RESUMEN
BACKGROUND: Both animals and plants respond rapidly to pathogens by inducing the expression of defense-related genes. Whether such an inducible system of innate immunity is present in the model nematode Caenorhabditis elegans is currently an open question. Among conserved signaling pathways important for innate immunity, the Toll pathway is the best characterized. In Drosophila, this pathway also has an essential developmental role. C. elegans possesses structural homologs of components of this pathway, and this observation raises the possibility that a Toll pathway might also function in nematodes to trigger defense mechanisms or to control development. RESULTS: We have generated and characterized deletion mutants for four genes supposed to function in a nematode Toll signaling pathway. These genes are tol-1, trf-1, pik-1, and ikb-1 and are homologous to the Drosophila melanogaster Toll, dTraf, pelle, and cactus genes, respectively. Of these four genes, only tol-1 is required for nematode development. None of them are important for the resistance of C. elegans to a number of pathogens. On the other hand, C. elegans is capable of distinguishing different bacterial species and has a tendency to avoid certain pathogens, including Serratia marcescens. The tol-1 mutants are defective in their avoidance of pathogenic S. marcescens, although other chemosensory behaviors are wild type. CONCLUSIONS: In C. elegans, tol-1 is important for development and pathogen recognition, as is Toll in Drosophila, but remarkably for the latter rôle, it functions in the context of a behavioral mechanism that keeps worms away from potential danger.
Asunto(s)
Conducta Animal/fisiología , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Proteínas de Drosophila , Proteínas del Helminto/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Actinomycetales/patogenicidad , Secuencia de Aminoácidos , Animales , Ascomicetos/patogenicidad , Secuencia de Bases , Caenorhabditis elegans/microbiología , Secuencia Conservada , Proteínas de Unión al ADN , Genes de Helminto , Genes Letales , Proteínas del Helminto/genética , Inmunidad Innata , Hongos Mitospóricos/patogenicidad , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Fosfoproteínas , Proteínas Serina-Treonina Quinasas , Pseudomonas aeruginosa/patogenicidad , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
BACKGROUND: The study evaluates the incidence of ocular compromise in the Kaposi's sarcoma associated with AIDS. METHODS: We revised the clinical histories of HIV seropositives patients seen in the ophthalmology department from January, 1994 to December, 1998. All patients were examined by direct visually and dilated fundus examination with the use of either a direct or an indirect ophthalmoscope. RESULTS: In 6,552 patients, ocular Kaposi's sarcoma was diagnosed in 17 (0.25%), predominantly in male sex (88.23%). The lesions predominated in eyelids, and the inferior has been the most affected. In only one female, the ocular compromise was the first neoplastic manifestation. CONCLUSIONS: The ocular compromise in the Kaposi's sarcoma is an alternative to be considered in AIDS patients with previous or simultaneous cutaneous or visceral involvement. Due to the few clinical signs of these lesions, a thorough ocular study is recommended in these patients.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Neoplasias del Ojo/epidemiología , Sarcoma de Kaposi/epidemiología , Adulto , Argentina/epidemiología , Recuento de Linfocito CD4 , Neoplasias de la Conjuntiva/epidemiología , Neoplasias de la Conjuntiva/etiología , Neoplasias de la Conjuntiva/patología , Técnicas de Diagnóstico Oftalmológico , Neoplasias del Ojo/diagnóstico , Neoplasias del Ojo/etiología , Neoplasias del Ojo/patología , Neoplasias de los Párpados/diagnóstico , Neoplasias de los Párpados/epidemiología , Neoplasias de los Párpados/etiología , Neoplasias de los Párpados/patología , Femenino , Humanos , Incidencia , Linfedema/etiología , Masculino , Neoplasias Primarias Múltiples/epidemiología , Neoplasias Primarias Múltiples/etiología , Neoplasias Primarias Múltiples/patología , Oftalmoscopía , Estudios Retrospectivos , Factores de Riesgo , Sarcoma de Kaposi/diagnóstico , Sarcoma de Kaposi/etiología , Sarcoma de Kaposi/patología , Neoplasias Cutáneas/epidemiología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patologíaRESUMEN
A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.
Asunto(s)
Aparato de Golgi/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Células Cultivadas , Aparato de Golgi/ultraestructura , Datos de Secuencia Molecular , Neuronas/química , Octoxinol , Polietilenglicoles/química , Ratas , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Proteínas de Unión al GTP rab/análisis , Proteínas de Unión al GTP rab/químicaRESUMEN
Advances in mass spectrometry combined with accelerated progress in genome sequencing projects have facilitated the rapid identification of proteins by enzymatic digestion, mass analysis, and sequence database searching. Applications for this technology range from the surveillance of protein expression in cells, tissues, and whole organisms, to the identification of proteins and posttranslational modifications. Here we consider practical aspects of the application of mass spectrometry in cell biology and illustrate these with examples from our own laboratories.
Asunto(s)
Espectrometría de Masas/métodos , Análisis de Secuencia de Proteína/métodos , Secuencia de Aminoácidos , Bases de Datos Factuales , Glicoproteínas/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.
Asunto(s)
Vesículas Cubiertas/metabolismo , Desarrollo Embrionario y Fetal/fisiología , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/fisiología , Receptores Citoplasmáticos y Nucleares , Alelos , Animales , Transporte Biológico , Northern Blotting , Western Blotting , Proteína Coatómero/metabolismo , Desarrollo Embrionario y Fetal/genética , Marcación de Gen , Genes Letales , Genotipo , Aparato de Golgi/ultraestructura , Sustancias Macromoleculares , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Familia de Multigenes , Fracciones Subcelulares/químicaRESUMEN
In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.
Asunto(s)
Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Insectos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calorimetría , Cartilla de ADN , Hidrólisis , Proteínas de Insectos/química , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Receptores de Superficie Celular/química , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de Superficie , Receptores Toll-Like , UltracentrifugaciónRESUMEN
Six new members of the yeast p24 family have been identified and characterized. These six genes, named ERP1-ERP6 (for Emp24p- and Erv25p-related proteins) are not essential, but deletion of ERP1 or ERP2 causes defects in the transport of Gas1p, in the retention of BiP, and deletion of ERP1 results in the suppression of a temperature-sensitive mutation in SEC13 encoding a COPII vesicle coat protein. These phenotypes are similar to those caused by deletion of EMP24 or ERV25, two previously identified genes that encode related p24 proteins. Genetic and biochemical studies demonstrate that Erp1p and Erp2p function in a heteromeric complex with Emp24p and Erv25p.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Levaduras/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Genes Fúngicos , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Fenotipo , Levaduras/genéticaAsunto(s)
Supervivencia de Injerto , Trasplante de Islotes Pancreáticos/fisiología , Trasplante Heterólogo/fisiología , Animales , Bovinos , Células Cultivadas , Cromatografía Líquida de Alta Presión , Femenino , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/citología , Trasplante de Islotes Pancreáticos/métodos , Trasplante de Islotes Pancreáticos/patología , Porcinos , Timo , Trasplante Heterólogo/métodos , Trasplante Heterólogo/patología , Trasplante HeterotópicoAsunto(s)
Diabetes Mellitus Tipo 1/cirugía , Insulina/sangre , Trasplante de Islotes Pancreáticos/fisiología , Trasplante Heterólogo/fisiología , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Glucemia/metabolismo , Bovinos , Ciclosporina/uso terapéutico , Diabetes Mellitus Tipo 1/sangre , Electrólitos/sangre , Femenino , Supervivencia de Injerto , Inmunosupresores/uso terapéutico , Trasplante de Islotes Pancreáticos/inmunología , Pancreatectomía , Porcinos , Timo , Factores de Tiempo , Trasplante Heterólogo/inmunología , Trasplante HeterotópicoRESUMEN
Phospholipase D (PLD) activity has been implicated in the regulation of membrane trafficking [1,2], superoxide generation and cytoskeletal remodelling [3,4]. Several PLD genes have now been identified and it is probable that different isoforms regulate distinct functions. Defining the subcellular localisation of each isoform would facilitate understanding of their roles. Previous PLD localisation studies have been based largely on enzyme activity measurements, which cannot distinguish between isoforms [2,5]. We have cloned the cDNAs encoding human PLD1a and PLD1b from an HL60 cell cDNA library and expressed them as catalytically active fusion proteins with green fluorescent protein (GFP) in COS-1 cells and RBL-2H3 cells, a mast cell model which degranulates upon cross-linking of the high-affinity immunoglobulin E (IgE) receptor. In unstimulated cells, GFP-PLD1b colocalised with secretory granule and lysosomal markers; it was not found at the plasma membrane or nucleus and did not colocalise with markers for the Golgi. Stimulation or RBL-2H3 cells through IgE receptor cross-linking caused plasma membrane recruitment of GFP-PLD1b. Inhibition of IgE-receptor-stimulated, PLD-catalysed phosphatidate formation suppressed secretion of granule and lysosomal contents, but did not affect translocation of GFP-PLD1b. These experiments suggest that PLD1 plays a role in regulated exocytosis rather than endoplasmic reticulum (ER) to Golgi membrane transport.
Asunto(s)
Gránulos Citoplasmáticos/enzimología , Lisosomas/enzimología , Fosfolipasa D/metabolismo , Animales , Células COS , Membrana Celular/enzimología , Clonación Molecular , Aparato de Golgi/enzimología , Proteínas Fluorescentes Verdes , Células HL-60 , Humanos , Leucemia Basofílica Aguda , Proteínas Luminiscentes/biosíntesis , Mastocitos/inmunología , Mastocitos/fisiología , Fosfolipasa D/genética , Ratas , Receptores de IgE/fisiología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
Calcitonin-gene-related peptide (CGRP) and adrenomedullin are related peptides with distinct pharmacological profiles. Here we show that a receptor with seven transmembrane domains, the calcitonin-receptor-like receptor (CRLR), can function as either a CGRP receptor or an adrenomedullin receptor, depending on which members of a new family of single-transmembrane-domain proteins, which we have called receptor-activity-modifying proteins or RAMPs, are expressed. RAMPs are required to transport CRLR to the plasma membrane. RAMP1 presents the receptor at the cell surface as a mature glycoprotein and a CGRP receptor. RAMP2-transported receptors are core-glycosylated and are adrenomedullin receptors.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Receptores de Péptido Relacionado con el Gen de Calcitonina/metabolismo , Receptores de Calcitonina/metabolismo , Receptores de Péptidos , Células 3T3 , Adrenomedulina , Secuencia de Aminoácidos , Animales , Transporte Biológico , Calcitonina/química , Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Proteína Similar al Receptor de Calcitonina , Línea Celular , Clonación Molecular , Reactivos de Enlaces Cruzados , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Expresión Génica , Glicosilación , Humanos , Péptidos y Proteínas de Señalización Intracelular , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/metabolismo , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores , Proteína 2 Modificadora de la Actividad de Receptores , Proteínas Modificadoras de la Actividad de Receptores , Receptores de Adrenomedulina , Receptores de Péptido Relacionado con el Gen de Calcitonina/genética , Alineación de Secuencia , Células Tumorales Cultivadas , XenopusRESUMEN
Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatially N-linked oligosaccharide modifications with the productive folding of newly synthesized glycoproteins. Calnexin was originally identified as a major type I integral membrane protein substrate of kinase(s) associated with the ER. Casein kinase II (CK2) was subsequently identified as an ER-associated kinase responsible for the in vitro phosphorylation of calnexin in microsomes (Ou, W-J., Thomas, D. Y., Bell, A. W., and Bergeron, J. J. M. (1992) J. Biol. Chem. 267, 23789-23796). We now report on the in vivo sites of calnexin phosphorylation. After 32PO4 labeling of HepG2 and Madin-Darby canine kidney cells, immunoprecipitated calnexin was phosphorylated exclusively on serine residues. Using nonradiolabeled cells, we subjected calnexin immunoprecipitates to in gel tryptic digestion followed by nanoelectrospray mass spectrometry employing selective scans specific for detection of phosphorylated fragments. Mass analyses identified three phosphorylated sites in calnexin from either HepG2 or Madin-Darby canine kidney cells. The three sites were localized to the more carboxyl-terminal half of the cytosolic domain: S534DAE (CK2 motif), S544QEE (CK2 motif), and S563PR. We conclude that CK2 is a kinase that phosphorylates calnexin in vivo as well as in microsomes in vitro. Another yet to be identified kinase (protein kinase C and/or proline-directed kinase) is directed toward the most COOH-terminal serine residue. Elucidation of the signaling cascade responsible for calnexin phosphorylation at these sites in vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Calnexina , Quinasa de la Caseína II , Línea Celular , Perros , Humanos , Espectrometría de Masas/métodos , Datos de Secuencia Molecular , Mapeo Peptídico , Fosforilación , Proteínas Quinasas Dirigidas por Prolina , Homología de Secuencia de Aminoácido , Serina/metabolismo , Tripsina/metabolismo , Células Tumorales CultivadasRESUMEN
BACKGROUND: Acute cutaneous miliary tuberculosis is a rare form of Mycobacterium tuberculosis infection which has been described in only 25 published cases in the last 15 years. The appearance of serious disseminated forms of tuberculosis is enhanced by the severe immunodeficiency which characterizes AIDS. METHODS: Four cases are described of disseminated tuberculosis with cutaneous localization clinically described as the acute miliary form. Patients had AIDS and CD4 lymphocyte levels of < 100 cells/mm3. Diagnosis was established in all cases by scraping of cutaneous lesions and direct examination with Ziehl-Neelsen coloration. RESULTS: The 4 patients presented signs and symptoms of disseminated tuberculosis with cutaneous manifestations, without a definite pattern and with localization predominantly on the thorax. Three had BAAR-positive sputum. Multiresistant strains of Mycobacterium tuberculosis were confirmed by antibiogram. One patient infected by a strain sensitive to first-line drugs responded favorably to conventional treatment for disseminated tuberculosis. In the remaining three with multiresistant strains, the evolution of the disease was rapid and unfavorable. CONCLUSIONS: Abrupt appearance of cutaneous lesions in a patient with disseminated tuberculosis and advanced HIV-disease should raise the suspicion of its etiology. Scraping and direct examination using Ziehl-Neelsen coloration is practical, economical and non-invasive diagnostic method.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/complicaciones , Tuberculosis Cutánea/complicaciones , Tuberculosis Miliar/complicaciones , Adulto , Humanos , Masculino , Tuberculosis Cutánea/diagnóstico , Tuberculosis Miliar/diagnósticoRESUMEN
Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.
Asunto(s)
Eosinófilos/inmunología , Interleucina-5/inmunología , Células Cultivadas , Humanos , Interleucina-5/genética , Mutación , Receptores de Interleucina/inmunología , Receptores de Interleucina-5 , Transducción de Señal/genética , Transducción de Señal/inmunologíaAsunto(s)
Trasplante de Islotes Pancreáticos/métodos , Trasplante Heterólogo/métodos , Trasplante Heterotópico/métodos , Animales , Bovinos , Femenino , Supervivencia de Injerto , Inmunohistoquímica , Insulina/análisis , Insulina/sangre , Trasplante de Islotes Pancreáticos/inmunología , Trasplante de Islotes Pancreáticos/fisiología , Radioinmunoensayo , Porcinos , Timo , Trasplante Heterólogo/inmunología , Trasplante Heterotópico/inmunologíaRESUMEN
The rat basophilic leukaemia (RBL) cell line has been widely used as a convenient model system to study regulated secretion in mast cells. Activation of these cells through the high-affinity receptor for IgE (Fcepsilon-RI) results in degranulation and the extracellular release of mediators. There is good evidence of a role for GTPases in mast cell degranulation, and a number of studies with peptides derived from the Rab3a effector domain have suggested that Rab3a may function in this process. However, in neuroendocrine cells, overexpression of Rab3a can act as a negative regulator of stimulated exocytosis [Holz, Brondyk, Senter, Kuizon and Macara (1994) J. Biol. Chem. 269, 10229-10234; Johanes, Lledo, Roa, Vincent, Henry and Darchen (1994) EMBO J. 13, 2029-2037]. In order to study the function of Rab3a in RBL degranulation, we have generated clones of RBL cells stably expressing Rab3a, and show that in these haematopoietic cells Rab3a can also function as a negative regulator of exocytosis. Overexpression of a mutant form of Rab3a (Asn-135 to Ile), which is predicted to be predominantly GTP-bound, also inhibited degranulation. However, overexpression of a mutant form of Rab3a that was truncated at the C-terminus to remove the sites for geranylgeranylation failed to inhibit degranulation. The effect of Rab3a is specific to secretion, and we observe no effect of Rab3a on receptor-mediated endocytosis. The Rab3a-induced block in degranulation can be bypassed by stimulation of streptolysin-O-permeabilized cells with guanosine 5'-[gamma-thio]triphosphate. We conclude from these studies that Rab3a is implicated in an early stage of granule targeting, whereas fusion of granules with the plasma membrane is regulated by a distinct downstream GTP-binding protein or proteins.
Asunto(s)
Degranulación de la Célula , Exocitosis , Proteínas de Unión al GTP/metabolismo , Mastocitos/fisiología , Fusión de Membrana , Animales , Proteínas Bacterianas , Western Blotting , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ratas , Estreptolisinas/farmacología , Transfección , Células Tumorales Cultivadas , Proteínas de Unión al GTP rab3RESUMEN
Chemokines are chemotactic proteins which play a central role in immune and inflammatory responses. Chemokine receptors are members of the seven transmembrane G-protein coupled family and have recently been shown to be involved in the entry of human immunodeficiency virus (HIV) into target cells. To study chemokine endocytosis in detail we have used novel site-specific chemistry to make a fluorescently labeled CC-chemokine agonist (rhodamine-MIP-1alpha) and antagonist (NBD-RANTES). We have also generated a CHO cell line stably expressing a hemagglutinin-tagged version of the CC-chemokine receptor 1 (CCR1), and using these reagents we have examined the receptor-mediated endocytosis of CC-chemokines by confocal microscopy. Our studies reveal that the agonist was internalized and accumulated in transferrin receptor-positive endosomes whereas the antagonist failed to internalize. However, receptor-bound antagonist could be induced to internalize by co-administration of agonist. Analysis of receptor redistribution following chemokine addition confirmed that sequestration was induced by agonists but not by antagonists.