RESUMEN
We describe the cloning of SC1, a novel cDNA that was selected from a rat brain expression library using a mixed polyclonal antibody directed against synaptic junction glycoproteins. SC1 detects a 3.2 kb mRNA expressed throughout postnatal development of the brain and present at high levels in the adult. In situ hybridization reveals that the SC1 mRNA is expressed widely in the brain and is present in many types of neurons. DNA sequence data suggest that the SC1 product is a secreted, calcium binding glycoprotein. Strikingly, the carboxy-terminal region of the SC1 protein shows substantial similarity to the extracellular matrix glycoprotein osteonectin/BM40/SPARC. These data are consistent with the hypothesis that SC1 is an extracellular matrix glycoprotein in the brain.
Asunto(s)
Química Encefálica , ADN/genética , Glicoproteínas/genética , Proteínas del Tejido Nervioso/genética , Osteonectina/genética , Molécula de Adhesión Celular del Leucocito Activado , Secuencia de Aminoácidos , Animales , Northern Blotting , Southern Blotting , Clonación Molecular , Expresión Génica , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Osteonectina/inmunología , Proteínas/inmunología , Proteínas/metabolismo , ARN/genética , RatasRESUMEN
The composition and biosynthesis of fucosyl glycoproteins present in rat brain synaptic membranes and synaptic junctions were investigated. Reaction with 125I-labelled fucose-binding protein (Lotus tetragonolobus) following sodium dodecyl sulphate gel electrophoresis identified 6--8 fucosyl glycoproteins in synaptic membranes but only three major high molecular classes (Mr = 180 000, 130 000 and 110 000) in synaptic junctions. Affinity chromatography on concanavalin A-Sepharose resolved each of the synaptic junctional fucosyl glycoproteins into concanavalin A-positive and negative components indicating the presence of at least six high molecular weight fucosyl glycoproteins in synaptic junctions. Following the administration of [3H]fucose synaptic membranes, synaptic junctions and post-synaptic densities incorporated isotope, the order of relative specific activities being synaptic membranes greater than synaptic junctions greater than post-synaptic densities. Fractionation of [3H]fucose-labelled synaptic junctions on concanavalin A-Sepharose revealed a time-dependent increase in the percentage of isotope associated with the concanavalin A-positive glycoproteins. The results demonstrate both molecular and biosynthetic heterogeneity of fucosyl glycoproteins associated with synaptic junctions.
Asunto(s)
Corteza Cerebral/metabolismo , Fucosa/metabolismo , Glicoproteínas/biosíntesis , Sinapsis/metabolismo , Membranas Sinápticas/metabolismo , Animales , Concanavalina A , Glicoproteínas/aislamiento & purificación , Peso Molecular , Ratas , Fracciones Subcelulares/metabolismoRESUMEN
Synaptic plasma membranes were prepared from cortices of rats varying in post-natal age between 4 and 30 days. Sialic acid associated with synaptic plasma membrane glycoproteins and gangliosides increased 75% and 50%, respectively, between 4 and 30 days. The amount of sialic acid released from these membrane constituents by intrinsic synaptic sialidase increased 2-4-fold over the same period. Incubation of synaptic plasma membranes with exogenous gangliosides or glycopeptides demonstrated a 2-3-fold increase in sialidase activity during development. The major gangliosides present in synaptic plasma membranes at all ages were GT1, GD1a, GD1b and GM1. Intrinsic sialidase hydrolyzed 50-70% of endogenous GT1 and GD1a gangliosides at all ages. Endogenous GD1b ganglioside was poorly hydrolyzed in young rats and its susceptibility to enzymic hydrolysis increased during development. When exogenous GD1a and GD1b were used as substrates a preferential increase in activity against GD1b occurred during development, the ratio of activity (GD1a/GD1b) decreasing from 3.6 to 1.6 between 7 and 30 days. 10- and 30-day-old synaptic plasma membranes contained complex mixtures of sialoglycoproteins, an increase in the relative concentrations of lower molecular weight sialoglycoproteins occurring during development. Intrinsic sialidase present in 10- and 30-day-old synaptic plasma membranes acted upon all molecular weight classes of sialoglycoproteins.
Asunto(s)
Corteza Cerebral/crecimiento & desarrollo , Neuraminidasa/metabolismo , Membranas Sinápticas/enzimología , Animales , Corteza Cerebral/enzimología , Gangliósidos/metabolismo , ARN/análisis , Ratas , Ácidos Siálicos/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Distribución TisularRESUMEN
Cerebral ischemia results in activation of the mitogen-activated protein kinase pathway and increased tyrosine phosphorylation of proteins associated with postsynaptic densities (PSDs). The authors investigated the possible relation between these events by determining the effect of ischemia on tyrosine phosphorylation of the brain-specific, PSD-enriched, Ras-GTPase activating protein, SynGAP. Transient (15 minutes) global ischemia was produced in rats by 4-vessel occlusion and PSDs prepared from forebrains immediately after ischemia or at 20 minutes, 1 hour, or 24 hours of reperfusion. Tyrosine phosphorylation of SynGAP was elevated relative to sham-operated controls by 20 minutes of reperfusion and remained elevated for at least 24 hours. Tyrosine phosphorylation of SynGAP also increased in CA1 and CA3/DG subfields of the hippocampus. Enhanced tyrosine phosphorylation of SynGAP was not accompanied by a change in PSD RasGAP activity. SynGAP bound to the SH2 domains of Src and Fyn in a tyrosine phosphorylation-dependent fashion, and this interaction increased after ischemia. SynGAP binds to the PDZ domains of PSD-95/SAP90 and coimmunoprecipitated with PSD-95. The coimmunoprecipitation of SynGAP with PSD-95 decreased after ischemia. The results indicate that changes in the properties and interactions of SynGAP may be involved in the neuropathology of ischemia.
Asunto(s)
Ataque Isquémico Transitorio/metabolismo , Proteínas ras/metabolismo , Animales , Masculino , Fosforilación , Ratas , Ratas Wistar , Sinapsis/metabolismo , TirosinaRESUMEN
Transient cerebral ischemia results in an increase in the tyrosine phosphorylation of proteins associated with postsynaptic densities (PSDs). The authors investigated the possible mechanisms behind this increase by analyzing isolated PSDs for protein tyrosine kinase activity and for the presence of specific tyrosine kinases. Transient (15 minutes) global ischemia was produced in adult rats by four-vessel occlusion, and PSDs were isolated immediately after ischemia or after 20 minutes or 6 hours of reperfusion. Tyrosine phosphorylation of several PSD proteins, including the N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, was enhanced relative to shams after 20 minutes of reperfusion and underwent a further increase between 20 minutes and 6 hours. The ability of intrinsic PSD tyrosine kinase to phosphorylate PSD proteins, including the NMDA receptor, increased threefold after ischemia. Whereas PSD-associated proline-rich tyrosine kinase 2 (PYK2) and gp145TrkB were elevated immediately after the ischemic event, increases in Src and Fyn were not apparent until 6 hours of reperfusion. The level of PSD-associated pp125FAK decreased after ischemia. The results demonstrate that ischemia results in selective changes in the association of protein tyrosine kinases with the PSD which may account for ischemia-induced increases in the tyrosine phosphorylation of PSD proteins.
Asunto(s)
Encéfalo/enzimología , Ataque Isquémico Transitorio/enzimología , Proteínas Tirosina Quinasas/metabolismo , Sinapsis/enzimología , Animales , Ácido Desoxicólico/farmacología , Masculino , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Ratas , Ratas Wistar , Solubilidad , Sinaptosomas/enzimología , Tirosina/metabolismoRESUMEN
Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase-SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.
Asunto(s)
Hipocampo/fisiopatología , Ataque Isquémico Transitorio/fisiopatología , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Glutatión Transferasa/genética , Hipocampo/metabolismo , Ataque Isquémico Transitorio/metabolismo , Masculino , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fyn , Proteínas Proto-Oncogénicas pp60(c-src)/química , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/química , Proteínas Recombinantes de Fusión/metabolismo , Dominios Homologos srcRESUMEN
The authors investigated the gene expression of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptor and the functional electrophysiologic activity of NMDA receptor complexes in the vulnerable CA1 and less vulnerable dentate gyrus subfields of the rat hippocampus at different times after transient cerebral ischemia. Decreased expression for both subtypes was observed in both the CA1 subfield and dentate granule cell layer at early times after challenge; however, the decreased expression in the dentate granule cell layer was reversible because mRNA levels for both the NR2A and NR2B subtypes recovered to, or surpassed, sham-operated mRNA levels by 3 days postchallenge. No recovery of expression for either subtype was observed in the CA1 subfield. The functional activity of NMDA receptor complexes, as assessed by slow field excitatory postsynaptic potentiations (slow f-EPSP) in CA1 pyramidal neurons, was maintained at 6 hours postchallenge; however, this activity was diminished greatly by 24 hours postchallenge, and absent at 7 days postchallenge. A similar pattern was observed for the non-NMDA receptor-mediated fast f-EPSP. In dentate granule neurons, however, no significant change in NMDA receptor-mediated slow f-EPSP from sham control was observed at any time after insult. The non-NMDA receptor-generated fast f-EPSPs also were maintained at all times postinsult in the dentate gyrus. These results illustrate that the activity of NMDA receptors remains functional in dentate granule neurons, but not in the pyramidal neurons of the CA1 subfield, at early and intermediate times after transient cerebral ischemia, and suggest that there is a differential effect of ischemia on the glutamatergic transmission systems in these two hippocampal subfields.
Asunto(s)
Giro Dentado/metabolismo , Hipocampo/metabolismo , Ataque Isquémico Transitorio/metabolismo , Células Piramidales/metabolismo , Receptores de N-Metil-D-Aspartato/biosíntesis , Animales , Giro Dentado/fisiopatología , Regulación hacia Abajo , Potenciales Postsinápticos Excitadores , Hipocampo/fisiopatología , Hibridación in Situ , Ataque Isquémico Transitorio/fisiopatología , Masculino , Reacción en Cadena de la Polimerasa , Células Piramidales/fisiología , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiología , Transcripción GenéticaRESUMEN
In order to study the mechanisms of synaptogenesis in the rat cerebellar cortex, a library of monoclonal antibodies has been generated against proteins of the isolated synapse. One recognizes a glycosylated 38 kDa protein that is concentrated in the synaptic vesicle fraction and resembles synaptophysin biochemically in its molecular weight, charge, and pattern of glycosylation. In the adult cerebellar cortex, the antisynaptophysin(mabQ155) immunoreactivity is codistributed with synapses. Immunoreactivity is strongest in the molecular layer where punctate deposits of reaction product outline the Purkinje cell dendrites. Discrete small profiles, consistent with the distribution of basket cell axon terminals, surround the Purkinje cells, and in the granular layer the synaptic glomeruli are intensely stained. There is no immunoreactivity in the white matter axon tracts. Electron microscope immunocytochemistry confirms the synaptic location of the antigen and suggests that the reaction product is associated with synaptic vesicles. Both round and flat vesicle populations are immunoreactive. Antisynaptophysin(mabQ155) has been used to follow synaptogenesis in the developing rat cerebellum. In the newborn rat (P0), despite the paucity of synapses, there is some specific immunoreactivity, especially in the subcortical white matter. Electron microscopy shows that the antigenicity is associated with vesicles within growth cones, filopodia, and immature axon profiles. During development, antisynaptophysin immunoreactivity increases progressively, along with the maturing cell populations, for both the granule cell-Purkinje cell and the mossy fiber-granule cell synapses. Quantitative biochemical analysis confirms the cytochemical results. These data suggest that neuronal growth cones express a synapse-specific antigen before complete morphological synapses are present.
Asunto(s)
Envejecimiento/metabolismo , Corteza Cerebelosa/metabolismo , Proteínas de la Membrana/metabolismo , Sinapsis/metabolismo , Animales , Anticuerpos Monoclonales , Corteza Cerebelosa/crecimiento & desarrollo , Inmunohistoquímica , Proteínas de la Membrana/fisiología , Ratas , Ratas Endogámicas Lew , Fracciones Subcelulares/metabolismo , Sinapsis/fisiología , Sinapsis/ultraestructura , SinaptofisinaRESUMEN
The monoclonal antibody PAC 1 (postsynaptic density and cytoskeleton enriched) recognizes an epitope present on two postsynaptic density-enriched glycoproteins of 130,000 (postsynaptic density-enriched glycoprotein 130) and 117,000 mol. wt (postsynaptic density-enriched glycoprotein 117), and a cytoskeleton-enriched polypeptide of 155,000 mol. wt (cp155). The PAC 1 antibody has been used to study the development of the PAC 1 antigens in the developing rat forebrain in vivo and in tissue culture. cp155 is detected by embryonic day 14 and its level continues to rise until the sixth postnatal week. Postsynaptic density-enriched glycoproteins 130 and 117 are also expressed in embryonic brain although the level of postsynaptic density-enriched glycoprotein 130 initially increases more rapidly than that of postsynaptic density-enriched glycoprotein 117. Peak values are observed at postnatal days 4 (postsynaptic density-enriched glycoprotein 117) and 9 (postsynaptic density-enriched glycoprotein 130). The level of post synaptic density-enriched glycoprotein 117 subsequently decreases to some 50% of the peak value by postnatal day 42. Immunocytochemical studies show that PAC 1 immunoreactivity in developing cerebral cortex, detectable by postnatal day 0, is primarily associated with the perikarya and dendrites of pyramidal cells. The immunoreactivity develops as patches of PAC 1-positive neurons, uniform staining of the cortex only being fully established after postnatal day 9. Double-immunofluorescence labelling studies of forebrain cultures prepared from embryonic day 18 animals shows that many, but not all, growth-associated protein 43-positive neurons exhibit PAC 1 immunoreactivity. Some non-neuronal cells also stain with the PAC 1 monoclonal antibody. The growth cones of cultured neurons exhibit PAC 1 immunoreactivity and the PAC 1 antigens are detected on immunodeveloped western blots of isolated growth cones. The PAC 1 epitope is intracellular, but immunoreactivity does not co-localize with F-actin as detected by rhod-amine-phalloidin or with tubulin immunoreactivity. Postsynaptic density-enriched glycoprotein 130 is readily detected on PAC 1 immunodeveloped western blots of forebrain cultures maintained for up to 14 days in vitro. Postsynaptic density-enriched glycoprotein 117 is only poorly expressed by these cultures. The PAC 1 glycoproteins are present in forebrain synaptic membranes and postsynaptic densities at an early stage of development. The synaptic membrane level of postsynaptic density-enriched glycoprotein 130 and postsynaptic density-enriched glycoprotein 117 increases markedly between postnatal days 3 and 8. The level of both glycoproteins detected in postsynaptic densities remain virtually constant from postnatal days 9-90. These results are consistent with functional roles for these molecules in neuronal and synapse development.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Citoesqueleto/química , Epítopos/inmunología , Glicoproteínas de Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Prosencéfalo/citología , Sinapsis/química , Animales , Antígenos/biosíntesis , Diferenciación Celular , Células Cultivadas , Expresión Génica , Glicoproteínas de Membrana/biosíntesis , Peso Molecular , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/metabolismo , Neuronas/ultraestructura , Prosencéfalo/embriología , Prosencéfalo/crecimiento & desarrollo , Ratas , Ratas Wistar , Membranas Sinápticas/químicaRESUMEN
A cDNA clone designated NGB, which was isolated from a rat brain expression library, detected two mRNA species of 1.8 and 4.0 kb which are highly enriched in brain tissue. cDNAs NGB1 and NGB2 corresponding to these two mRNAs have been isolated and characterized. Sequence data showed that both mRNA species contain the same open reading frames but differ in their 3' untranslated regions. The open reading frame encodes a calmodulin protein of 148 amino acids. Both mRNA species are derived from the rat CaMI gene by utilization of different polyadenylation addition sites. Analysis of the 3' untranslated sequence which is unique to the larger mRNA species revealed a putative AU-rich 'destabilizer' sequence which is thought to be involved in mechanisms of selective mRNA breakdown. In situ hybridization studies revealed that the two calmodulin mRNAs are expressed strongly in neuronal cells in the adult rat brain. Levels of the two mRNA species increased during early postnatal development.
Asunto(s)
Encéfalo/fisiología , Calmodulina/genética , Neuronas/fisiología , Prosencéfalo/fisiología , ARN Mensajero/genética , Sinapsis/fisiología , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/crecimiento & desarrollo , Bovinos , Cerebelo/crecimiento & desarrollo , Cerebelo/fisiología , Clonación Molecular/métodos , Sondas de ADN , Escherichia coli/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Peso Molecular , Oligodesoxirribonucleótidos , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas EndogámicasRESUMEN
Systemic administration of kainic acid (KA) induces status epilepticus (SE) that causes neurodegeneration and may subsequently lead to spontaneous recurrent seizures. We investigated the effects of KA-induced SE on tyrosine phosphorylation and solubility properties of the NMDA receptor. Following 1 h of SE, total protein tyrosine phosphorylation was elevated in both the hippocampus and frontal cortex relative to controls. Tyrosine phosphorylation of the NMDA receptor subunits NR2A and NR2B was also enhanced following SE. Animals that received KA but did not develop SE, did not exhibit increased tyrosine phosphorylation. SE resulted in a decrease in the solubility of NMDA receptor subunits and of PSD-95 in 1% deoxycholate. In contrast, the detergent solubility of AMPA and kainate receptors was not affected. These findings demonstrate that SE alters tyrosine phosphorylation of the NMDA receptor, and indicate that the interaction of the NMDA receptor with other components of the NMDA receptor complex are altered as a consequence of seizure activity.
Asunto(s)
Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Convulsiones/metabolismo , Tirosina/metabolismo , Animales , Western Blotting , Detergentes , Electroforesis en Gel de Poliacrilamida , Ácido Kaínico , Masculino , Fosforilación , Pruebas de Precipitina , Ratas , Ratas Long-Evans , Receptores de N-Metil-D-Aspartato/química , Convulsiones/inducido químicamente , SolubilidadRESUMEN
The phosphorylation of proteins on tyrosine residues, initially believed to be primarily involved in cell growth and differentiation, is now recognized as having a critical role in regulating the function of mature cells. The brain exhibits one of the highest levels of tyrosine kinase activity in the adult animal and the synaptic region is particularly rich in tyrosine kinases and tyrosine phosphorylated proteins. Recent studies have described the effects of tyrosine phosphorylation on the activities of a number of proteins which are potentially involved in the regulation of synaptic function. Furthermore, it is becoming apparent that tyrosine phosphorylation is involved in the modification of synaptic activity, such as occurs during depolarization, the induction of long-term potentiation or long-term depression, and ischemia. Changes in the activities of tyrosine kinases and/or protein tyrosine phosphatases which are associated with synaptic structures may result in altered tyrosine phosphorylation of proteins located at the synapse leading to both short-term and long-lasting changes in synaptic and neuronal function.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Fosfotirosina/metabolismo , Sinapsis/metabolismo , Sinapsis/fisiología , Animales , Humanos , FosforilaciónRESUMEN
Incubation of postsynaptic densities (PSDs) with [gamma-32P]adenosine triphosphate (ATP) results in the phosphorylation of a number of proteins. Of these, phosphoproteins with apparent molecular weights (Mr) of 51,000, 180,000, 300,000, 320,000 and 370,000 contain 32P which is resistant to digestion with hot KOH suggesting the presence of [32P]phosphotyrosine residues. Phosphoamino acid analysis of total 32P-labelled PSDs identified [32P]phosphotyrosine as well as phosphoserine and phosphothreonine as products of the phosphorylation reaction. The PSD-specific glycoprotein gp180 was isolated from 32P-labelled PSDs and shown to contain [32P]phosphotyrosine. The results identify tyrosine kinase as a component of purified PSDs and gp180 as an endogenous substrate for this enzyme.
Asunto(s)
Encéfalo/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana , Proteínas del Tejido Nervioso/metabolismo , Proteínas Quinasas/metabolismo , Animales , Técnicas In Vitro , Fosforilación , Proteínas Tirosina Quinasas , Ratas , Sinapsis/metabolismoRESUMEN
Rats with chronic electrode implants to region CA3 of the hippocampus were rapidly kindled by stimulation with a 10 s, 10 Hz train of biphasic square waves presented every 5 min, until generalized seizures developed (60-70 stimulations). The hippocampi were isolated from the brains of control animals (implanted but not stimulated), and experimental animals which had developed generalized seizures. Synaptic membranes (SM) were prepared. SM were incubated with [gamma-32P]ATP and the incorporation of 32P into proteins and glycoproteins isolated by affinity chromatography on concanavalin-A-agarose was investigated. There was no difference in the phosphorylation pattern of total SM proteins between groups. In contrast, the phosphorylation of a glycoprotein with an apparent molecular weight of 180,000 was decreased 20-40% in kindled animals. This result was replicated in three independent experiments. The results suggest that the phosphorylation of glycoprotein 180 may be related to neuroplastic events.
Asunto(s)
Glicoproteínas/metabolismo , Hipocampo/metabolismo , Excitación Neurológica , Proteínas de la Membrana/metabolismo , Membranas Sinápticas/metabolismo , Animales , Estimulación Eléctrica , Hipocampo/fisiología , Masculino , Peso Molecular , Fosforilación , Ratas , Ratas Endogámicas , Convulsiones/metabolismoRESUMEN
Synaptic junctions (SJs) isolated from rat brain are associated with protein kinase activity and a unique complement of high molecular weight gglycoproteins. Incubation of SJs with [gamma-32P]A+ glycoproteins which were retained by concanavalin A agarose (con A+ glycoproteins). Three major (apparent mol. wt. 180 K, 130 K and 110 K) and 2 minor (apparent mol. wt. 230 K and 145 K) glycoproteins were identified in the con A+ fraction. Of these, GP180 incorporated the most 32P and GP145 was not labeled. Peptide mapping experiments showed that each molecular weight class of glycoprotein was associated with a unique set of phosphorylated peptides. Cyclic AMP stimulated the incorporation of 32P into total SJ proteins and con A+ lycoproteins by 38% and 58%, respectively. GP130 showed the greatest increase in labelling in the presence of cyclic AMP (198% of control levels) although incorporation into all 4 glycoproteins was increased. Cyclic AMP selectively stimulated the incorporation of 32P into only 2 of the 6 phosphorylated peptides derived from GP130. These studies demonstrate that endogenous glycoproteins serve as substrates for intrinsic SJ protein kinases and identify this reaction as a potential means of modifying postsynaptic membrane function.
Asunto(s)
Encéfalo/metabolismo , AMP Cíclico/farmacología , Glicoproteínas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Quinasas/metabolismo , Sinapsis/metabolismo , Animales , Fenómenos Químicos , Química , Peso Molecular , Fosforilación , Ratas , Ratas EndogámicasRESUMEN
The expression and properties of the neuron-specific glycoprotein, GP50, by neonatal rat granule cells grown in primary tissue culture were studied using the monoclonal antibody MabSM-GP50. GP50 was expressed by granule cells in culture but not by astrocytes or PC12 cells that had been induced to differentiate with nerve growth factor. Immunocytochemical staining of granule cell cultures demonstrated that immunoreactivity was concentrated in the cell body. Although the amount of GP50 increased slowly throughout the culture period the maximum level of expression in vitro was markedly lower than that observed in cerebellum of the comparable age. Radiolabelling of cell surface proteins by lactoperoxidase-catalyzed iodination demonstrated that GP50 was expressed on the surface of granule cells. Following phase-partitioning of granule cells in the presence of Triton X-114, 75% of GP50 was found to be present in the detergent phase, indicating that it is an integral membrane protein. Sucrose density gradient centrifugation of Triton X-100 extracts of granule cells resolved GP50 into two components with sedimentation coefficients of 3.6S and 7.3S. The 3.6S species (Mr 42,000 Da) accounted for greater than 80% of the total, indicating that GP50 is present predominantly in a monomeric form within the membrane. These properties are similar to those of GP50 expressed in P12 cerebellum but contrast with the behavior of GP50 from mature brain, in which the 7.3S, hydrophilic form is the predominant species. The results suggest that the mature expression of GP50 may be dependent upon the histotypic pattern of development which occurs in vivo.
Asunto(s)
Cerebelo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Células Cultivadas , Cerebelo/citología , Inmunohistoquímica , RatasRESUMEN
Although extracellular matrix (ECM) components have been shown to play important roles in the development of the CNS, expression generally decreases in the adult brain. This study examines the expression of the SPARC-related glycoprotein SC1 in the rat brain during postnatal development and in the adult. In situ hybridization analysis indicates that expression of SC1 mRNA increases in a caudal to rostral manner as postnatal neural development proceeds and is found at near maximal levels in the adult brain. SC1 mRNA is expressed in glial-enriched areas of the brain at postnatal day 1 (P1) and P5. Between P10 and P20, SC1 mRNA increases in neuron-enriched regions of the hippocampus, dentate gyrus, and cerebral cortex. Immunohistochemistry in the adult shows that SC1 protein is localized to neurons in these regions and to scattered glial cells. Subcellular fractionation demonstrates that the SC1 116/120 kDa doublet is associated with synaptosomes. SC1 is present in the aqueous phase following extraction of membranes with TX-114, suggesting that it is not a transmembrane protein, a property consistent with other adult brain ECM components. Furthermore in cerebellar granule cells grown in culture, high levels of the 120 kDa component are secreted into the media. These results are consistent with the hypothesis that SC1 is an ECM glycoprotein expressed in both the developing and adult brain.
Asunto(s)
Animales Recién Nacidos/crecimiento & desarrollo , Encéfalo/crecimiento & desarrollo , Matriz Extracelular/efectos de los fármacos , Glicoproteínas/fisiología , ARN Mensajero/biosíntesis , Animales , Encéfalo/metabolismo , Cerebelo/metabolismo , Inmunohistoquímica , Hibridación in Situ , Ratas , Ratas WistarRESUMEN
In the nervous system, extracellular matrix (ECM) molecules have been shown to have effects on cell migration, process outgrowth and the survival of neurons. Recently we have described the molecular cloning of SC1, a putative brain extracellular matrix glycoprotein, showing partial similarity to the ECM glycoprotein SPARC/osteonectin. We have now examined the expression of SC1 during the development of the rat cerebellum at both the protein and mRNA levels. Our results indicate that SC1 is both temporally and spatially regulated during this process. Bergmann glial cells express SC1 mRNA and the resultant protein is deposited along the length of their radial fibres during the process of granule cell migration in the developing cerebellum. SC1 mRNA and protein is also found in the adult cerebellum, concentrated in the Bergmann glial cells and their radial processes, indicating that this putative ECM molecule continues to play roles in the central nervous system after migration and proliferative events have ceased.
Asunto(s)
Cerebelo/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Glicoproteínas/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Molécula de Adhesión Celular del Leucocito Activado , Animales , Movimiento Celular , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Femenino , Inmunohistoquímica , Hibridación in Situ , Neuroglía/metabolismo , Neuronas/fisiología , Embarazo , ARN Mensajero/biosíntesis , Ratas , Ratas WistarRESUMEN
Synaptic fractions of decreasing morphological complexity were prepared by phase partitioning of synaptic membranes in an aqueous two-phase polymer system containing increasing concentrations of the neutral detergent n-octylglucoside (OG). The morphology, distribution of concanavalin A binding sites (CABS) and protein and glycoprotein composition of the resultant fractions were examined. The lowest concentration of OG employed (0.5% w/w) gave fractions enriched in relatively intact junctions retaining both pre- and postsynaptic structures. Increasing the detergent concentration resulted in the stepwise solubilization of pre- and postsynaptic structures until purified postsynaptic densities (PSDs) were obtained with 1% (w/w) OG. CABS were generally distributed on all membrane structures present in the 0.5% OG fraction, were restricted to synaptic structures in the fraction obtained with 0.75% OG, and were localized to the convex (outer) surface of purified PSDs. Gel electrophoretic analysis showed that the restriction of CABS to the region of the synapse was associated with a marked increase in the concentration of glycoproteins with apparent molecular weights of 180,000 and 130,000. These glycoproteins were retained, and further concentrated in the purified PSD fraction.
Asunto(s)
Encéfalo/inmunología , Receptores de Concanavalina A/metabolismo , Membranas Sinápticas/inmunología , Animales , Encéfalo/ultraestructura , Microscopía Electrónica , Peso Molecular , Ratas , Ratas Endogámicas , Receptores de Concanavalina A/aislamiento & purificación , Membranas Sinápticas/ultraestructuraRESUMEN
Carbohydrate-containing macromolecules have been localized in the outer plexiform layer of the embryonic and hatchling chick retina by horseradish peroxidase-conjugated wheat germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) lectins. Both WGA and RCA binding sites are present along developing photoreceptor synaptic membranes in the embryonic retina and the plasma membranes of developing neurites and glia. After photoreceptor synapse formation, RCA staining is restricted to non-synaptic membranes, but WGA staining is present on the pre- and post-synaptic membranes of receptor ribbon synapses in addition to non-synaptic membranes. These differing results between the accessibility of RCA and WGA binding sites on mature synaptic membranes in the chick retina suggests that RCA receptors on synaptic membranes are somehow masked after synapse formation and maturation, but that WGA receptors remain accessible. The effects of enzymatic digestion on WGA and RCA binding has been studied after prior treatment with neuraminidase. RCA staining of developing synaptic and non-synaptic membranes in the embryo remains the same after treatment with the enzyme, but in the hatchling RCA staining of non-synaptic membranes is enhanced, which suggests that galactosyl residues are relatively exposed on immature membranes but inaccessible to the lectin on mature membranes until neuraminidase acts to expose them by removing the terminal sialic acid residues. WGA staining on developing synaptic and non-synaptic membranes in the embryo is greatly diminished after neuraminidase pretreatment which suggests that a considerable amount of staining at this time is due to sialic acid in addition to N-acetylglucosamine. In the hatchling, photoreceptor synaptic membranes are no longer labeled with WGA and non-synaptic membrane staining is reduced after neuraminidase digestion, which suggests that after synapse formation synaptic membrane WGA labeling is primarily to sialyl residues, whereas most of the non-synaptic labeling is to N-acetylglucosamine residues.