RESUMEN
Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20 nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artifact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artifacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Antígenos CD4/metabolismo , Membrana Celular/metabolismo , Imagen Individual de Molécula/métodos , Fijación del Tejido/métodos , Animales , Artefactos , Células COS , Chlorocebus aethiops , Errores Diagnósticos/prevención & control , Formaldehído/farmacología , Microfluídica , Polímeros/farmacología , Conformación Proteica/efectos de los fármacos , Agregación de Receptores/efectos de los fármacos , Reproducibilidad de los ResultadosRESUMEN
Several different receptor proteins have been identified that bind monomeric, oligomeric, or fibrillar forms of amyloid-ß (Aß). "Good" receptors internalize Aß or promote its transcytosis out of the brain, whereas "bad" receptors bind oligomeric forms of Aß that are largely responsible for the synapticloss, memory impairments, and neurotoxicity that underlie Alzheimer disease. The prion protein both removes Aß from the brain and transduces the toxic actions of Aß. The clustering of distinct receptors in cell surface signaling platforms likely underlies the actions of distinct oligomeric species of Aß. These Aß receptor-signaling platforms provide opportunities for therapeutic intervention in Alzheimer disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptores de Superficie Celular/agonistas , Transducción de Señal , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/prevención & control , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Animales , Apoptosis/efectos de los fármacos , Humanos , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/agonistas , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Terapia Molecular Dirigida , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Neuronas/efectos de los fármacos , Neuronas/patología , Nootrópicos/farmacología , Nootrópicos/uso terapéutico , Proteínas PrPC/agonistas , Proteínas PrPC/antagonistas & inhibidores , Proteínas PrPC/metabolismo , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/prevención & control , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Agregación de Receptores/efectos de los fármacos , Receptor para Productos Finales de Glicación Avanzada/agonistas , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal/efectos de los fármacos , Transcitosis/efectos de los fármacosRESUMEN
The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated by ado-trastuzumab emtansine (Kadcyla(TM)). While selecting a suitable antibody for an ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics of the optimal antibody candidate remain poorly understood. We studied a large panel of human HER2 antibodies to identify the characteristics that make them most suitable for an ADC approach. As a model toxin, amenable to in vitro high-throughput screening, we employed Pseudomonas exotoxin A (ETA') fused to an anti-kappa light chain domain antibody. Cytotoxicity induced by HER2 antibodies, which were thus non-covalently linked to ETA', was assessed for high and low HER2 expressing tumor cell lines and correlated with internalization and downmodulation of HER2 antibody-target complexes. Our results demonstrate that HER2 antibodies that do not inhibit heterodimerization of HER2 with related ErbB receptors internalize more efficiently and show greater ETA'-mediated cytotoxicity than antibodies that do inhibit such heterodimerization. Moreover, stimulation with ErbB ligand significantly enhanced ADC-mediated tumor kill by antibodies that do not inhibit HER2 heterodimerization. This suggests that the formation of HER2/ErbB-heterodimers enhances ADC internalization and subsequent killing of tumor cells. Our study indicates that selecting HER2 ADCs that allow piggybacking of HER2 onto other ErbB receptors provides an attractive strategy for increasing ADC delivery and tumor cell killing capacity to both high and low HER2 expressing tumor cells.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/terapia , Diseño de Fármacos , Inmunoterapia/métodos , Anticuerpos Monoclonales Humanizados/farmacología , Neoplasias de la Mama/inmunología , Línea Celular Tumoral , Dimerización , Endocitosis , Factor de Crecimiento Epidérmico/metabolismo , Femenino , Humanos , Inmunotoxinas/uso terapéutico , Agregación de Receptores/efectos de los fármacos , Receptor ErbB-2/inmunología , Transducción de Señal/efectos de los fármacos , TrastuzumabRESUMEN
BACKGROUND: Chronic inflammatory and autoimmune diseases are largely due to inappropriate response of hyperactive or autoreactive B cells. These autoreactive B cells can evade central tolerance checkpoints and migrate to the periphery, where they would be silenced by anergy. Such anergic cells are characterized by B-cell receptor (BCR) desensitization and altered downstream signaling. OBJECTIVE: We sought to determine whether intravenous immunoglobulin (IVIg) induces a nonresponsive state of B cells and to address the similarities of this mechanism to those described in anergy. METHODS: Human B cells were stimulated with anti-IgM antibody, and effects of IVIg on several parameters, such as calcium release, tyrosine phosphorylation, BCR aggregation, BCR internalization, or transcriptional activity, were studied by using flow cytometry, confocal microscopy, Western blotting, and a quantitative PCR array. RESULTS: IVIg-treated B cells show defects in activating coreceptor expression, calcium signaling, and BCR aggregation on engagement by antigen. IVIg also induces suppression of phosphoinositide 3-kinase signaling, which plays a central role in determining B-cell fate. All these events ultimately lead to profound modifications in gene expression, resulting in long-term functional but reversible silencing of IVIg-treated B cells. CONCLUSION: Our findings provide insights into the effectiveness of IVIg in treating autoimmune or inflammatory pathologies associated with the loss of B-cell tolerance. Furthermore, these data provide a model to explore the complexity of positive versus negative selection in B cells.
Asunto(s)
Enfermedades Autoinmunes/terapia , Linfocitos B/efectos de los fármacos , Inmunoglobulinas Intravenosas/farmacología , Inmunosupresores/farmacología , Receptores de Antígenos de Linfocitos B/metabolismo , Anticuerpos Antiidiotipos/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos B/inmunología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/inmunología , Células Cultivadas , Niño , Anergia Clonal/efectos de los fármacos , Humanos , Tolerancia Inmunológica , Activación de Linfocitos/efectos de los fármacos , Agregación de Receptores/efectos de los fármacosRESUMEN
Acute inflammatory responses are important in host defense, whereas dysregulated inflammation results in life-threatening complications. Here we found that paired immunoglobulin-like type 2 receptor alpha (PILRα), an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), negatively regulated neutrophil infiltration during inflammation. Pilra(-/-) mice had increased neutrophil recruitment to inflammatory sites and were highly susceptible to endotoxin shock. Pilra(-/-) neutrophils showed enhanced transmigration ability and increased adhesion to the ß(2) integrin ligand ICAM-1. PILRα expressed on neutrophils constitutively associated in cis with its ligands, resulting in clustering of PILRα during stimulation with a chemoattractant. Clustering of PILRα enhanced ITIM-mediated signaling, thus modulating ß(2) integrin inside-out activation. These data demonstrate that neutrophil recruitment in inflammatory responses is regulated by PILRα via modulation of integrin activation.
Asunto(s)
Inflamación/inmunología , Integrinas/metabolismo , Neutrófilos/inmunología , Receptores Inmunológicos/fisiología , Animales , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Adhesión Celular/inmunología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Cultivadas , Predisposición Genética a la Enfermedad , Inflamación/genética , Integrinas/genética , Integrinas/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Ratones Noqueados , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/genética , Receptores Inmunológicos/genética , Choque Séptico/genética , Choque Séptico/inmunologíaRESUMEN
With the advent of the key discovery in the mid-1980s that the amyloid ß-protein (Aß) is the core constituent of the amyloid plaque pathology found in Alzheimer disease (AD), an intensive effort has been underway to attempt to mitigate its role in the hope of treating the disease. This effort fully matured when it was clarified that the Aß is a normal product of cleavage of the amyloid precursor protein, and well-defined proteases for this process were identified. Further therapeutic options have been developed around the concept of anti-Aß aggregation inhibitors and the surprising finding that immunization with Aß itself leads to reduction of pathology in animal models of the disease. Here we review the progress in this field toward the goal of targeting Aß for treatment and prevention of AD and identify some of the major challenges for the future of this area of medicine.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Enfermedad de Alzheimer/prevención & control , Vacunas contra el Alzheimer , Péptidos beta-Amiloides/uso terapéutico , Barrera Hematoencefálica/efectos de los fármacos , Ensayos Clínicos como Asunto , Descubrimiento de Drogas , Predicción , Humanos , Inmunoterapia/métodos , Agregación de Receptores/efectos de los fármacos , VacunaciónRESUMEN
The capacity of opioids to alleviate inflammatory pain is negatively regulated by the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). Increased activity of this receptor complicates the clinical use of opioids to treat persistent neuropathic pain. Immunohistochemical and ultrastructural studies have demonstrated the coexistence of both receptors within single neurons of the CNS, including those in the mesencephalic periaqueductal gray (PAG), a region that is implicated in the opioid control of nociception. We now report that mu-opioid receptors (MOR) and NMDAR NR1 subunits associate in the postsynaptic structures of PAG neurons. Morphine disrupts this complex by protein kinase-C (PKC)-mediated phosphorylation of the NR1 C1 segment and potentiates the NMDAR-CaMKII, pathway that is implicated in morphine tolerance. Inhibition of PKC, but not PKA or GRK2, restored the MOR-NR1 association and rescued the analgesic effect of morphine as well. The administration of N-methyl-D-aspartic acid separated the MOR-NR1 complex, increased MOR Ser phosphorylation, reduced the association of the MOR with G-proteins, and diminished the antinociceptive capacity of morphine. Inhibition of PKA, but not PKC, CaMKII, or GRK2, blocked these effects and preserved morphine antinociception. Thus, the opposing activities of the MOR and NMDAR in pain control affect their relation within neurons of structures such as the PAG. This finding could be exploited in developing bifunctional drugs that would act exclusively on those NMDARs associated with MORs.
Asunto(s)
Dolor/fisiopatología , Sustancia Gris Periacueductal/metabolismo , Agregación de Receptores/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Opioides mu/metabolismo , Animales , Células CHO , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Tolerancia a Medicamentos/fisiología , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/fisiología , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos ICR , Morfina/administración & dosificación , Morfina/antagonistas & inhibidores , Morfina/farmacología , N-Metilaspartato/farmacología , Neuronas/metabolismo , Dolor/prevención & control , Sustancia Gris Periacueductal/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Agregación de Receptores/efectos de los fármacosRESUMEN
Depression, or major depressive disorder (MDD), is a serious mental illness that causes substantial worldwide disability. Current antidepressant medications mostly target the serotonin and norepinephrine neurotransmitter systems. These drugs are ineffective in many patients, and there are limited options for treatment-resistant depression. The dopamine neurotransmitter system has recently been identified as another modulator of mood and depressive symptoms, and a recently discovered interaction between the dopamine D1 and D2 receptor may be a novel antidepressant target.
Asunto(s)
Antidepresivos/farmacología , Sistemas de Liberación de Medicamentos/psicología , Descubrimiento de Drogas/métodos , Agregación de Receptores/efectos de los fármacos , Receptores de Dopamina D2/metabolismo , Animales , Antidepresivos/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Humanos , Modelos Biológicos , Receptores de Dopamina D1/efectos de los fármacos , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/efectos de los fármacosRESUMEN
UNLABELLED: Duchenne muscular dystrophy (DMD) is caused by the absence of a functional transcript of the protein dystrophin. DMD is associated with a range of cognitive deficits that are thought to result from a lack of the protein dystrophin in brain structures involved in cognitive functions. The CNS involvement extends to an impairment of cognitive abilities, with many DMD boys having significant reduction in IQ. In the cerebellum, dystrophin is normally localized at the postsynaptic membrane of GABAergic synapses on Purkinje cells. Here, we investigate the effect of an absence of dystrophin on the number of GABA(A) channels located at the synapse in cerebellar Purkinje cells of the dystrophin-deficient mdx mouse. Whole-cell patch-clamp recordings of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) were performed in cerebellar slices from mdx and littermate control mice. Our results showed that the number of receptors at GABAergic synapses in the cerebellar Purkinje cell was significantly reduced in mdx mice (38.38 ± 2.95) compared to littermate controls (53.03 ± 4.11). Furthermore, when gaboxadol was added to the bath, the change in holding current in mdx mice was significantly enhanced (65.01 ± 5.89pA) compared to littermate controls (37.36 ± 3.82pA). The single channel unitary conductance and the rise and decay time of mIPSCs were not significantly different in these two groups of mice, indicating that those GABA(A) channels located at the postsynaptic sites in the mdx mice function normally. CONCLUSION: There is a reduction in the number of functional receptors localized at GABAergic synapses in the cerebellar Purkinje cells of dystrophin-deficient mdx mice and an increase in a gaboxadol induced holding current, which is evidence for an increase in extrasynaptic GABA(A) receptors in mdx mice. We hypothesize that the absence of dystrophin, from mdx Purkinje cells, reduces the number of post-synaptic GABA(A) receptors and as a result there is an increase in extrasynaptic receptors. If similar changes occur in the CNS in boys with DMD, it will impact on the function of neural networks and may contribute to some of the motor, behavioral and cognitive impairment apparent in many boys with DMD.
Asunto(s)
Antagonistas del GABA/farmacología , Isoxazoles/farmacología , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatología , Células de Purkinje/patología , Receptores de GABA-A/deficiencia , Animales , Modelos Animales de Enfermedad , Femenino , Agonistas del GABA/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Técnicas de Placa-Clamp/métodos , Células de Purkinje/efectos de los fármacos , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/genética , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Potenciales Sinápticos/efectos de los fármacos , Potenciales Sinápticos/genéticaRESUMEN
At the mammalian skeletal neuromuscular junction, cycling of nicotinic ACh receptors (nAChRs) is critical for the maintenance of a high postsynaptic receptor density. However, the mechanisms that regulate nAChRs recycling in living animals remain unknown. Using in vivo time-lapse imaging, fluorescence recovery after photobleaching, and biochemical pull down assays, we demonstrated that recycling of internalized nAChRs into fully functional and denervated synapses was promoted by both direct muscle stimulation and pharmacologically induced intracellular calcium elevations. Most of internalized nAChRs are recycled directly into synaptic sites. Chelating of intracellular calcium below resting level drastically decreased cycling of nAChRs. Furthermore we found that calcium-dependent AChR recycling is mediated by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Inhibition of CaMKII selectively blocked recycling and caused intracellular accumulation of internalized nAChRs, whereas internalization of surface receptors remained unaffected. Electroporation of CaMKII-GFP isoforms into the sternomastoid muscle showed that muscle-specific CaMKIIßm isoform is highly expressed at the neuromuscular junction (NMJ) and precisely colocalized with nAChRs at crests of synaptic folds while the CaMKIIγ and δ isoforms are poorly expressed in synaptic sites. These results indicate that Ca(2+) along with CaMKII activity are critical for receptor recycling and may provide a mechanism by which the postsynaptic AChR density is maintained at the NMJ in vivo.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/fisiología , Unión Neuromuscular/metabolismo , Agregación de Receptores/fisiología , Receptores Colinérgicos/metabolismo , Animales , Bencilaminas/farmacología , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/biosíntesis , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Femenino , Recuperación de Fluorescencia tras Fotoblanqueo , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/fisiología , Ratones , Unión Neuromuscular/citología , Unión Neuromuscular/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Agregación de Receptores/efectos de los fármacos , Sulfonamidas/farmacologíaRESUMEN
Peripheral blood natural killer (NK) cell therapy for acute myeloid leukemia has shown promise in clinical trials after allogeneic stem cell transplantation. Cord blood (CB) is another potentially rich source of NK cells for adoptive immune therapy after stem cell transplantation. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. However, despite expressing normal surface activating and inhibitory NK receptors, CB-derived NK cells have poor cytolytic activity. In this study, we investigate the cellular mechanism and demonstrate that unmanipulated CB-NK cells exhibit an impaired ability to form F-actin immunologic synapses with target leukemia cells compared with peripheral blood-derived NK cells. In addition, there was reduced recruitment of the activating receptor CD2, integrin leukocyte function-associated antigen-1, and the cytolytic molecule perforin to the CB-NK synapse site. Exvivo interleukin (IL)-2 expansion of CB-NK cells enhanced lytic synapse formation including CD2 and leukocyte function-associated antigen-1 polarization and activity. Furthermore, the acquired antileukemic function of IL-2-expanded CB-NK cells was validated using a nonobese diabetic severe combined immunodeficient IL-2 receptor common γ-chain null mouse model. We believe our results provide important mechanistic insights for the potential use of IL-2-expanded CB-derived NK cells for adoptive immune therapy in leukemia.
Asunto(s)
Inmunoterapia Adoptiva , Subunidad gamma Común de Receptores de Interleucina/genética , Células Asesinas Naturales/metabolismo , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Actinas/metabolismo , Animales , Antígenos CD2/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citotoxicidad Inmunológica/efectos de los fármacos , Citotoxicidad Inmunológica/genética , Sangre Fetal/citología , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/genética , Sinapsis Inmunológicas/inmunología , Interleucina-2/farmacología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Ratones Noqueados , Ratones SCID , Perforina/metabolismo , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/genética , Agregación de Receptores/inmunologíaRESUMEN
Statins, HMG-CoA reductase inhibitors, are used widely in the treatment of hypercholesterolemia. Apart from lowering lipid levels, statins have been shown to have anti-inflammatory effects. Previously we showed that atorvastatin inhibits NF-kappaB activation, dose and time dependently, in LPS-TLR4 signaling pathway. In this study, we investigated the anti-inflammatory mechanism of atorvastatin via Toll-like receptor 4 (TLR4) in murine pro-B cell lines transfected with TLR4. Co-treatment of LPS-stimulated cells with both atorvastatin and mevalonate rescued NF-kappaB activation and TLR4 blockade demonstrated that atorvastatin does not exert its inhibitory effect via TLR4 receptor-ligand binding mechanism. Further investigation into the anti-inflammatory mechanism has shown that atorvastatin causes an impairment of TLR4 recruitment into the lipid raft thereby affecting anti-inflammatory responses. In contrast, mevalonate repaired lipid raft function leading to TLR4 clustering in the lipid raft. Together, these data suggest that atorvastatin exerts its anti-inflammatory effect via lipid raft modification. This novel finding offers another insight into the pleiotropic effects of atorvastatin and may be applicable to other pattern recognition receptors that utilize membrane lipid raft as a platform for signal transduction.
Asunto(s)
Antiinflamatorios/farmacología , Ácidos Heptanoicos/farmacología , Microdominios de Membrana/efectos de los fármacos , Células Precursoras de Linfocitos B/efectos de los fármacos , Pirroles/farmacología , Receptor Toll-Like 4/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Anticuerpos Bloqueadores/farmacología , Atorvastatina , Línea Celular , Ácidos Heptanoicos/uso terapéutico , Humanos , Hipercolesterolemia/tratamiento farmacológico , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Microdominios de Membrana/metabolismo , Microdominios de Membrana/patología , Ácido Mevalónico/farmacología , Ratones , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Células Precursoras de Linfocitos B/patología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/inmunología , Pirroles/uso terapéutico , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunologíaRESUMEN
The epidermal growth factor family of receptor tyrosine kinases (ErbBs) plays essential roles in tumorigenesis and cancer disease progression, and therefore has become an attractive target for structure-based drug design. ErbB receptors are activated by ligand-induced homo- and heterodimerization. Structural studies have revealed that ErbB receptor dimers are stabilized by receptor-receptor interactions, primarily mediated by a region in the second extracellular domain, termed the "dimerization arm". The present study is the first biological characterization of a peptide, termed Inherbin3, which constitutes part of the dimerization arm of ErbB3. Inherbin3 binds to the extracellular domains of all four ErbB receptors, with the lowest peptide binding affinity for ErbB4. Inherbin3 functions as an antagonist of epidermal growth factor (EGF)-ErbB1 signaling. We show that Inherbin3 inhibits EGF-induced ErbB1 phosphorylation, cell growth, and migration in two human tumor cell lines, A549 and HN5, expressing moderate and high ErbB1 levels, respectively. Furthermore, we show that Inherbin3 inhibits tumor growth in vivo and induces apoptosis in a tumor xenograft model employing the human non-small cell lung cancer cell line A549. The Inherbin3 peptide may be a useful tool for investigating the mechanisms of ErbB receptor homo- and heterodimerization. Moreover, the here described biological effects of Inherbin3 suggest that peptide-based targeting of ErbB receptor dimerization is a promising anti-cancer therapeutic strategy.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Fragmentos de Péptidos/farmacología , Mucosa Respiratoria/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Receptores ErbB/biosíntesis , Receptores ErbB/genética , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones SCID , Trasplante de Neoplasias , Fragmentos de Péptidos/química , Agregación de Receptores/efectos de los fármacos , Receptor ErbB-3/química , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Carga Tumoral/efectos de los fármacosRESUMEN
The effects of low and high concentrations of cocaine have been studied in vitro on the trafficking of plasma membrane A(2A) and D(2) immunoreactivities in previously characterized A(2A)-D(2) CHO cell lines. Receptor double immunofluorescence staining was performed with D(2) and A(2A) antibodies, planar lipid rafts immunolabeling with biotinylated cholera toxin subunit B and membrane invaginations with an anti-caveolin-1 antibody. A computer-assisted image analysis demonstrated a substantial and highly significant rise of membrane-associated D(2) immunoreactivity (IR) after 8 h of exposure to a low concentration of cocaine (150 nM). At this low concentration of cocaine, there was also an increase of membrane associated A(2A) immunoreactivity but smaller and less significant. However, this increase became considerably larger and highly significant at 150 microM at which concentration the rise of D(2) immunoreactivity had begun to disappear. It may be suggested that an allosteric action of cocaine at 150 nM on the D(2) receptors may primarily increase the insertion of D(2) monomers, homomers and also of a subpopulation of A(2A)-D(2) heteromers from the cytoplasm into the plasma membrane due to the conformational change induced by cocaine in the D(2) receptor. The planar lipid rafts and the caveolae are only affected by the higher concentrations of cocaine. It is proposed that changes in D(2) and A(2A)-D(2) trafficking induced by allosteric actions of cocaine at D(2) receptors may contribute to the alterations of D(2) signaling found in cocaine abusers.
Asunto(s)
Cocaína/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/fisiología , Microdominios de Membrana/efectos de los fármacos , Agregación de Receptores/efectos de los fármacos , Receptor de Adenosina A2A/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Células CHO , Cocaína/metabolismo , Cricetinae , Cricetulus , Inhibidores de Captación de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Microdominios de Membrana/metabolismo , Agregación de Receptores/fisiología , TransfecciónRESUMEN
Lipid raft, a specialized membrane structure enriched with cholesterol and glycosphingolipid, contains molecules that convey environmental stimuli to the intracellular systems. Authors investigated the effects of raft cholesterol depletion on non-small cell lung cancer (NSCLC) cell migration. Incubation of NSCLC cells in media containing lovastatin resulted in inhibition of cell migration by 63.1-83.3%, whereas raft cholesterol depletion with successive treatment using methyl-beta cyclodextrin (MbetaCD) followed by lovastatin further suppressed their migration by 35.0-57.8%. Raft cholesterol depletion partially inhibited EGF-induced phosphorylation of EGFR and FAK, however, no change was observed in other molecules comprising focal adhesion complex. It resulted in disappearance of filopodia, inhibition of EGF-induced pY397 FAK aggregation, and its destabilization. Cholesterol depletion inhibited phosphorylation of Src on Y416 in the detergent-insoluble fraction followed by decreased localization of total and pY397 FAK in the detergent-insoluble fraction. Minimal changes in these molecules were observed in the detergent-soluble fraction and interactions between FAK and other molecules of the focal adhesion complex were not influenced. Immunocytochemical analysis confirmed translocation of Src from the raft into cytoplasm and disappearance of EGF-induced membrane ruffling by raft cholesterol depletion. In cholesterol-depleted cells, EGF-induced phosphorylation of Src, Akt, and p44/42 in the detergent-insoluble fraction were inhibited whereas phosphorylation of GSK-3beta was unaffected. We conclude that raft cholesterol depletion inhibited NSCLC migration through inhibition of phosphorylation of raft associated Src and dislocation of molecules comprising focal adhesion complexes from raft rather than by inhibiting their recruitment to Src and interaction.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Colesterol/deficiencia , Receptores ErbB/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Microdominios de Membrana/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Colesterol/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Quinasa 1 de Adhesión Focal/genética , Adhesiones Focales/efectos de los fármacos , Humanos , Lovastatina/farmacología , Microdominios de Membrana/efectos de los fármacos , Proteína Oncogénica pp60(v-src)/metabolismo , Fosforilación , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas , Seudópodos/genética , Agregación de Receptores/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor cross-talk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30-60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.
Asunto(s)
Receptores ErbB/metabolismo , Gonadotropinas/farmacología , Células Intersticiales del Testículo/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Receptores de HL/metabolismo , Testosterona/biosíntesis , Animales , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Gonadotropinas/metabolismo , Humanos , Células Intersticiales del Testículo/citología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/metabolismo , Ratones , Mitocondrias/metabolismo , Ovario/citología , Ovario/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/fisiología , Factores de Tiempo , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/fisiologíaRESUMEN
The alpha4beta2 subtype is the most abundant nicotinic acetylcholine receptor (nAChR) in the brain and possesses the high-affinity binding site for nicotine. The alpha4 and beta2 nAChR subunits assemble into two alternate stoichiometries, (alpha4)(2)(beta2)(3) and (alpha4)(3)(beta2)(2), which differ in their functional properties and sensitivity to chronic exposure to nicotine. Here, we investigated the sensitivity of both receptor stoichiometries to modulation by Zn2+. We show that Zn2+ exerts an inhibitory modulatory effect on (alpha4)(2)(beta2)(3) receptors, whereas it potentiates or inhibits, depending on its concentration, the function of (alpha4)(3)(beta2)(2) receptors. Furthermore, Zn2+ inhibition on (alpha4)(2)(beta2)(3) nAChRs is voltage-dependent, whereas it is not on (alpha4)(3)(beta2)(2) receptors. We used molecular modeling in conjunction with alanine substitution and functional studies to identify two distinct sets of residues that determine these effects and may coordinate Zn(2+). Zn(2+) inhibition is mediated by a site located on the beta2(+)/alpha4(-) subunit interfaces on both receptor stoichiometries. alpha4(H195) and beta2(D218) are key determinants of this site. Zn2+ potentiation on (alpha4)(3)(beta2)(2) nAChRs is exerted by a site that resides on the alpha4(+)/alpha4(-) of this receptor stoichiometry. alpha4(H195) on the (-) side of the ACh-binding alpha4 subunit and alpha4(E224) on the (+) side of the non-ACh-binding alpha4 subunit critically contribute to this site. We also identified residues within the beta2 subunit that confer voltage dependency to Zn2+ inhibition on (alpha4)(2)(beta2)(3), but not on (alpha4)(3)(beta2)(2) nAChRs.
Asunto(s)
Membrana Celular/metabolismo , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Zinc/farmacología , Acetilcolina/química , Acetilcolina/metabolismo , Alanina/química , Alanina/metabolismo , Secuencia de Aminoácidos/fisiología , Sustitución de Aminoácidos/fisiología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Membrana Celular/efectos de los fármacos , Femenino , Humanos , Ligandos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Moleculares , Oocitos , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/metabolismo , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/fisiología , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Receptores Nicotínicos/química , Transmisión Sináptica/fisiología , Xenopus laevis , Zinc/químicaRESUMEN
Phencyclidine (PCP), a non-competitive NMDA/glutamate receptor antagonist, is a psychotomimetic drug that produces a syndrome in normal humans that resembles schizophrenia. The present study investigated the mechanisms of PCP actions by examining the density of glutamate and muscarinic receptors in the rat brain 4h after a single injection of PCP. We used receptor autoradiography and [3H]MK801, [3H]AMPA, [3H]pirenzepine and [3H]AFDX384 to target glutamate NMDA, glutamate AMPA and muscarinic M1 and M2 receptors, respectively. The major outcome from the present study was an overall decrease in levels of the glutamate AMPA receptor density (F=14.5, d.f.=1, p<0.001) in the PCP treated rats. More specifically, PCP-treated animals displayed decreased AMPA receptor density in hippocampus CA1 (-16%), hippocampus CA2 (-25%), dentate gyrus (-27%), parietal cortex layers III-VI (-19%), central nucleus of the amygdala (-40%), and basolateral amygdala (-19%). Other brain regions examined were unaffected. PCP administration did not significantly affect glutamate NMDA, muscarinic M1 and M2 receptor density. The present study demonstrates the limbic system as the anatomical locus of alterations in AMPA receptor density after acute administration of PCP and may have implications for models of schizophrenia that focus on glutamatergic dysfunction in limbic cortical regions.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Ácido Glutámico/metabolismo , Sistema Límbico/efectos de los fármacos , Fenciclidina/farmacología , Receptores AMPA/efectos de los fármacos , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiopatología , Giro Dentado/efectos de los fármacos , Giro Dentado/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Sistema Límbico/metabolismo , Sistema Límbico/fisiopatología , Antagonistas Muscarínicos/farmacología , Ratas , Ratas Sprague-Dawley , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/fisiología , Receptor Muscarínico M1/efectos de los fármacos , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/efectos de los fármacos , Receptor Muscarínico M2/metabolismo , Receptores AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatología , Factores de TiempoRESUMEN
In the hypoglossal nucleus of wild-type mice, early mixed glycinergic-GABAergic inhibitory transmission becomes mainly glycinergic during postnatal maturation. In spastic mice (SPA), a model of human hyperekplexic syndrome, an insertion into the gene of the glycine receptor (GlyR) beta subunit results in a decreased accumulation of GlyRs at postsynaptic sites and an impaired glycinergic neurotransmission. In SPA mice displaying a mild phenotype (B6C3Fe strain), a compensatory process involving an increased aggregation of GABA(A) receptors (GABA(A)Rs) at postsynaptic sites was proposed to explain survival of mutant animals until adulthood. However, C57BL/6J strain SPA mice which express a lower amount of GlyR beta subunit die 2-3 weeks after birth, suggesting that GABAergic compensation does not necessarily take place. We performed a morphofunctional study of inhibitory synapses in the developing hypoglossal nucleus of C57BL/6J SPA mice. In this mutant, the inhibitory synaptic activity was mainly GABAergic. Accordingly, we observed a developmental loss of glycinergic presynaptic terminals and an increase in the density of GABAergic presynaptic terminals during the first two postnatal weeks. In addition, while C57BL/6J SPA mice displayed a strong impairment in GlyR aggregation at postsynaptic loci, the proportion of inhibitory presynaptic terminals facing diffuse GABA(A)Rs significantly increased during development. Our results suggest crosstalk between postsynaptic and presynaptic elements, leading to the developmental regulation of the presynaptic terminal neurotransmitter content according to the level of postsynaptic GlyR aggregation. They also indicate that GABAergic neurotransmission does not compensate for defects in GlyR postsynaptic aggregation leading to spastic syndrome in C57BL/6J SPA mice.
Asunto(s)
Nervio Hipogloso/metabolismo , Neuronas Motoras/metabolismo , Inhibición Neural/genética , Receptores de Glicina/metabolismo , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Envejecimiento/fisiología , Animales , Diferenciación Celular/genética , Nervio Hipogloso/citología , Nervio Hipogloso/crecimiento & desarrollo , Bulbo Raquídeo/citología , Bulbo Raquídeo/crecimiento & desarrollo , Bulbo Raquídeo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes Neurológicos , Neuronas Motoras/efectos de los fármacos , Espasticidad Muscular/genética , Espasticidad Muscular/metabolismo , Espasticidad Muscular/fisiopatología , Inhibición Neural/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/metabolismo , Agregación de Receptores/efectos de los fármacos , Agregación de Receptores/genética , Receptor Cross-Talk/fisiología , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Receptores de Glicina/efectos de los fármacos , Receptores de Glicina/genética , Sinapsis/efectos de los fármacos , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genéticaRESUMEN
Fluorescence resonance energy transfer (FRET) experiments at neuromuscular junctions in the mouse tibialis anterior muscle show that postsynaptic acetylcholine receptors (AChRs) become more tightly packed during the first month of postnatal development. Here, we report that the packing of AChRs into postsynaptic aggregates was reduced in 4-week postnatal mice that had reduced amounts of the AChR-associated protein, rapsyn, in the postsynaptic membrane (rapsyn(+/-) mice). We hypothesize that nerve-derived agrin increases postsynaptic expression and targeting of rapsyn, which then drives the developmental increase in AChR packing. Neural agrin treatment elevated the expression of rapsyn in C2 myotubes by a mechanism that involved slowing of rapsyn protein degradation. Similarly, exposure of synapses in postnatal muscle to exogenous agrin increased rapsyn protein levels and elevated the intensity of anti-rapsyn immunofluorescence, relative to AChR, in the postsynaptic membrane. This increase in the rapsyn-to-AChR immunofluorescence ratio was associated with tighter postsynaptic AChR packing and slowed AChR turnover. Acute blockade of synaptic AChRs with alpha-bungarotoxin lowered the rapsyn-to-AChR immunofluorescence ratio, suggesting that AChR signaling also helps regulate the assembly of extra rapsyn in the postsynaptic membrane. The results suggest that at the postnatal neuromuscular synapse agrin signaling elevates the expression and targeting of rapsyn to the postsynaptic membrane, thereby packing more AChRs into stable, functionally-important AChR aggregates.