RESUMEN
F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoproteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE). F1-20 was cloned and characterized because of its synapse specificity. AP-3 was purified and studied biochemically because of its function as a clathrin assembly protein. Here we present evidence that establishes the identity of F1-20 and AP-3. Monoclonal antibodies against F1-20 and AP-3 both specifically recognize a single protein from mouse brain with an apparent molecular mass of 190 kDa on SDS-PAGE. These monoclonal antibodies also specifically recognize the cloned F1-20 protein expressed in Escherichia coli. The anti-F1-20 monoclonal antibody (mAb) stains a bovine protein with an apparent molecular mass on SDS-PAGE of 190 kDa that copurifies with brain clathrin-coated vesicles (CCVs) and that can be extracted from the brain CCVs under conditions that extract AP-3. The anti-F1-20 and anti-AP-3 mAbs specifically recognize the same spot on a two-dimensional gel run on a bovine brain clathrin-coated vesicle extract. AP-3 purified from bovine brain CCVs is recognized by both the anti-F1-20 and anti-AP-3 mAbs. Purified preparations of bovine AP-3 and bacterially expressed mouse F1-20 give identical patterns of protease digestion with bromelain and subtilisin. Sequence analyses reveal that F1-20 has an essentially neutral 30-kDa NH2-terminal domain with an amino acid composition typical of a globular structure and an acidic COOH-terminal domain rich in proline, serine, threonine, and alanine. This is consistent with proteolysis experiments that suggested that AP-3 could be divided into a 30-kDa globular uncharged clathrin-binding domain and an acidic, anomalously migrating domain.
Asunto(s)
Química Encefálica , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/química , Fosfoproteínas/química , Proteínas Adaptadoras del Transporte Vesicular , Empalme Alternativo , Aminoácidos/análisis , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Western Blotting , Bovinos , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Ratones , Datos de Secuencia Molecular , Peso Molecular , Proteínas del Tejido Nervioso/biosíntesis , Oligodesoxirribonucleótidos , Sistemas de Lectura Abierta , Fosfoproteínas/biosíntesis , Reacción en Cadena de la Polimerasa , ARN/metabolismoRESUMEN
The NP185 polypeptide (AP3) is a multifunctional component isolated from brain endocytic vesicles, which binds to tubulin and clathrin light chains, decoated vesicles, synaptic vesicles, and the synaptosomal plasma membrane (Su et al., 1991). The NP185 molecules are expressed during avian cerebellar synaptogenesis and appear to function in CNS regions rich in synaptic terminals (Perry et al., 1991). In this report we describe double-labelling experiments with avian embryonic striated muscle fibers demonstrating the exclusive presence of the brain-specific protein at the neuromuscular junction. We used indirect rhodamine immunofluorescence labeling with a monoclonal antibody (mAb-8G8) to mark the location of NP185 in muscle combined with fluorescein-alpha-bungarotoxin to mark the postsynaptic location of the acetylcholine receptors (AChRs). We show that the distribution of both NP185 and AChRs has an overall correlation, but the location of NP185 is circumscribed to presynaptic structures adjacent but not overlapping with postsynaptic structures displaying the AchRs. To confirm the identity of NP185, the molecule was extracted from both tissues, partially purified, immunoprecipitated, and identified in Western blots with the mAb 8G8. The mAb reacted with an identical 185 kD protein band purified from both tissues. Based on its properties and specific neuronal location, the NP185 molecule may function in motor nerve terminals by screening membrane proteins, identifying areas of the synaptic plasma membrane, and to anchor these elements with structural proteins for their recycling and transport within the neuronal cellular compartments.
Asunto(s)
Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/metabolismo , Sinapsis/metabolismo , Complejo 3 de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Anticuerpos Monoclonales , Western Blotting , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Embrión de Pollo , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Músculos/química , Terminaciones Nerviosas/inmunología , Terminaciones Nerviosas/metabolismo , Proteínas del Tejido Nervioso/inmunología , Receptores Colinérgicos/inmunología , Receptores Colinérgicos/metabolismoRESUMEN
An antibody was detected in the sera from patients with systemic lupus erythematosus (SLE) and central nervous system (CNS) involvement that reacted with a 50-kD antigen in the plasma membrane of brain synaptic terminals. The 50-kD antigen was solubilized with Triton X-100 from preparations enriched with synaptic plasma membranes, and was partially purified by molecular sieve filtration column chromatography. The sera of 19 of 20 CNS-SLE patients showed strong to moderate immunoreactivity with the 50-kD protein in Western blots. Immunoreactivity with the 50-kD protein was also detected in the cerebrospinal fluid of CNS-SLE patients. Control sera from healthy individuals did not react with the 50-kD protein. Low to background reactivity was detected in 35% of a group of SLE patients without CNS manifestations, and in 3% of patients displaying other connective tissue diseases. A total of 100 individuals were tested in this study. Purified autoantibodies to the 50-kD protein from CNS-SLE patients were used for immunofluorescent labeling of neuroblastoma cells. The immunofluorescent staining revealed a distinct macular distribution pattern on the surface of the cell membrane. Taken together, the data suggest that the 50-kD protein may be an important target for autoantibodies, preponderantly found in CNS-SLE patients, and that the antigen may play a role in the pathogenesis of some neurological manifestations in SLE.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades del Sistema Nervioso Central/inmunología , Lupus Eritematoso Sistémico/inmunología , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Neuronas/inmunología , Adolescente , Adulto , Anciano , Animales , Western Blotting , Bovinos , Enfermedades del Sistema Nervioso Central/complicaciones , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Lupus Eritematoso Sistémico/complicaciones , Masculino , Membranas Sinápticas/inmunologíaRESUMEN
Evidence is presented here that demonstrates the presence of NP185 (AP3) in neuronal cells, specifically within syn-aptic terminals of the central nervous system and in the peripheral nervous system, particularly in the neuro-muscular junction of adult chicken muscle. Biochemical results obtained in our laboratories indicate that NP185 is associated with brain synaptic vesicles, with clathrin-coated vesicles, and with the synaptosomal plasma membrane. Also, NP185 binds to tubulin and clathrin light chains and the binding is regulated by phosphorylation (Su et al., 1991). Based on these properties and the data reported here, we advance the postulate that NP185 fulfills multiple functions in synaptic terminals. One function is that of a plasma membrane docking or channel protein, another of a signaling molecule for brain vesicles to reach the synaptic terminal region, and a third is that of a recycling molecule by binding to protein components on the lipid bilayer of the synaptic plasma membrane during the process of endocytosis. In support of these premises, a thorough study of NP185 using the developing chick brain, adult mouse brain, and chicken straited muscle was begun by temporally and spatially mapping the expression and localization of NP185 in evolving and mature nerve endings. To achieve these objectives, monoclonal antibodies to NP185 were used for immunocytochemistry in tissue sections of chicken and mouse cerebella. The distribution of NP185 was compared with those of other cytoskeletal and cytoplasmic proteins of axons and synapses, namely synaptophysin, vimentin, neurofilament NF68, and the intermediate filaments of glial cells (GFAP). The data indicate that expression of NP185 temporally coincides with synaptogenesis, and that the distribution of this protein is specific for synaptic terminal buttons of the CNS and the PNS.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/fisiología , Cerebelo/fisiología , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Sinapsis/fisiología , Complejo 3 de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Embrión de Pollo , Pollos , Terminaciones Nerviosas/metabolismo , Terminaciones Nerviosas/fisiología , Sinapsis/metabolismoRESUMEN
The neuronal protein NP185 is a neural tissue-specific protein isolated from clathrin-coated vesicles in brain. Using 8G8, a monoclonal antibody (MAb) characterized in our laboratory, we studied the expression and distribution of neuronal protein NP185 in developing avian cerebellum and in mature murine cerebellum. Furthermore, we compared these parameters to that of synapse-specific neuronal protein, synaptophysin, and an axon-specific (i.e., non-synaptic) neuronal protein, neurofilament NF68. We found that NP185 expression temporally and spatially corresponds to avian cerebellar synaptogenesis. In addition, NP185 distribution parallels synaptophysin distribution throughout development, while differing from that of either unassembled or filamentous forms of NF68. The evidence also suggests that embryonic NP185 expression coincides with synaptogenesis, and that NP185 remains concentrated in the terminal boutons of mature neurons. The synapse specificity of NP185 and the recent biochemical properties reported for this protein support the postulate that this molecule may trigger synaptic events and distinguish structurally and functionally active synapses.
Asunto(s)
Cerebelo/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Terminaciones Nerviosas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Animales , Anticuerpos Monoclonales , Cerebelo/crecimiento & desarrollo , Embrión de Pollo , Inmunohistoquímica , Ratones , Microscopía Fluorescente , Proteínas del Tejido Nervioso/inmunología , Sinaptofisina/metabolismoRESUMEN
The neuronal specific protein NP185, found associated with brain clathrin-coated vesicles, formed a complex with unphosphorylated, but not with phosphorylated, clathrin light chains. The NP185-clathrin light chain complex was associated with casein kinase II activity, which, in the presence of polylysine, phosphorylated clathrin light chain b but not the NP185. The dissociation of this complex with 50% ethylene glycol pH 11.5 suggests that NP185 binds to hydrophobic domains of clathrin light chains. When NP185 molecules were retained by monoclonal antibody-linked Sepharose beads, they bound synaptic vesicles, decoated vesicles and synaptosomal plasma membrane. Immunohistochemistry on mouse cerebellar tissue sections using 8G8, a monoclonal antibody raised against NP185, showed neuronal specific labeling closely following synaptic distribution. In immunoblots, NP185 shares similar epitopes to those detected in another assembly polypeptide, AP-180, an indication that both proteins are identical. It appears that NP185 plays a specific role in nerve ending functions through its ability to induce clathrin to polymerize into cages, its interaction with synaptic vesicles, with the plasma membrane and with clathrin coat components.
Asunto(s)
Encéfalo/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptosomas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Animales , Anticuerpos Monoclonales , Encéfalo/citología , Encéfalo/ultraestructura , Bovinos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Cromatografía de Afinidad , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Electroforesis en Gel de Poliacrilamida , Sustancias Macromoleculares , Microscopía Electrónica , Peso Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/aislamiento & purificación , Unión Proteica , Vesículas Sinápticas/ultraestructura , Sinaptosomas/ultraestructuraRESUMEN
Three independently isolated clathrin-associated proteins have been reported that have molecular weights of approximately 155,000-185,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: the 155K protein (Keen, J. H., and Black, M. M. (1986) J. Cell Biol. 102, 1325-1333), AP 180 (Ahle, S., and Ungewickell, E. (1986) EMBO J. 5, 3143-3149), and NP185 (Kohtz, D. S., and Puszkin, S. (1988) J. Biol. Chem. 263, 7418-7425). Using two-dimensional isoelectric focusing polyacrylamide gel electrophoresis and one- and two-dimensional immunoblots with two different monoclonal antibodies, we show that these three proteins are identical. The term AP-3 is used to denote this protein. A preliminary analysis of the domain structure of AP-3 was done by controlled proteolysis. Trypsin treatment of AP-3 yields two distinct classes of products. The larger fragments obtained (100,000-135,000 apparent Mr) are acidic and behave anomalously on gel electrophoresis, yielding aberrantly high Mr and exhibiting poor dye binding; these characteristics are shared with intact AP-3. Trypsin also generates a smaller neutral species of approximately 30,000 Da which migrates appropriately on sodium dodecyl sulfate-gel electrophoresis, binds dye comparatively strongly, and behaves as a monomeric globular species in solution. In addition, this species, which is also released by a variety of other proteases, binds specifically and reversibly to clathrin-Sepharose, identifying it as a clathrin recognition domain.
Asunto(s)
Clatrina/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Fosfoproteínas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Animales , Anticuerpos Monoclonales/inmunología , Bovinos , Electroforesis en Gel Bidimensional , Técnicas Inmunológicas , Técnicas In Vitro , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Fosfoproteínas/química , Fosfoproteínas/inmunología , Tripsina/metabolismoRESUMEN
A monoclonal antibody, S-11D9, detected a group of novel polypeptides whose expression was induced 6 hr after incubation of PC12 cells with nerve growth factor. The antigens also were visualized by immune precipitation and Western blotting in synaptic vesicles, clathrin-coated vesicles, and synaptic plasma membrane prepared from bovine brain. A large polypeptide was detected on the synaptic plasma membrane; an intermediate size protein was visualized on the plasma membrane and on both synaptic and clathrin-coated vesicles, while a smaller molecule was found only on brain Golgi-enriched membrane preparations. Immunofluorescence labeling with S-11D9 on nerve growth factor-stimulated PC12 cells showed the antigens distributed in the cytoplasm and concentrated in discrete areas on the tip of most neurites. The particular distribution of these proteins in vesicles and synaptic plasma membrane and the finding that the different antigens share a common epitope recognized by a single monoclonal antibody open the possibility that these molecules are related markers for organelle transport pathways in nerve cells.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/patología , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/biosíntesis , Química Encefálica , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/biosíntesis , Feocromocitoma/patología , Animales , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Bovinos , Diferenciación Celular/efectos de los fármacos , Epítopos/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Ratones , Terminaciones Nerviosas/química , Proteínas del Tejido Nervioso/inmunología , Ratas , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
A monoclonal antibody, A-7C11, was generated which reacts with two polypeptides of 40 kDa and 80 kDa associated with the coat proteins of purified brain clathirn-coated vesicles. The 40-kDa antigen was purified and found to display actin-binding properties. Negative-staining electron microscopy showed that one of the antigens reactive with A-7C11 appears to mediate the association of isolated clathrin-coated vesicles with assembling actin filaments in vitro. Immunofluorescence microscopy of cultured fibroblasts with A-7C11 revealed the antigens aligned with both actin filaments and as punctate structures near the plasma membrane. The data suggest that the interaction between clathrin-coated vesicles and the actin cytoskeleton is mediated by antigens identified by monoclonal antibody A-7C11.
Asunto(s)
Actinas/metabolismo , Encéfalo/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Músculos/metabolismo , Actinas/ultraestructura , Animales , Anticuerpos Monoclonales , Bovinos , Línea Celular , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Electroforesis en Gel de Poliacrilamida , Sustancias Macromoleculares , Proteínas de la Membrana/aislamiento & purificación , Peso MolecularRESUMEN
An equilibrium kinetics model is proposed to described some of the enzymatic properties of the cyclic GMP-stimulated phosphodiesterase activity associated with brain clathrin coated vesicles. The model assumes the presence of pharmacologically distinct regulatory and catalytic domains in the enzyme. The model contemplates that random fashion occupancy of the regulatory site by the substrate, cyclic GMP, induces a conformational change which leads to the generation of an actived catalytic state. Therefore, cyclic GMP is a positive allosteric modulator of the coated vesicle enzyme. Experimental data revealed that occupancy or activation of the regulatory site was not essential for catalysis to occur since hydrolysis occurred after loss (200%) of the activation by cyclic GMP. This constitutes an example of non-essential substrate activation. Analysis of this PDE following activation by cGMP and after loss of the regulation, activation capacity of the enzyme allows the calculation of the various kinetic parameters inherent in the model.
Asunto(s)
3',5'-GMP Cíclico Fosfodiesterasas/metabolismo , Encéfalo/enzimología , Invaginaciones Cubiertas de la Membrana Celular/enzimología , Modelos Biológicos , Regulación Alostérica , Animales , Encéfalo/ultraestructura , Bovinos , GMP Cíclico/metabolismo , Activación Enzimática , CinéticaRESUMEN
Monoclonal antibodies prepared against clathrin-coated vesicles cargo molecules were screened for their ability to precipitate the mAChR binding activity present in a light-density smooth membrane fraction from bovine brain called peak I. Only monoclonal antibody 10C7 was able to selectively sediment 38% (+/- 4.2) of the mAChR binding activity of peak I. Analysis by competition experiments of the supernatant obtained after the immunoprecipitation step reveals that neither the ratio of high/low affinity sites, nor the affinity ratio KH/KL was significantly altered for either of the muscarinic antagonists. The data implies that the bovine brain smooth-membrane compartment(s) expressing the mAb 10C7 antigen is also enriched in M1 and M2 mAChR.
Asunto(s)
Anticuerpos Monoclonales , Encéfalo/metabolismo , Pruebas de Precipitina , Receptores Muscarínicos/inmunología , Animales , Unión Competitiva , Bovinos , Pirenzepina/metabolismo , Quinuclidinil Bencilato/metabolismo , Receptores Muscarínicos/metabolismo , Fracciones Subcelulares/análisisRESUMEN
The 50-kilodalton (kDa) assembly polypeptide of bovine brain clathrin coated vesicles (CCVs) is phosphorylated in a cyclic nucleotide- and Ca2+-independent manner and is dephosphorylated by a Mg2+-ATP-dependent CCV phosphatase. This report provides evidence for modulation of the phosphorylation reaction of the 50-kDa assembly polypeptide by phosphorylated clathrin light chain beta (pLC beta). In vitro, phosphorylated LC beta inhibits phosphorylation of the 50-kDa polypeptide in CCVs. Furthermore, incubation of previously phosphorylated 50-kDa polypeptide in CCVs with phosphorylated LC beta results in a rapid dephosphorylation of the 50-kDa assembly polypeptide. Both phenomena are time and concentration dependent. Monoclonal antibodies to LC beta prevent the modulatory effect of phosphorylated LC beta on the 50-kDa assembly polypeptide phosphorylation in CCVs. The results obtained indicate for the first time, to our knowledge, that phosphorylated LC beta has a modulatory role in CCVs. The data also suggest that phosphorylated LC beta promotes activation of a coated vesicle phosphatase.
Asunto(s)
Encéfalo/metabolismo , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Bovinos , Clatrina/aislamiento & purificación , Homeostasis , Sustancias Macromoleculares , Magnesio/metabolismo , Peso Molecular , Fosfoproteínas Fosfatasas/metabolismo , FosforilaciónRESUMEN
A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton. Analysis of radioiodinated brain CVs immunoprecipitated with a tubulin antibody revealed that all brain CVs contained tubulin. The mAb A-7C11 recognized a 40-kilodalton (kDa) polypeptide on the clathrin coat and immunoprecipitated one-quarter of the total brain CVs. The mAb S-11D9 reacted with a 44-kDa antigen and immunoprecipitated 25% of the CVs. This antigen (44 kDa) was present in synaptic vesicles and synaptosomal membrane as well. Moreover, this mAb (S-11D9) reacted with a polypeptide of 56 kDa detected only in synaptosomal membrane. A mAb (C-10B2) that reacted with one of the clathrin light chains (LCb) immunoprecipitated 90% of the brain CVs. One of the mAbs immunoprecipitated a CV subtype that displayed a reversed ratio of the clathrin LCs (LCa greater than LCb). Each of the mAbs yielded different immunofluorescent staining patterns of vesicles in culture cell types that included nerve growth factor-differentiated PC12 cells, neuroblastoma cells, and Madin Darby bovine kidney cells. The data suggest that in brain tissue there is a heterogeneous population of CVs with different polypeptide compositions and subcellular distributions and that each of these subtypes performs a different role in nerve cells.
Asunto(s)
Anticuerpos Monoclonales , Encéfalo/ultraestructura , Clatrina/metabolismo , Neuropéptidos/metabolismo , Pruebas de Precipitina , Animales , Encéfalo/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Immunoblotting , Radioisótopos de Yodo , Ratones , Ratones Endogámicos BALB C , Orgánulos/metabolismo , Péptidos/metabolismoRESUMEN
We recently described a new protein associated exclusively with neuronal clathrin-coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S-8G8 and S-6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV-associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.
Asunto(s)
Encéfalo/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/metabolismo , Proteínas Quinasas/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Animales , Encéfalo/ultraestructura , Caseína Quinasas , Clatrina/metabolismo , Electroforesis en Gel de Poliacrilamida , Ratones , Ratones Endogámicos BALB C , FosforilaciónRESUMEN
Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.9 M NaCl extracts of CCVs were diluted and immunoprecipitated by either S-8G8 or S-6G7, NP185 precipitated as a complex with a fraction of the CCV assembly polypeptides. Immunofluorescence microscopy of PC12 cells cultured in nerve growth factor (NGF) revealed that NP185 was distributed in a punctate manner throughout the mature neurites. Immunoblot analysis of PC12 cell extracts, taken at various times during NGF-induced differentiation, revealed that steady-state accumulation of NP185 reaches significant levels 3 days after the addition of NGF and returns to undetectable levels when NGF is removed from the cultures. Significantly, the quantity of NP185 detected in differentiated PC12 cells exceeded the quantity of clathrin. These data indicate that while NP185 may be a specialized component of neuronal CCVs, its function in neuronal cells cannot be associated exclusively with these organelles.
Asunto(s)
Clatrina/aislamiento & purificación , Invaginaciones Cubiertas de la Membrana Celular/análisis , Endosomas/análisis , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas Adaptadoras del Transporte Vesicular , Animales , Anticuerpos Monoclonales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Clatrina/inmunología , Epítopos/análisis , Femenino , Técnica del Anticuerpo Fluorescente , Hibridomas/inmunología , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/inmunologíaRESUMEN
The N-terminal segment of human interleukin-2 (hIL-2) appears to mediate binding of the beta hIL-2 receptor (R. Robb, C. Rusk, J. Yodoi, and W. Greene, Proc. Natl. Acad. Sci. USA 84:2002-2006, 1987). An affinity-purified antibody prepared against this peptide segment (p81) is shown here to cross-react with a homologous region of the human T-cell leukemia virus type I (HTLV-I) envelope glycoprotein, raising the interesting possibility that the envelope glycoprotein of HTLV-I can interact with the beta hIL-2 receptor.
Asunto(s)
Deltaretrovirus/inmunología , Glicoproteínas/inmunología , Interleucina-2/inmunología , Receptores Inmunológicos/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Reacciones Cruzadas , Deltaretrovirus/genética , Técnica del Anticuerpo Fluorescente , Glicoproteínas/genética , Humanos , Fragmentos de Péptidos/inmunología , Receptores de Interleucina-2 , Homología de Secuencia de Ácido Nucleico , Proteínas del Envoltorio Viral/genéticaRESUMEN
Two basic polypeptides, bee venom melittin and poly-L-lysine, induced concentration-dependent destabilization of bovine brain coated vesicles. Ultrastructurally the changes observed were aggregation of clathrin coats and segregation of the vesicle membrane, concomitant with the appearance of elongated cisternae of various sizes. Changes in coated vesicle morphology induced by melittin and poly-L-lysine were concurrent with stimulation of phosphate incorporation in proteins of the coat lattice: Mr 33,000 and 100,000. Melittin-stimulated phosphorylation was Ca2+ sensitive and inhibited by EGTA. The initiation of vesicle membrane segregation by melittin, followed by fusion and formation of elongated membrane cisternae, paralleled an increase of endogenous phospholipase A2 activity. The data suggest that a correlation exists between the state of assembly of the coat proteins on coated vesicles and protein phosphorylation.
Asunto(s)
Encéfalo/ultraestructura , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Endosomas/ultraestructura , Fosfatos/metabolismo , Animales , Calcio/farmacología , Bovinos , Invaginaciones Cubiertas de la Membrana Celular/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Meliteno/farmacología , Microscopía Electrónica , Peso Molecular , Fosfolipasas A/biosíntesis , Fosfolipasas A2 , Fosforilación , Polilisina/farmacologíaRESUMEN
Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components.
Asunto(s)
Encéfalo/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endosomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Quinasas/metabolismo , Animales , Encéfalo/ultraestructura , Bovinos , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad/métodos , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Cinética , Rayos Láser , Fosforilación , Proteínas Quinasas/aislamiento & purificación , Dispersión de Radiación , Especificidad por SustratoRESUMEN
The two forms of clathrin light chains (LCA and LCB) or clathrin-associated proteins (CAP1 and CAP2) have presented an immunochemical paradox. Biochemically similar, both possess two known functional parameters: binding the clathrin heavy chain and mediating the action of an uncoating ATPase. All previously reported anti-CAP mAbs, however, react specifically with only CAP1 (Brodsky, F. M., 1985, J. Cell Biol., 101:2047-2054; Kirchhausen, T., S. C. Harrison, P. Parham, and F. M. Brodsky, 1983, Proc. Natl. Acad. Sci. USA, 80:2481-2485). Four new anti-CAP mAbs are reported here: two, C-7H12 and C-6C1, react with both forms; two others, C-10B2 and C-4E5, react only with the lower form. Sandwich ELISAs indicated that C-10B2, C-4E5, C-6C1, and C-7H12 react with distinct epitopes. Monoclonal antibodies C-10B2 and C-4E5 immunoprecipitate clathrin-coated vesicles (CCVs) and react with CAP2 epitopes accessible to chymotrypsin on the vesicle. These mAbs inhibit phosphorylation of CAP2 by endogenous CCV casein kinase II. In contrast, C-6C1 and C-7H12 react with epitopes that are relatively insensitive to chymotrypsin. CAP peptide fragments containing these epitopes remain bound to reassembled cages or CCVs after digestion. Immunoprecipitation and ELISAs demonstrate that C-7H12 and C-6C1 react with unbound CAPs but not with CAPs bound to triskelions or CCVs. The data indicate that the CAPs consist of at least two discernible structural domains: a nonconserved, accessible domain that is relevant to the phosphorylation of CAP2 and a conserved, inaccessible domain that mediates the binding of CAPs to CCVs.
Asunto(s)
Anticuerpos Monoclonales , Clatrina/análisis , Animales , Complejo Antígeno-Anticuerpo/análisis , Línea Celular , Quimotripsina , Clatrina/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Femenino , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/análisis , Fosforilación , PlasmacitomaRESUMEN
Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957).