RESUMEN
Transcytosis of the polymeric immunoglobulin receptor (pIgR) is stimulated by binding of its ligand, dimeric IgA (dIgA). During this process, dIgA binding at the basolateral surface of the epithelial cell transmits a signal to the apical region of the cell, which in turn stimulates the transport of dIgA-pIgR complex from a postmicrotubule compartment to the apical surface. We have previously reported that the signal of stimulation was controlled by a protein-tyrosine kinase (PTK) activated upon dIgA binding. We now show that this signal of stimulation moves across the cell independently of pIgR movement or microtubules and acts through the tyrosine kinase activity by releasing Ca++ from inositol trisphosphate-sensitive intracellular stores. Surprisingly we have found that a second independent signal is required to achieve dIgA-stimulated transcytosis of pIgR. This second signal depends on dIgA binding to the pIgR solely at the basolateral surface and the ability of pIgR to dimerize. This enables pIgR molecules that have bound dIgA at the basolateral surface to respond to the signal of stimulation once they reach the postmicrotubule compartment. We propose that the use of two signals may be a general mechanism by which signaling receptors maintain specificity along their signaling and trafficking pathways.
Asunto(s)
Células Epiteliales/metabolismo , Receptores de Inmunoglobulina Polimérica/metabolismo , Transducción de Señal , Animales , Calcio/metabolismo , Compartimento Celular , Línea Celular , Dimerización , Perros , Células Epiteliales/efectos de los fármacos , Prueba de Complementación Genética , Inmunoglobulina A/metabolismo , Inmunoglobulina A/farmacología , Microtúbulos/metabolismo , Mutación , Proteínas Tirosina Quinasas/metabolismo , Receptores de Inmunoglobulina Polimérica/química , Receptores de Inmunoglobulina Polimérica/genéticaRESUMEN
The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-gammal. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-gammal, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727-736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-gammal and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-gammal activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-gammal that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.
Asunto(s)
Receptores de Inmunoglobulina Polimérica/metabolismo , Tirosina/metabolismo , Animales , Transporte Biológico , Línea Celular , Citoplasma/enzimología , Citoplasma/fisiología , Dimerización , Perros , Endocitosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Genisteína/farmacología , Inmunoglobulina A/metabolismo , Inmunoglobulina A/fisiología , Fosfatos de Inositol/antagonistas & inhibidores , Fosfatos de Inositol/biosíntesis , Isoenzimas/metabolismo , Riñón/citología , Ligandos , Fragmentos de Péptidos/fisiología , Fosfolipasa C gamma , Fosforilación , Unión Proteica/efectos de los fármacos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Receptores de Inmunoglobulina Polimérica/genética , Eliminación de Secuencia , Fosfolipasas de Tipo C/metabolismoRESUMEN
Recent studies have demonstrated that the CD3-zeta subunit of the T cell antigen receptor (TCR) complex is involved in signal transduction. However, the function of the remaining invariant subunits, CD3-gamma, -delta, and epsilon, is still poorly understood. To examine their role in TCR function, we have constructed TCR/CD3 complexes devoid of functional zeta subunit and showed that they are still able to trigger the production of interleukin-2 in response to antigen or superantigen. These data, together with previous results, indicate that the TCR/CD3 complex is composed of at least two parallel transducing units, made of the gamma delta epsilon and zeta chains, respectively. Furthermore, the analysis of partially truncated zeta chains has led us to individualize a functional domain that may have constituted the building block of most of the transducing subunits associated with antigen receptors and some Fc receptors.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Complejo CD3 , Antígenos CD8/genética , Línea Celular , Quimera , Deleción Cromosómica , Citometría de Flujo , Expresión Génica , Interleucina-2/biosíntesis , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Timoma , Neoplasias del Timo , TransfecciónRESUMEN
Ligand engagement of the TCR/CD3 complex leads to its internalization and modulation from the cell surface. In the present study, we analyzed the intracellular fate of internalized TCR/CD3 complexes following activation of a CTL clone with an anti-clonotypic mAb (anti-TCR mAb). Confocal microscopy using fluorescent anti-TCR mAb showed that after 15 min the TCR/CD3 complex colocalized with the transferrin receptor within endosomes, whereas at later times (2 h) it migrated in late endocytic compartments devoid of transferrin receptor. Using a cell fractionation technique, CD3 components could be detected in early endosomes in the absence of ligand-induced internalization, but were detected in late endosomes only after 2-h anti-TCR-induced internalization. In late endosomes, the internalized TCR/CD3 complex was found to be associated with an active protein kinase, distinct from p56(lck) and p59(fyn), which were mainly present in early endosomes, and ZAP-70, which was only present in the postnuclear supernatant. Phosphoamino acid analysis following an in vitro kinase assay of CD3 immunoprecipitates from early and late endosome fractions showed that the CD3 zeta- and epsilon-chains were phosphorylated exclusively on tyrosine, whereas the CD3 gamma- and delta-chains were phosphorylated on serine and tyrosine, as were 40-kDa and 60-kDa associated proteins. Furthermore, the serine phosphorylation was increased in late endosomes compared with early endosomes. These results suggest that the TCR/CD3 may be associated with different kinase activities during its intracellular pathway following ligand triggering.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Línea Celular , Endosomas/enzimología , Endosomas/metabolismo , Cinética , Ligandos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Fracciones Subcelulares/metabolismo , Proteína Tirosina Quinasa ZAP-70 , Familia-src Quinasas/metabolismoRESUMEN
Transcytosis of polymeric immunoglobulin A (pIgA) across epithelial cells is mediated by the polymeric immunoglobulin receptor (pIgR). Binding of pIgA to pIgR stimulates transcytosis of the pIgA-pIgR complex via a signal transduction pathway that is dependent on a protein tyrosine kinase (PTK) of the SRC family. Here we identify the PTK as p62yes. We demonstrate the specific physical and functional association of the pIgR with p62yes in rodent liver. Analysis of p62yes knockout mice revealed a dramatic reduction in the association of tyrosine kinase activity with the pIgR and in transcytosis of pIgA. We conclude that p62yes controls pIgA transcytosis in vivo.
Asunto(s)
Inmunoglobulina A/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Asialoglicoproteínas/metabolismo , Transporte Biológico , Fetuínas , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteína Oncogénica pp60(v-src)/genética , Pruebas de Precipitina , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-yes , Ratas , Ratas Sprague-Dawley , Receptores de Inmunoglobulina Polimérica/metabolismo , Transducción de Señal , alfa-Fetoproteínas/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Since TCR/CD3 modulation may be involved in induction of T cell tolerance to self antigens, we compared ligand-induced TCR/CD3 internalization by a CTL clone and by immature thymocytes and mature T cells from mice bearing the same TCR alpha beta as transgene. The ligand used is a monoclonal antibody (mAb) specific for the receptor expressed by the clone and transgenic mice (anti-Ti mAb). CD8+ splenocytes triggered by anti-Ti mAb internalize the ligand-TCR/CD3 complex at a low rate, through a mechanism inhibited by the protein tyrosine kinase (PTK) inhibitor genistein and by staurosporine, a potent but non selective protein kinase C (PKC) inhibitor. This pattern of inhibition was similar to that observed in the CTL clone. Anti-Ti mAb induced TCR/CD3 internalization in CD4+CD8+ thymocytes at a high rate, through a mechanism which was insensitive to either genistein or staurosporine. In the CTL clone, genistein was shown to inhibit TCR/CD3 surface redistribution preceeding internalization. To characterize the PTK possibly involved in this step, we analyzed TCR/CD3 associated kinases in mature T splenocytes and thymocytes. Kinase activities present in anti-Ti mAb immunoprecipitates phosphorylated the CD3 components gamma, delta, epsilon, and zeta in both cell types although the intensity was stronger in splenic than in thymocyte extracts, whereas the phosphorylation of 70, 14 and 12kD substrates was more pronounced in thymocytes than in splenocytes. Comparable amounts of CD3 components were coprecipitated with and phosphorylated by p56lck and p59fyn respectively, in both cell types.
Asunto(s)
Diferenciación Celular/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Alcaloides/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Células Clonales , Genisteína , Isoflavonas/farmacología , Ratones , Ratones Transgénicos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Estaurosporina , Subgrupos de Linfocitos T/enzimología , Linfocitos T Citotóxicos/enzimologíaRESUMEN
TCR engagement leads to down-modulation of TCR/CD3 complexes from the T cell surface. The importance of this effect in T cell physiology is unknown. Here, we characterized a CTL clone deficient in TCR/CD3 surface expression that had lost both CD3delta and CD3gamma mRNA, allowing us to address the role of these chains in the assembly, signaling, and dynamics of the TCR/CD3 complex. Expression of either CD3delta or CD3gamma alone failed to reconstitute surface expression of the TCR/CD3 complex, but reconstitution with a cytoplasmically truncated CD3delta (delta t) and a native (gamma) or cytoplasmically truncated (gamma t) human CD3gamma led to reexpression of TCR/CD3 complexes in both cases. This indicated that CD3delta and CD3gamma assume specific functions in TCR/CD3 assembly independently of their cytoplasmic domains. The delta t gamma t variant specifically killed target cells, expressed the IFN-gamma gene in response to Ag, and produced TNF-alpha in response to anti-CD3 mAb, but it was affected in CD3 ligand-induced TCR/CD3 down-modulation. Both PMA- and CD3 ligand-induced TCR/CD3 down-modulation were defective in the delta t gamma t variant, whereas the delta t gamma variants were unaffected, and previously described delta gamma t variants were affected only in PMA-induced down-modulation. Specific protein kinase C (PKC) inhibitors indicated that PMA- but not CD3 ligand-induced down-modulation was dependent on PKC activity. Thus, amino acid sequences present in either the CD3delta or CD3gamma cytoplasmic domain control ligand-induced TCR/CD3 down-modulation, and neither these sequences nor this property are required for cytolysis and IFN-gamma gene expression in response to Ag.
Asunto(s)
Complejo CD3/fisiología , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Animales , Línea Celular , Separación Celular , Citoplasma/fisiología , Regulación hacia Abajo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Microscopía Confocal , Proteína Quinasa C/fisiología , Linfocitos T Citotóxicos/ultraestructura , Acetato de Tetradecanoilforbol/farmacología , Factores de TiempoRESUMEN
Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.
Asunto(s)
Complejo CD3/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Citoesqueleto/ultraestructura , Regulación hacia Abajo , Endocitosis , Recubrimiento Inmunológico , Técnicas In Vitro , Ligandos , Ratones , Linfocitos T Citotóxicos/metabolismoRESUMEN
The fate of the T cell receptor (TcR)/CD3 complex was examined on a cytotoxic T lymphocyte (CTL) clone (KB5.C20) activated either via binding of an anti-TcR monoclonal antibody (mAb) or by a Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA). After binding of the anti-TcR mAb, electron microscopy revealed internalization through coated vesicles followed by slow degradation of the antibody as shown by use of radiolabeled mAb. The influence of activation on TcR/CD3 internalization was analyzed. The Ca2+ ionophore alone had no effect on internalization, whereas PMA induced an accelerated internalization of anti-TcR mAb. PMA-induced internalization was dependent on protein kinase C (PKC) as shown by its absence in PKC-depleted cells or in the presence of the PKC inhibitor staurosporine. Anti-TcR mAb-induced internalization was maintained in PKC-depleted cells, but unexpectedly remained sensitive to inhibition by staurosporine. The monovalent anti-TcR mAb Fab fragment is non-stimulatory for the CTL. It was poorly internalized but its internalization was induced by PMA. Surprisingly, on PKC-depleted cells, the Fab was internalized more readily than in untreated cells and this internalization was sensitive to inhibition by staurosporine. Inhibition of PMA-induced phosphorylation of gamma and epsilon subunits of CD3 was demonstrated after depletion of PKC or in the presence of staurosporine, confirming that PKC function was inhibited in those conditions. Cross-linking of the TcR via plastic-coated anti-TcR mAb led to phosphorylation of CD3 gamma and epsilon and also of zeta, known to be phosphorylated on tyrosines. All of these phosphorylation events were inhibited by treatment with staurosporine. Our results indicate that staurosporine inhibits the receptor internalization induced by anti-TcR mAb by means other than inhibition of PKC, suggesting that other kinases may control a step of this internalization process.
Asunto(s)
Alcaloides/farmacología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Activación de Linfocitos , Proteína Quinasa C/fisiología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos Monoclonales , Complejo CD3 , Calcio/metabolismo , Células Clonales , Fluorescencia , Antígenos H-2/metabolismo , Radioisótopos de Yodo , Ratones , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The TCR is composed of two chains (alpha/beta) containing variable regions associated at the cell surface with invariant chains (CD3 gamma-, delta-, epsilon-, and zeta/eta chains). The latter control assembly and surface expression of the TCR/CD3 complex, as well as its cytoplasmic association with signal transduction relays. In differentiated CTL, stimulation through the TCR leads to the transcriptional activation of genes coding secreted cytokines such as gamma-IFN as well as transcription-independent activation of the lytic machinery. It is not known which of the CD3 components is necessary to transduce the required signals. CD3 gamma- and delta-chains have high sequence homology, in particular in their cytoplasmic domain, and it has been proposed that alpha beta gamma epsilon zeta and alpha beta delta, epsilon zeta may be expressed and function in signal transduction independently. Here, we characterize a CTL clone that has selectively lost expression of the CD3 delta mRNA. This results in expression of partial CD3 complexes devoid of TCR alpha beta chains at the surface of the clone, which are not functional for activation of cytolysis or for gamma-IFN production. Transfection of the clone with either the native or a cytoplasmic exon-deleted CD3 delta gene restores full TCR/CD3 surface expression as well as Ag- or CD3-mediated activation for killing and for gamma-IFN production, indicating that the CD3 delta chain is essential for surface expression of the TCR alpha beta, but that the CD3 delta cytoplasmic portion is not required either for complex assembly or for signal transduction involved in the functions studied.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T Citotóxicos/fisiología , Secuencia de Aminoácidos , Animales , Complejo CD3 , Línea Celular , Citoplasma/ultraestructura , Técnicas In Vitro , Interferón gamma/biosíntesis , Activación de Linfocitos , Sustancias Macromoleculares , Proteínas de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Transducción de Señal , Relación Estructura-Actividad , TransfecciónRESUMEN
Primary biliary cirrhosis (PBC) is a chronic autoimmune liver disease of unknown etiology characterized by high-titer anti-mitochondrial antibodies. The major autoantigen has been identified as the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2). The fact that PDC-E2 is present in all nucleated cells, but autoimmune damage is confined to biliary epithelial cells, prompted us to investigate the possibility that mucosally-derived IgA may be pathogenic for biliary epithelial cells. Serum IgA was purified from six patients with PBC and its localization and ability to penetrate cells was studied using Madine-Darby canine kidney (MDCK) cells transfected with the human IgA receptor (MDCK-pIgR). The potential of IgA to be transported through the cells was studied by a combination of immunohistochemistry and dual color fluorescent microscopy. Interestingly, IgA from all PBC patients co-localized with PDC-E2 (the major autoantigen of PBC) inside the cells; this was demonstrated by dual staining with anti-human IgA and a mouse monoclonal antibody directed to PDC-E2. In contrast, no co-localization was observed for IgA controls. Furthermore, dual staining of liver sections from PBC patients demonstrated co-localization of IgA and PDC-E2, both cytoplasmically and at the apical surface. We postulate that there may be a direct effect of these autoantibodies on the mitochondrial function of biliary epithelial cells.