided.
Asunto(s)
Modelos Biológicos , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células CHO , Cricetinae , Regulación hacia Abajo , Citometría de Flujo , Proteínas de Unión al GTP/metabolismo , Isoproterenol/farmacología , Matemática , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pindolol/metabolismo , Propanolaminas/metabolismo , Ensayo de Unión Radioligante , Receptores Adrenérgicos beta 2/efectos de los fármacos , Receptores Adrenérgicos beta 2/genéticaRESUMEN
We studied two children with recurrent cancer, who during treatment with ifosfamide developed severe encephalopathy characterized by coma of several days duration. Ifosfamide was used as a single drug without mesna as an uroprotector. A 1-hour infusion of ifosfamide (1.8 g/m2) was given on days one and two of a planned 5-day course. Encephalopathy was associated with severe electroencephalographic abnormalities, e.g. slowing in delta range in one patient and electrographic seizures in another. Both, clinical and electroencephalographic features of encephalopathy were reversible.
Asunto(s)
Coma/inducido químicamente , Ifosfamida/efectos adversos , Neoplasias Encefálicas/tratamiento farmacológico , Preescolar , Electroencefalografía , Femenino , Humanos , Neoplasias Renales/tratamiento farmacológico , Masculino , Neuroblastoma/tratamiento farmacológico , Tumor de Wilms/tratamiento farmacológicoRESUMEN
An active fluorescent derivative of the actin-binding mushroom toxin phallacidin has been synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells and actively streaming algal cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained stress fibers, ruffles, the cellular geodome, and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with nitrobenzoxadiazole (NBD)-phallacidin did not inhibit cytoplasmic streaming. NBD-phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living systems.
Asunto(s)
Actinas/metabolismo , Citoplasma/ultraestructura , Citoesqueleto/ultraestructura , Micotoxinas , Animales , Permeabilidad de la Membrana Celular , Supervivencia Celular , Células Cultivadas , Eucariontes/ultraestructura , Fibroblastos/ultraestructura , Ratones , Microscopía FluorescenteRESUMEN
Agonist-dependent desensitization and internalization of G protein-coupled receptors (GPCR) are mediated by the binding of arrestins to phosphorylated receptors. The affinity of arrestins for the phosphorylated GPCR regulates the ability of the internalized receptor to be dephosphorylated and recycled back to the plasma membrane. In this study, we show that the naturally occurring loss of function vasopressin receptor mutation R137H, which is associated with familial nephrogenic diabetes insipidus, induces constitutive arrestin-mediated desensitization. In contrast to the wild-type vasopressin receptor, the nonsignaling R137H receptor is phosphorylated and sequestered in arrestin-associated intracellular vesicles even in the absence of agonist. Eliminating molecular determinants on the receptor that promote high affinity arrestin-receptor interaction reestablishes plasma membrane localization and the ability of the mutated receptors to signal. These findings suggest that unregulated desensitization can contribute to the etiology of a GPCR-based disease, implying that pharmacological targeting of GPCR desensitization may be therapeutically beneficial.
Asunto(s)
Arrestinas/metabolismo , Diabetes Insípida Nefrogénica/genética , Mutación/genética , Receptores de Vasopresinas/metabolismo , Adenilil Ciclasas/metabolismo , Sustitución de Aminoácidos , Arginina Vasopresina/farmacología , Línea Celular , Membrana Celular/metabolismo , Dinaminas , Endocitosis , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Microscopía Fluorescente , Fenotipo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Receptores de Vasopresinas/genética , Proteínas Recombinantes , Transducción de Señal , Transfección , beta-ArrestinasRESUMEN
The process of agonist-promoted internalization (sequestration) of G protein-coupled receptors (GPCRs) is intimately linked to the regulation of GPCR responsiveness. Following agonist-mediated desensitization, sequestration of GPCR is presumably associated with the dephosphorylation and recycling of functional receptors. However, the exact mechanisms responsible for GPCR sequestration, even for the prototypic beta2-adrenergic receptor (beta2AR), have remained controversial. We demonstrate here that dynamin, a GTPase that regulates the formation and internalization of clathrin-coated vesicles, is essential for the agonist-promoted sequestration of the beta2AR, suggesting that the beta2AR internalizes via the clathrin-coated vesicle-mediated endocytic pathway. In contrast, internalization of the angiotensin II type 1A receptor (AT1AR), another typical GPCR, does not require dynamin. In addition, the AT1AR internalizes independent of the function of beta-arrestin, a critical component for beta2AR cellular trafficking, but additional AT1ARs are mobilized to the dynamin-dependent pathway upon overexpression of beta-arrestin. These findings demonstrate that GPCRs can utilize distinct endocytic pathways, distinguishable by dynamin and beta-arrestin, and that beta-arrestins function as adaptor proteins specifically targeting GPCRs for dynamin-dependent endocytosis via clathrin-coated vesicles.