RESUMEN
SAM68 (Src-associated in mitosis 68 kDa) is a member of the signal transduction of activator RNA novel gene family coding for proteins postulated to be involved in signal transduction and activation of RNA. It has been implicated through its phosphorylation status in the control of the transition from the G(1) to the S phases during mitosis. However, the implication and role of SAM68 in nonproliferative cells are unknown. The present study was initiated to examine the role of SAM68 in the phagocytic responses of the terminally differentiated human neutrophils. The results obtained show that SAM68 is present in human neutrophils and that it is tyrosine phosphorylated in response to stimulation by monosodium urate crystals or by ligation of CD32. Stimulation of neutrophils by these agonists decreases the association of SAM68 with Sepharose-conjugated poly-U beads. Additionally, the amount of immunoprecipitable SAM68 was modulated differentially after stimulation by monosodium urate crystals or by CD32 engagement indicating that the posttranslational modifications and/or protein associations of SAM68 induced by these two agonists differed. The results of this study provide evidence for an involvement of SAM68 in signal transduction by phagocytic agonists in human neutrophils and indicate that SAM68 may play a role in linking the early events of signal transduction to the posttranscriptional modulation of RNA.
Asunto(s)
Neutrófilos/inmunología , Neutrófilos/metabolismo , Proteínas de Unión al ARN/fisiología , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Ácido Úrico/farmacología , Proteínas Adaptadoras Transductoras de Señales , Adulto , Cristalización , Proteínas de Unión al ADN , Humanos , Isoflurofato/farmacología , Ligandos , Microesferas , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Fagocitosis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Poli U/metabolismo , Pruebas de Precipitina , Inhibidores de Proteasas/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , Tirosina/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.
Asunto(s)
Proteínas de Caenorhabditis elegans , Proteínas de Unión al ADN/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al ADN/genética , Dimerización , Células HeLa , Proteínas del Helminto/metabolismo , Humanos , Proteínas de Insectos/metabolismo , Ratones , Fosfoproteínas/genética , Mutación Puntual , Poli U/metabolismo , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn , ARN/fisiología , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de FusiónRESUMEN
Interleukin 8 (IL-8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL-8 receptors and the alpha subunit of guanine nucleotide regulatory protein (Gi(alpha)2) upon stimulation of human neutrophils by IL-8. In the present study, we identified by site-directed mutagenesis key residues within the three intracellular loops of the IL-8RA receptor involved in the interaction with Gi(alpha)2. We first systematically mutated, in groups of two to four, all the residues in the three intracellular loops of the IL-8 type A receptor to alanine and analyzed the mutant receptors transiently expressed in 293 cells. Four residues in the second intracellular loop (Y136, L137, I139, V140) and one residue in the third intracellular loop (M241) were shown to be crucial for mediating calcium signaling in response to IL-8. Other residues in the second and third intracellular loops were also found to affect IL-8RA-mediated signaling, but to a lesser extent. These effects were not due to lower expression or low IL-8 binding affinities to the mutated receptors. Mutagenesis of the residues in the first intracellular loop had only weak effects on the mobilization of calcium induced by IL-8. We then used a coimmunoprecipitation protocol with anti-Gi(alpha)2 antibodies to determine the involvement of the two regions defined above in Gi(alpha)2 coupling to IL-8 type A receptors. Whereas the anti-Gi(alpha)2 antibodies coimmunoprecipitated IL-8 receptors in the wild-type cells, this interaction was lost in cells expressing mutated receptors that affected intracellular calcium mobilization. The peptides corresponding to the regions of the type A receptor found to be critical for Gi(alpha)2 coupling and induction of intracellular calcium mobilization were next introduced into cells expressing wild-type IL-8RA or IL-8RB to assess their role in coupling Gi(alpha)2 to both IL-8 receptors. The results obtained in the latter experiments suggest that the same regions of the second intracellular loop (Y136, L137, I139, V140) and of the third intracellular loop (M241) are critically involved in the coupling of both IL-8RA and IL-8 RB to Gi(alpha)2 as well as to a downstream effector (or effectors) involved in calcium mobilization.
Asunto(s)
Antígenos CD/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Interleucina/metabolismo , Adulto , Secuencia de Aminoácidos , Antígenos CD/química , Sitios de Unión , Línea Celular Transformada , Humanos , Datos de Secuencia Molecular , Mutagénesis , Pruebas de Precipitina , Receptores de Interleucina/química , Receptores de Interleucina-8A , TransfecciónRESUMEN
Interleukin 8 (IL-8) and Gro-alpha are members of the CXC branch of a family of cytokines recently designated the "chemokine" superfamily. Recent evidence indicates that, contrary to previously held beliefs, IL-8 and Gro-alpha may not be perceived equivalently by neutrophils. In this study, we have evaluated the effects of IL-8 and Gro-alpha on the rate of calcium influx in human neutrophils and in 293 cells transfected with type A or type B IL-8 receptors. Of these two chemokines, only Gro-alpha induced an influx of calcium in neutrophils as judged by the sensitivity of the mobilization of calcium to the extracellular calcium chelator EGTA and to the nonselective divalent cation channel inhibitor SK&F 96365, as well as by manganese quenching experiments. IL-8 was similarly without effect on the rate of Mn2+ influx in 293 cells transfected with IL-8 receptor A (IL-8RA) or IL-8RB. On the other hand, Gro-alpha induced an SK&F 96365-sensitive increase of the rate of Mn+2 influx in IL-8RB-, but not in IL-8RA-transfected 293 cells. These results indicate not only that neutrophils respond differently to IL-8 than they do to Gro-alpha but, furthermore, that the consequences of the binding of IL-8 and Gro-alpha to IL-8RB are distinct.
Asunto(s)
Antígenos CD/fisiología , Calcio/fisiología , Quimiocinas CXC , Factores Quimiotácticos/fisiología , Sustancias de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intercelular , Interleucina-8/fisiología , Neutrófilos/fisiología , Receptores de Interleucina/fisiología , Membrana Celular/metabolismo , Quelantes/farmacología , Quimiocina CXCL1 , Citoplasma/metabolismo , Ácido Egtácico/farmacología , Humanos , Imidazoles/farmacología , Manganeso/metabolismo , Receptores de Interleucina-8A , Transducción de Señal , TransfecciónRESUMEN
Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (neutrophil-activating peptide-2) and Gro-alpha and whose biological activities are directed to a great extent toward neutrophils. Two distinct receptors have been described with overlapping, but not identical, binding affinities for IL-8, NAP-2, and Gro-alpha. This study was designed to examine the intracellular pathways activated upon the occupation of each of the IL-8 receptors (IL-8R). The formation of a physical coupling between IL-8 receptors and the alpha-subunit of heterotrimeric G proteins was tested in neutrophils by examining the presence of the former in anti-Galpha immune precipitates. The addition of IL-8 to a suspension of human neutrophils led to a time-dependent detection of IL-8 in anti-Gi2alpha (raised against amino acids 159-168 (LERIAQSDYI) of Gi2alpha) and anti-Gtalpha (raised against the COOH-terminal 10 amino acids (KENLKDCGLF) of Gtalpha), but not anti-Gq, immunoprecipitates. Similar results were obtained in human 293 cells stably transfected with IL-8RA or IL-8RB. The peptide derived from the COOH-terminal sequence of Gt inhibited the co-immunoprecipitation of IL-8R and Gi observed in response to the anti-Gtalpha and anti-Gi2alpha antibodies. On the other hand, the Gi2alpha peptide only inhibited the immunoprecipitation induced by the anti-Gi2alpha antibody. Peptides derived from Gi1alpha or Gi3alpha had no effect in this assay. The introduction of the anti-Gi2alpha or anti-Gtalpha antibodies or their neutralizing peptides, but not the Gi1alpha or Gi3alpha peptides, into 293 IL-8RA or 293 IL-8RB cells completely blocked the calcium responses obtained upon stimulation with IL-8. These results demonstrate that the occupation of either type of IL-8 receptor leads to a physical coupling to the alpha-subunit of Gi2. In addition, the use of the subunit-specific peptides identified two functionally important but distinct regions of Gialpha, one involved in receptor/Gialpha interaction (KENLKDCGLF) and the other mediating downstream signal transmission (LERIAQSDYI). Finally, the results of this study also validate the use of the transfected 293 cell line as a model for the study of the signal transduction pathway(s) initiated by IL-8.