RESUMO
Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.
Assuntos
Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Domínios de Homologia de src/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linhagem Celular , Citometria de Fluxo , Regulação da Expressão Gênica , Rearranjo Gênico , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transdução de Sinais/imunologiaRESUMO
SLP-76 is an adapter protein expressed in T cells and myeloid cells that is a substrate for ZAP-70 and Syk. SLP-76-deficient mice exhibit a profound block in T-cell development. We found that although SLP-76 is expressed in mouse mast cells, SLP-76(-/-) mice have normal numbers of mast cells in their skin and bronchi. SLP-76(-/-) mice are resistant to IgE-mediated passive anaphylaxis. SLP-76(-/-) mice sensitized with IgE anti-dinitrophenyl (DNP) and then challenged with DNP-HSA developed only mild and transient tachycardia, failed to increase their plasma histamine level, and all survived the antigen challenge. Bone marrow-derived mast cells (BMMCs) from SLP76(-/-) mice failed to release beta-hexosaminidase and to secrete IL-6 after FcepsilonRI cross-linking. Tyrosine phosphorylation of phospholipase C-gamma1 (but not of Syk) and calcium mobilization in response to IgE cross-linking were reduced in SLP-76-deficient BMMCs. These results suggest that SLP-76 plays an important role in FcepsilonRI-mediated signaling in mast cells.
Assuntos
Mastócitos/metabolismo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Receptores de IgE/fisiologia , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal , Transferência Adotiva , Animais , Sítios de Ligação/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Degranulação Celular/genética , Diferenciação Celular/genética , Células Cultivadas , Citocinas/metabolismo , Imunoglobulina E/administração & dosagem , Imunoglobulina E/fisiologia , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Anafilaxia Cutânea Passiva , Fosfoproteínas/biossíntese , Receptores de IgE/genética , Receptores de IgE/imunologiaRESUMO
SLP-76 and LAT are two recently identified adapter proteins that are involved in the signal transduction cascade initiated by engagement of the TCR. The role of these two molecules in thymocyte development has become clearer following studies of gene targeted mice. The data indicate that SLP-76 and LAT are each critical for the expansion and differentiation of double-negative thymocytes and that SLP-76 is essential for allelic exclusion at the TCRbeta locus.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/fisiologia , Proteínas de Membrana , Fosfoproteínas/fisiologia , Subpopulações de Linfócitos T/citologia , Alelos , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem da Célula , DNA Nucleotidiltransferases/metabolismo , Retroalimentação , Regulação da Expressão Gênica no Desenvolvimento , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Hematopoese , Humanos , Ativação Linfocitária , Camundongos , Camundongos Knockout , Fenótipo , Fosfoproteínas/química , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transdução de Sinais , Relação Estrutura-Atividade , Timo/citologia , Transgenes , VDJ RecombinasesRESUMO
How platelet shape change initiated by a collagen-related peptide (CRP) specific for the GPVI/FcR gamma-chain complex (GPVI/FcR gamma-chain) is coupled to SLP-76, phosphoinositide (PI) 3-kinase, and gelsolin is reported. As shown by video microscopy, platelets rapidly round and grow dynamic filopodial projections that rotate around the periphery of the cell after they contact a CRP-coated surface. Lamellae subsequently spread between the projections. All the actin-driven shape changes require SLP-76 expression. SLP-76 is essential for the Ca(++) mobilization induced by CRP, whereas PI 3-kinase only modulates it. The extension of lamellae requires net actin assembly and an exposure of actin filament barbed ends downstream of PI 3-kinase. Gelsolin expression is also required for the extension of lamellae, but not for the formation of filopodia. Altogether, the data describe the role of SLP-76 in the platelet activation initiated by GPVI/FcR gamma-chain and the roles of PI 3-kinase and gelsolin in lamellae spreading. (Blood. 2000;96:3786-3792)
Assuntos
Plaquetas/efeitos dos fármacos , Gelsolina/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores de IgG/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/efeitos dos fármacos , Actinas/metabolismo , Actinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Plaquetas/citologia , Plaquetas/ultraestrutura , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Colágeno/química , Colágeno/metabolismo , Colágeno/farmacologia , Gelsolina/farmacologia , Humanos , Integrinas/metabolismo , Integrinas/fisiologia , Camundongos , Camundongos Mutantes , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Colágeno , Receptores de IgG/metabolismo , Receptores de Trombina/fisiologia , Transdução de SinaisRESUMO
The SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kDa) adapter protein is expressed in T cells and myeloid cells, whereas its homologue BLNK (B cell linker protein) is expressed in B cells. SLP-76 and BLNK link immunoreceptor tyrosine-based activation motif-containing receptors to signaling molecules that include phospholipase C-gamma, mitogen-activated protein kinases, and the GTPases Ras and Rho. SLP-76 plays a critical role in T cell receptor, FcvarepsilonRI and gpVI collagen receptor signaling, and participates in signaling via FcgammaR and killer cell inhibitory receptors. BLNK plays a critical role in B cell receptor signaling. We show that murine bone marrow-derived macrophages express both SLP-76 and BLNK. Selective ligation of FcgammaRI and FcgammaRII/III resulted in tyrosine phosphorylation of both SLP-76 and BLNK. SLP-76(-/-) bone marrow-derived macrophages display FcgammaR-mediated tyrosine phosphorylation of Syk, phospholipase C-gamma2, and extracellular signal regulated kinases 1 and 2, and normal FcgammaR-dependent phagocytosis. These data suggest that both SLP-76 and BLNK are coupled to FcgammaR signaling in murine macrophages.