RESUMEN
We compared self-collected oral fluid swab specimens with and without clinician supervision, clinician-supervised self-collected anterior nasal swab specimens, and clinician-collected nasopharyngeal swab specimens for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Supervised oral fluid and nasal swab specimens performed similarly to clinician-collected nasopharyngeal swab specimens. No sample type could detect SARS-CoV-2 infections amongst all positive participants.
Asunto(s)
COVID-19 , Humanos , Nasofaringe , SARS-CoV-2 , Saliva , Manejo de EspecímenesAsunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteolípidos/metabolismo , Baculoviridae/genética , Línea Celular , Dinaminas , GTP Fosfohidrolasas/aislamiento & purificación , GTP Fosfohidrolasas/ultraestructura , Humanos , Luz , Microscopía Electrónica , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/ultraestructura , Unión Proteica , Proteolípidos/aislamiento & purificación , Proteolípidos/ultraestructura , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/ultraestructura , Dispersión de RadiaciónRESUMEN
Amphiphysin 1 is a phosphoprotein expressed at high levels in neurons, where it participates in synaptic vesicle endocytosis and neurite outgrowth. It is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton. The yeast homologue of amphiphysin, Rvs167, functions in endocytosis and actin dynamics, is phosphorylated by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit Pcl2. We show here that amphiphysin 1 interacts with the cdk5-activating subunit p35 and that this interaction is mediated by the conserved NH2-terminal region of amphiphysin. Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts. Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285. Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells. These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Encéfalo/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Quinasa 5 Dependiente de la Ciclina , Mitosis , Fosforilación , RatasRESUMEN
We report the identification of betaIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The betaIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of betaIV spectrin generates at least four distinct isoforms, numbered betaIVSigma1-betaIVSigma4 spectrin. The longest isoform (betaIVSigma1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. betaIVSigma2 and betaIVSigma3 spectrin encompass the NH(2)- and COOH-terminal halves of betaIVSigma1 spectrin, respectively, while betaIVSigma4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of betaIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, betaIVSigma1 spectrin is recognized as a protein of 250 kD. Anti-betaIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from betaIVSigma1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for betaIV spectrin is more prominent in alpha than in beta cells. In brain, betaIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrin(G) 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, betaIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, betaIVSigma1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that betaIVSigma1 spectrin interacts with ankyrin(G) 480/270-kD and participates in the clustering of voltage-gated Na(+) channels and cell-adhesion molecules at initial segments and nodes of Ranvier.
Asunto(s)
Axones/química , Química Encefálica/fisiología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Nódulos de Ranvier/química , Nervio Ciático/química , Espectrina/análisis , Espectrina/genética , Secuencia de Aminoácidos , Animales , Ancirinas/metabolismo , Autoantígenos , Axones/fisiología , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Células COS , Cromosomas , Clonación Molecular , Citoplasma/química , Citoplasma/metabolismo , Citoesqueleto/química , Citoesqueleto/metabolismo , Neuropatías Diabéticas/fisiopatología , Expresión Génica/fisiología , Hipocampo/química , Hipocampo/citología , Hipocampo/fisiología , Humanos , Islotes Pancreáticos/química , Islotes Pancreáticos/fisiología , Masculino , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Fosfoproteínas/química , Fosfoproteínas/genética , Estructura Terciaria de Proteína , Proteínas Tirosina Fosfatasas/análisis , Proteínas Tirosina Fosfatasas/metabolismo , ARN Mensajero/análisis , Nódulos de Ranvier/fisiología , Ratas , Ratas Sprague-Dawley , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores , Nervio Ciático/citología , Nervio Ciático/fisiología , Transducción de Señal/fisiología , Canales de Sodio/metabolismo , Espectrina/químicaRESUMEN
Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.
Asunto(s)
Actinas/metabolismo , Adhesión Celular/fisiología , Línea Celular Transformada/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , GTP Fosfohidrolasas/metabolismo , Temperatura Corporal/fisiología , Adhesión Celular/efectos de los fármacos , Línea Celular Transformada/efectos de los fármacos , Línea Celular Transformada/ultraestructura , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Ciclosporina/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Dinamina I , Dinaminas , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación/fisiología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/ultraestructuraRESUMEN
Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.
Asunto(s)
Clatrina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Ensamble de Clatrina Monoméricas , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Células CHO , Cricetinae , Glutatión Transferasa , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Clathrin-mediated endocytosis is a special form of vesicle budding important for the internalization of receptors and extracellular ligands, for the recycling of plasma membrane components, and for the retrieval of surface proteins destined for degradation. In nerve terminals, clathrin-mediated endocytosis is crucial for synaptic vesicle recycling. Recent structural studies have provided molecular details of coat assembly. In addition, biochemical and genetic studies have identified numerous accessory proteins that assist the clathrin coat in its function at synapses and in other systems. This review summarizes these advances with a special focus on accessory factors and highlights new aspects of clathrin-mediated endocytosis revealed by the study of these factors.
Asunto(s)
Clatrina/fisiología , Endocitosis/fisiología , Vesículas Sinápticas/fisiología , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Animales , Humanos , Proteínas de la Membrana/fisiología , Vesículas Sinápticas/ultraestructuraRESUMEN
Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH(2)-terminal region, which represents a module (epsin NH(2) terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.
Asunto(s)
Proteínas Portadoras/genética , Clatrina/metabolismo , Neuropéptidos/genética , Proteínas de Transporte Vesicular , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Vesículas Cubiertas/metabolismo , Cricetinae , ADN Complementario/química , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/genética , Luciferasas/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/química , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Filogenia , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Amphiphysin, a protein that is highly concentrated in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphological evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.
Asunto(s)
Clatrina/metabolismo , Endocitosis/fisiología , GTP Fosfohidrolasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Encéfalo/metabolismo , Bovinos , Sistema Libre de Células , Clatrina/química , Clatrina/ultraestructura , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Dimerización , Dinamina I , Dinaminas , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/ultraestructura , Humanos , Cinética , Liposomas , Microscopía Electrónica , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/ultraestructura , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
Exocytosis of synaptic vesicles is calcium-dependent, with synaptotagmin serving as the calcium sensor. Endocytosis of synaptic vesicles has also been postulated as a calcium-dependent process; however, an endocytic calcium sensor has not been found. We now report a physical association between the calcium-dependent phosphatase calcineurin and dynamin 1, a component of the synaptic endocytic machinery. The calcineurin-dynamin 1 interaction is calcium-dependent, with an EC(50) for calcium in the range of 0.1-0. 4 microM. Disruption of the calcineurin-dynamin 1 interaction inhibits clathrin-mediated endocytosis. Thus, the calcium-dependent formation of the calcineurin-dynamin 1 complex, delivered to the other endocytic coat proteins, provides a calcium-sensing mechanism that facilitates endocytosis.
Asunto(s)
Calcineurina/metabolismo , Calcio/metabolismo , Endocitosis , GTP Fosfohidrolasas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Clatrina/antagonistas & inhibidores , Clatrina/metabolismo , Dinamina I , Dinaminas , Glutatión Transferasa/metabolismo , Humanos , Inmunofilinas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión a TacrolimusRESUMEN
Clathrin-mediated endocytosis was shown to be arrested in mitosis due to a block in the invagination of clathrin-coated pits. A Xenopus mitotic phosphoprotein, MP90, is very similar to an abundant mammalian nerve terminal protein, epsin, which binds the Eps15 homology (EH) domain of Eps15 and the alpha-adaptin subunit of the clathrin adaptor AP-2. We show here that both rat epsin and Eps15 are mitotic phosphoproteins and that their mitotic phosphorylation inhibits binding to the appendage domain of alpha-adaptin. Both epsin and Eps15, like other cytosolic components of the synaptic vesicle endocytic machinery, undergo constitutive phosphorylation and depolarization-dependent dephosphorylation in nerve terminals. Furthermore, their binding to AP-2 in brain extracts is enhanced by dephosphorylation. Epsin together with Eps15 was proposed to assist the clathrin coat in its dynamic rearrangements during the invagination/fission reactions. Their mitotic phosphorylation may be one of the mechanisms by which the invagination of clathrin-coated pits is blocked in mitosis and their stimulation-dependent dephosphorylation at synapses may contribute to the compensatory burst of endocytosis after a secretory stimulus.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Terminaciones Nerviosas/metabolismo , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Transporte Vesicular , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Endocitosis , Exocitosis , Péptidos y Proteínas de Señalización Intracelular , Ratones , Mitosis , Fosforilación , Unión Proteica , RatasRESUMEN
During endocytosis, clathrin and the clathrin adaptor protein AP-2, assisted by a variety of accessory factors, help to generate an invaginated bud at the cell membrane. One of these factors is Eps15, a clathrin-coat-associated protein that binds the alpha-adaptin subunit of AP-2. Here we investigate the function of Eps15 by characterizing an important binding partner for its region containing EH domains; this protein, epsin, is closely related to the Xenopus mitotic phosphoprotein MP90 and has a ubiquitous tissue distribution. It is concentrated together with Eps15 in presynaptic nerve terminals, which are sites specialized for the clathrin-mediated endocytosis of synaptic vesicles. The central region of epsin binds AP-2 and its carboxy-terminal region binds Eps15. Epsin is associated with clathrin coats in situ, can be co-precipitated with AP-2 and Eps15 from brain extracts, but does not co-purify with clathrin coat components in a clathrin-coated vesicle fraction. When epsin function is disrupted, clathrin-mediated endocytosis is blocked. We propose that epsin may participate, together with Eps15, in the molecular rearrangement of the clathrin coats that are required for coated-pit invagination and vesicle fission.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Proteínas Portadoras/fisiología , Clatrina/fisiología , Endocitosis/fisiología , Neuropéptidos/fisiología , Fosfoproteínas/fisiología , Proteínas de Transporte Vesicular , Subunidades alfa de Complejo de Proteína Adaptadora , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Aminoácidos , Animales , Northern Blotting , Encéfalo/metabolismo , Células CHO , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/química , Cricetinae , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Neuropéptidos/química , Fosfoproteínas/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
Clathrin-mediated endocytosis involves cycles of assembly and disassembly of clathrin coat components and their accessory proteins. Dephosphorylation of rat brain extract was shown to promote the assembly of dynamin 1, synaptojanin 1, and amphiphysin into complexes that also included clathrin and AP-2. Phosphorylation of dynamin 1 and synaptojanin 1 inhibited their binding to amphiphysin, whereas phosphorylation of amphiphysin inhibited its binding to AP-2 and clathrin. Thus, phosphorylation regulates the association and dissociation cycle of the clathrin-based endocytic machinery, and calcium-dependent dephosphorylation of endocytic proteins could prepare nerve terminals for a burst of endocytosis.
Asunto(s)
Clatrina/metabolismo , Endocitosis , GTP Fosfohidrolasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora , Subunidades beta de Complejo de Proteína Adaptadora , Proteínas Adaptadoras del Transporte Vesicular , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Carbazoles/farmacología , Cromatografía de Afinidad , Ciclosporina/farmacología , Dimerización , Dinamina I , Dinaminas , Inhibidores Enzimáticos/farmacología , Alcaloides Indólicos , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Dominios Homologos srcRESUMEN
Clathrin-coated buds and dynamin-coated tubules morphologically similar to corresponding structures observed in synaptic membranes can be generated on protein-free liposomes by incubation with cytosol, or with clathrin coat proteins and purified dynamin, respectively. Dynamin- and clathrin-coated intermediates may form independently of each other, despite the coupling between the two processes typically observed in synaptic membranes. Formation of both structures on liposomes can occur in the absence of nucleotides. These findings indicate that interfaces between lipids and cytosolic proteins are fully sufficient to deform lipids bilayers into buds and tubules. They suggest that a main function of membrane proteins is to act as positive and negative regulators of coat assembly, therefore controlling these processes in time and space.
Asunto(s)
Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis , Liposomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Encéfalo , Bovinos , Compartimento Celular , Sistema Libre de Células/metabolismo , Citosol/metabolismo , Dinaminas , GTP Fosfohidrolasas/aislamiento & purificación , Humanos , Proteínas del Tejido Nervioso/aislamiento & purificación , Nucleótidos/farmacología , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Unión Proteica , Ratas , Membranas Sinápticas/metabolismoRESUMEN
Amphiphysin I is an SH3 domain-containing neuronal protein, enriched in axon terminals, which was reported to act as a physiological binding partner for dynamin I in synaptic vesicle endocytosis. Rvs167 and Rvs161, the yeast homologs of amphiphysin I, have been implicated in endocytosis, actin function, and cell polarity. Now we have explored the possibility that amphiphysin I also may have a role in actin dynamics and cell polarity by testing the effect of amphiphysin I suppression on neurite outgrowth. Freshly plated hippocampal neurons were exposed to antisense oligonucleotides via a new delivery system based on a polycationic amphipathic polymer, PS980. Western blot analysis revealed that amphiphysin I levels steadily increased with neuronal differentiation, whereas in antisense-treated cultures amphiphysin I levels were reduced to approximately 10% of control levels at 48 hr. Concomitantly, a collapse of growth cones and a severe inhibition of neurite outgrowth and axon formation were observed. A similar effect was observed previously after dynamin I suppression in the same culture system (). We also have found that amphiphysin I and dynamin I colocalize in developing neurons at all developmental stages and that a pool of both proteins is colocalized with actin patches at the leading edge of growth cones. Our findings suggest a conserved role of the amphiphysin protein family in the dynamics of the cortical cell cytoskeleton and provide new evidence for a close functional link between amphiphysin I and dynamin I.
Asunto(s)
Proteínas de Unión al Calcio , Hipocampo/citología , Proteínas del Tejido Nervioso/genética , Neuritas/fisiología , ARN Mensajero/farmacología , Actinas/análisis , Animales , Células Cultivadas , Dinamina I , Dinaminas , Endocitosis/fisiología , GTP Fosfohidrolasas/análisis , Expresión Génica/fisiología , Glicoproteínas de Membrana/análisis , Ratones , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Neuritas/química , Oligonucleótidos Antisentido/farmacología , Monoéster Fosfórico Hidrolasas/metabolismo , Conejos , Ratas , Vesículas Sinápticas/química , Vesículas Sinápticas/enzimología , Sinaptotagminas , Tubulina (Proteína)/análisisAsunto(s)
Vesículas Sinápticas/fisiología , Sinaptosomas/fisiología , Animales , Membrana Celular/fisiología , Clatrina/fisiología , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Dinaminas , Exocitosis/fisiología , GTP Fosfohidrolasas/fisiología , Proteínas de la Membrana/metabolismo , Microtúbulos/fisiología , Modelos Neurológicos , Terminaciones Nerviosas/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Amphiphysin is an SH3 domain-containing neuronal protein that is highly concentrated in nerve terminals where it interacts via its SH3 domain with dynamin I, a GTPase implicated in synaptic vesicle endocytosis. We show here that the SH3 domain of amphiphysin, but not a mutant SH3 domain, bound with high affinity to a single site in the long proline-rich region of human dynamin I, that this site was distinct from the binding sites for other SH3 domains, and that the mutation of two adjacent amino acids in dynamin I was sufficient to abolish binding. The dynamin I sequence critically required for amphiphysin binding (PSRPNR) fits in the novel SH3 binding consensus identified for the SH3 domain of amphiphysin via a combinatorial peptide library approach: PXRPXR(H)R(H). Our data demonstrate that the long proline-rich stretch present in dynamin I contained multiple SH3 domain binding sites that recognize interacting proteins with high specificity.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Dinamina I , Dinaminas , GTP Fosfohidrolasas/química , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Prolina/metabolismo , Conejos , Análisis de Secuencia , Dominios Homologos src/genéticaRESUMEN
Mss4 is a guanine nucleotide exchange factor that specifically binds to, and promotes GDP-GTP exchange on, a subset of the Rab GTPases (Burton, J. L., Burns, M. E., Gatti, E., Augustine, G. J., and De Camilli, P. (1994) EMBO J. 13, 5547-5558). In order to identify the domain(s) of the GTPase that is important for this interaction, protein chimeras were constructed between Rab3a, which binds Mss4, and Rab5a, which does not bind Mss4. We have identified the amino-terminal portion of Rab3a as the Mss4-binding region, with the effector domain being critically required for binding and the flanking regions further enhancing the interaction. Sequence comparisons have revealed that Mss4-binding Rabs share more homology with each other than with Rabs that do not bind Mss4. The region of highest homology between these Rabs, which defines them as members of the same evolutionary branch within the Rab subfamily, coincides with the domain shown here to be critical for Mss4 binding. A mutation in the zinc-binding domain of Mss4 (Mss4 D96H), a region that is highly conserved between Mss4 and its yeast homologue Dss4, completely abolished its property to bind to, and promote GDP-GTP exchange on, Rab3a. Thus, the preservation of the Mss4/Dss4-GTPase interaction appears to have been a critical factor in the evolution of this subset of Rab proteins.
Asunto(s)
Proteínas/química , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Proteínas de Unión al GTP/metabolismo , Factores de Intercambio de Guanina Nucleótido , Guanosina Difosfato/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Mutación Puntual , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab3RESUMEN
Synaptojanin is a nerve terminal protein of relative molecular mass 145,000 which appears to participate with dynamin in synaptic vesicle recycling. The central region of synaptojanin defines it as a member of the inositol-5-phosphatase family, which includes the product of the gene that is defective in the oculocerebrorenal syndrome of Lowe. Synaptojanin has 5-phosphatase activity and its amino-terminal domain is homologous with the yeast protein Sac1 (Rsd1), which is genetically implicated in phospholipid metabolism and in the function of the actin cytoskeleton. The carboxy terminus, which is of different lengths in adult and developing neurons owing to the alternative use of two termination sites, is proline-rich, consistent with the reported interaction of synaptojanin with the SH3 domains of Grb2 (refs 1, 2). Synaptojanin is the only other major brain protein besides dynamin that binds the SH3 domain of amphiphysin, a presynaptic protein with a putative function in endocytosis. Our results suggest a link between phosphoinositide metabolism and synaptic vesicle recycling.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Terminales Presinápticos/enzimología , Secuencia de Aminoácidos , Animales , Encéfalo/enzimología , Línea Celular , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Células PC12 , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Ratas , Homología de Secuencia de Aminoácido , Dominios Homologos srcRESUMEN
Artificial fatty acylation of proteins has attracted significant attention during the last decade as a method for modification of protein specificity and efficacy of action on mammalian cells (A. V. Kabanov and V. Yu. Alakhov (1994) J. Contr. Release 28, 15-35). Horse radish peroxidase (HRP) is used in this work to study the interaction of a fatty acylated protein with various mammalian cells. The HRP is modified with the chloranhydride of the stearic acid in the reversed micelles of sodium bis-(2-ethylhexyl)sulfosuccinate (Aerosol OT) in octane, a convenient protocol allowing production of protein molecules with a controlled, low modification degree (A.V. Kabanov et al. (1987) Ann. N. Y. Acad. Sci. 501, 63-66). The influence of the hydrophobic group on the binding and internalization of HRP in MDCK, P3-X63-Ag8, CHO, and HepG2 cells is examined. The major results are as follows: (i) the fatty acylation of a protein significantly enhances its binding to all tested mammalian cell lines, with a line-specific efficiency; (ii) the binding efficiency can be modified by changing growth conditions in a defined medium; (iii) along with the enhancement of protein adsorption on the plasma membrane, fatty acylation increases internalization of the protein during incubations at 37 degrees C; (iv) internalized protein was observed in endocytic vesicles; no evidence was obtained for a cytoplasmic distribution. These results are discussed in connection with previously observed effects of the fatty acylated proteins on cell activity.