RESUMO
Although high titers of neutralizing Abs in human serum are associated with protection from reinfection by SARS-CoV-2, there is considerable heterogeneity in human serum-neutralizing Abs against SARS-CoV-2 during convalescence between individuals. Standard human serum live virus neutralization assays require inactivation of serum/plasma prior to testing. In this study, we report that the SARS-CoV-2 neutralization titers of human convalescent sera were relatively consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Furthermore, we show that heat inactivation of human serum significantly lowered neutralization activity in a live virus SARS-CoV-2 neutralization assay. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of complement in SARS-CoV-2 neutralization was often >50% of the neutralizing activity of human sera without heat inactivation and could account for neutralizing activity when standard titers were zero after heat inactivation. This effect was also observed in COVID-19 vaccinees and could be abolished in individuals who were undergoing treatment with therapeutic anti-complement Abs. Complement activity was mainly dependent on the classical pathway with little contributions from mannose-binding lectin and alternative pathways. Our study demonstrates the importance of the complement pathway in significantly increasing viral neutralization activity against SARS-CoV-2 in spike seropositive individuals.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , Via Clássica do Complemento , Testes de Neutralização , SARS-CoV-2 , Humanos , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , COVID-19/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Via Clássica do Complemento/imunologia , Vacinas contra COVID-19/imunologia , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Convalescença , Idoso , Proteínas do Sistema Complemento/imunologiaRESUMO
SARS-CoV-2 is a respiratory pathogen that can cause severe disease in at-risk populations but results in asymptomatic infections or a mild course of disease in the majority of cases. We report the identification of SARS-CoV-2-reactive B cells in human tonsillar tissue obtained from children who were negative for coronavirus disease 2019 prior to the pandemic and the generation of mAbs recognizing the SARS-CoV-2 Spike protein from these B cells. These Abs showed reduced binding to Spike proteins of SARS-CoV-2 variants and did not recognize Spike proteins of endemic coronaviruses, but subsets reacted with commensal microbiota and exhibited SARS-CoV-2-neutralizing potential. Our study demonstrates pre-existing SARS-CoV-2-reactive Abs in various B cell populations in the upper respiratory tract lymphoid tissue that may lead to the rapid engagement of the pathogen and contribute to prevent manifestations of symptomatic or severe disease.
Assuntos
Tonsila Faríngea/imunologia , Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , COVID-19/imunologia , Mucosa/imunologia , Receptores de Antígenos de Linfócitos B/genética , Sistema Respiratório/imunologia , SARS-CoV-2/fisiologia , Anticorpos Antivirais/metabolismo , Criança , Células HEK293 , Humanos , Memória Imunológica , Ativação Linfocitária , Análise de Célula Única , Glicoproteína da Espícula de Coronavírus/imunologia , TranscriptomaRESUMO
Fc receptor-like (FCRL) 4 is an immunoregulatory receptor expressed on a subpopulation of human memory B cells of mucosa-associated lymphoid tissue. Fc receptor function of FCRL4 was demonstrated by binding of IgA to FCRL4 following heat aggregation of the Ig. In this study, we demonstrate that FCRL4 recognizes J chain-linked systemic IgA in the absence of heat aggregation. We further demonstrate that mucosal secretory IgA is not recognized by FCRL4 and that systemic IgA binding can be competitively inhibited by recombinant secretory component protein. Finally, we provide evidence that primary FCRL4-bearing human memory B cells are constitutively bound to IgA. Our study provides a mechanism for the negative regulatory activity of FCRL4 on AgR-mediated B cell activation.
Assuntos
Linfócitos B/imunologia , Imunoglobulina A/metabolismo , Mucosa/imunologia , Receptores Fc/metabolismo , Secreções Corporais , Adesão Celular , Células HEK293 , Temperatura Alta , Humanos , Memória Imunológica , Imunomodulação , Ligação Proteica , Agregação de Receptores , Receptores Fc/genética , Transdução de SinaisRESUMO
FCRL4, a low-affinity IgA Ab receptor with strong immunoregulatory potential, is an identifying feature of a tissue-based population of memory B cells (Bmem). We used two independent approaches to perform a comparative analysis of the Ag receptor repertoires of FCRL4+ and FCRL4- Bmem in human tonsils. We determined that FCRL4+ Bmem displayed lower levels of somatic mutations in their Ag receptors compared with FCRL4- Bmem but had similar frequencies of variable gene family usage. Importantly, Abs with reactivity to commensal microbiota were enriched in FCRL4+ cells, a phenotype not due to polyreactive binding characteristics. Our study links expression of the immunoregulatory FCRL4 molecule with increased recognition of commensal microbial Ags.
Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Linfócitos B/imunologia , Memória Imunológica/imunologia , Microbiota/imunologia , Receptores Fc/imunologia , Linhagem Celular , Expressão Gênica/imunologia , Células HEK293 , Humanos , Imunoglobulina A/imunologia , Ativação Linfocitária/imunologia , FenótipoRESUMO
FCRL4 is an immunoregulatory receptor expressed by a subpopulation of memory B cells. These tissue-based cells express increased levels of the src-family kinases HCK and FGR. In this study, we investigate the roles of these src-family kinases in FCRL4-mediated immunoregulation of B cells in the context of previously unrecognized palmitoylation of the receptor. We observed enhanced phosphorylation of FCRL4 on tyrosine residues in the presence of the HCK p59 or FGR. This phosphorylation was markedly reduced in assays using a palmitoylation-defective mutant of FCRL4. In reporter gene studies, we observe that FCRL4 expression enhances CpG-mediated activation of NF-κB signaling. Surprisingly, using a reporter gene linked to activation of the MAPK substrate Elk-1 in response to Ag receptor ligation, we find that FCRL4 has inhibitory activity in cells coexpressing FGR but an activating function in cells coexpressing HCK p59. We provide evidence that in primary memory B cells, expression of FCRL4 leads to increased expression of IL-10 in the presence of FGR or HCK p59 in response to CpG, but increased levels of IFN-γ only in the context of coexpression of FGR. Our study supports the specific requirement of HCK p59 and FGR src-family kinases for FCRL4-mediated immunomodulatory activity and indicates that palmitoylation serves as an additional level of regulatory control of FCRL4.
Assuntos
Imunomodulação , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Fc/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular , Expressão Gênica , Genes Reporter , Humanos , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Imunomodulação/genética , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-10/genética , Interleucina-10/metabolismo , Mutação , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica/imunologia , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Fc/química , Receptores Fc/genética , Proteínas Elk-1 do Domínio ets/metabolismoAssuntos
Proteínas de Peixes , Proteínas Ligadas por GPI , Linfócitos/imunologia , Petromyzon , Filogenia , Receptores Imunológicos , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Loci Gênicos/imunologia , Glicosilfosfatidilinositóis/genética , Glicosilfosfatidilinositóis/imunologia , Petromyzon/genética , Petromyzon/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Sequências Repetitivas de AminoácidosRESUMO
VH replacement provides a unique RAG-mediated recombination mechanism to edit nonfunctional IgH genes or IgH genes encoding self-reactive BCRs and contributes to the diversification of Ab repertoire in the mouse and human. Currently, it is not clear how VH replacement is regulated during early B lineage cell development. In this article, we show that cross-linking BCRs induces VH replacement in human EU12 µHC(+) cells and in the newly emigrated immature B cells purified from peripheral blood of healthy donors or tonsillar samples. BCR signaling-induced VH replacement is dependent on the activation of Syk and Src kinases but is inhibited by CD19 costimulation, presumably through activation of the PI3K pathway. These results show that VH replacement is regulated by BCR-mediated signaling in human immature B cells, which can be modulated by physiological and pharmacological treatments.
Assuntos
Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Células Precursoras de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Antígenos CD19/metabolismo , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular , Ativação Enzimática , Rearranjo Gênico , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Dados de Sequência Molecular , Tonsila Palatina/citologia , Células Precursoras de Linfócitos B/citologia , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Quinases da Família src/metabolismoRESUMO
The leucine-rich repeat-based variable lymphocyte receptor B (VLRB) antibody system of jawless vertebrates is capable of generating an antibody repertoire equal to or exceeding the diversity of antibody repertoires of jawed vertebrates. Unlike immunoglobulin-based immune repertoires, the VLRB repertoire diversity is characterized by variable lengths of VLRB encoding transcripts, rendering conventional immunoreceptor repertoire sequencing approaches unsuitable for VLRB repertoire sequencing. Here we demonstrate that long-read single-molecule real-time (SMRT) sequencing (PacBio) approaches permit the efficient large-scale assessment of the VLRB repertoire. We present a computational pipeline for sequence data processing and provide the first repertoire-based analysis of VLRB protein characteristics including properties of its subunits and regions of diversity within each structural leucine-rich repeat subunit. Our study provides a template to explore changes in the VLRB repertoire during immune responses and to establish large scale VLRB repertoire databases for computational approaches aimed at isolating monoclonal VLRB reagents for biomedical research and clinical applications.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Animais , Petromyzon/imunologia , Petromyzon/genética , Linfócitos/imunologia , Linfócitos/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologiaRESUMO
Fc receptor-like (FCRL) molecules comprise a family of imunoregulatory transmembrane proteins that are preferentially, but not exclusively expressed on B lineage cells. A strong regulatory potential on B cell activation has been characterized for the different FCRL proteins, but their biological roles are just beginning to be elucidated. We review recent advances in the understanding of FCRL1-6 expression and function, and indicate their potential roles in the pathogenesis of immunodeficiencies, lymphoid malignancies and autoimmune diseases.
Assuntos
Linfócitos B/imunologia , Regulação da Expressão Gênica/imunologia , Receptores Fc/metabolismo , Animais , Doenças Autoimunes/imunologia , Linfócitos B/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/metabolismo , Síndromes de Imunodeficiência/imunologia , Camundongos , Neoplasias/imunologia , Receptores Fc/química , Receptores Fc/imunologiaRESUMO
FcR-like (FCRL) 2 is a transmembrane protein with immunomodulatory potential that is preferentially expressed by memory B cells in humans. It has two consensus ITIMs in addition to a putative ITAM sequence in its cytoplasmic domain. We have confirmed the cellular distribution of FCRL2 and analyzed its functional potential to show that coligation with the BCR leads to tyrosine phosphorylation of its ITIM motifs and subsequent Src homology region 2 domain-containing phosphatase-1 recruitment to facilitate inhibition of BCR signaling. Mutational analysis indicates that the tyrosine residues in both inhibitory motifs of FCRL2 are required for complete inhibition of BCR signaling, whereas tyrosines in the putative activation motif are dispensable for signal modulation. These findings suggest a negative immunomodulatory function for FCRL2 in the regulation of memory B cells.
Assuntos
Linfócitos B/imunologia , Memória Imunológica/fisiologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Mutação , Fosforilação/genética , Fosforilação/imunologia , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos B/genética , Receptores de Superfície Celular/genética , Transdução de Sinais/genéticaRESUMO
The FcRH4 transmembrane molecule, a member of the Fc receptor homologue family, can potently inhibit B cell receptor (BCR) signaling. We show that cell surface expression of this immunoregulatory molecule is restricted to a subpopulation of memory B cells, most of which lack the classical CD27 marker for memory B cells in humans. The FcRH4+ and FcRH4- memory B cells have undergone comparable levels of immunoglobulin isotype switching and somatic hypermutation, while neither subpopulation expresses the transcription factors involved in plasma cell differentiation. The FcRH4+ memory cells are morphologically distinctive large lymphocytes that express the CD69, CD80, and CD86 cell activation markers. They are also shown to be poised to secrete high levels of immunoglobulins in response to stimulation with T cell cytokines, but they fail to proliferate in response either to BCR ligation or Staphylococcus aureus stimulation. A heightened expression of the CCR1 and CCR5 chemokine receptors may facilitate their preferential localization in lymphoid tissues near epithelial surfaces. Cell surface FcRH4 expression thus marks a unique population of memory B cells with distinctive morphology, functional capabilities, and tissue localization.
Assuntos
Subpopulações de Linfócitos B/imunologia , Memória Imunológica , Receptores Fc/biossíntese , Receptores Fc/genética , Anticorpos Monoclonais/biossíntese , Subpopulações de Linfócitos B/metabolismo , Células Cultivadas , Humanos , Região Variável de Imunoglobulina/genética , Memória Imunológica/genética , Ativação Linfocitária/imunologia , Receptores de Superfície Celular , Receptores de Quimiocinas/metabolismo , Receptores Fc/imunologia , Hipermutação Somática de ImunoglobulinaRESUMO
Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores. The recombinant VLR-B antibodies possess 8-10 uniform subunits that collectively bind antigen with high avidity. Sequence analysis, mutagenesis, and modeling studies show that antigen binding involves residues in the beta-sheets lining the VLR-B concave surface. EM visualization reveals tetrameric and pentameric molecules having a central core and highly flexible pairs of stalk-region "arms" with antigen-binding "hands." Remarkable antigen-binding specificity, avidity, and stability predict that these unusual LRR-based monoclonal antibodies will find many biomedical uses.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Antígenos/imunologia , Linhagem Celular , Dimerização , Humanos , Lampreias , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
SARS-CoV-2 is a newly emerged betacoronavirus and the causative agent for the COVID-19 pandemic. Antibodies recognizing the viral spike protein are instrumental in natural and vaccine-induced immune responses to the pathogen and in clinical diagnostic and therapeutic applications. Unlike conventional immunoglobulins, the variable lymphocyte receptor antibodies of jawless vertebrates are structurally distinct, indicating that they may recognize different epitopes. Here we report the isolation of monoclonal variable lymphocyte receptor antibodies from immunized sea lamprey larvae that recognize the spike protein of SARS-CoV-2 but not of other coronaviruses. We further demonstrate that these monoclonal variable lymphocyte receptor antibodies can efficiently neutralize the virus and form the basis of a rapid, single step SARS-CoV-2 detection system. This study provides evidence for monoclonal variable lymphocyte receptor antibodies as unique biomedical research and potential clinical diagnostic reagents targeting SARS-CoV-2.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Proteínas de Peixes/imunologia , Petromyzon/imunologia , SARS-CoV-2/fisiologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Neutralizantes/genética , Anticorpos Antivirais/genética , Evolução Biológica , Reações Cruzadas , Epitopos de Linfócito B/imunologia , Proteínas de Peixes/genética , HumanosRESUMO
A TCR-like molecule (TCRL) with two canonical ITIM has been identified in the sea lamprey. We show here that TCRL is preferentially expressed by lymphocytes bearing variable lymphocyte receptors. To examine the potential of the TCRL inhibitory motifs, chimeric proteins comprising the FcgammaRIIb extracellular and transmembrane domains and the TCRL intracellular domain were expressed in a mouse B-cell line. BCR co-ligation with the WT version of the FcgammaRIIb/TCRL chimeric protein resulted in its tyrosine phosphorylation and the inhibition of BCR-induced calcium mobilization, whole-cell protein tyrosine phosphorylation and Erk/Akt/JNK activation. Tyrosine to phenylalanine mutations in either or both ITIM compromised the inhibitory capacity of this receptor chimera. Analysis of receptor-associated proteins indicated that the inhibition is mediated by recruitment of the protein tyrosine kinases, SHP1 and SHP2. These findings demonstrate the inhibitory potential of TCRL and its expression by clonally diverse lymphocytes bearing the variable lymphocyte receptors, thereby implying an immunomodulatory role for this ancestral TCR relative in a jawless vertebrate.
Assuntos
Lampreias/imunologia , Linfócitos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de IgG/imunologia , Animais , Linhagem Celular , Lampreias/genética , Camundongos , Proteínas Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de IgG/genética , Transdução de SinaisRESUMO
Although jawless vertebrates are apparently capable of adaptive immune responses, they have not been found to possess the recombinatorial antigen receptors shared by all jawed vertebrates. Our search for the phylogenetic roots of adaptive immunity in the lamprey has instead identified a new type of variable lymphocyte receptors (VLRs) composed of highly diverse leucine-rich repeats (LRR) sandwiched between amino- and carboxy-terminal LRRs. An invariant stalk region tethers the VLRs to the cell surface by means of a glycosyl-phosphatidyl-inositol anchor. To generate rearranged VLR genes of the diversity necessary for an anticipatory immune system, the single lamprey VLR locus contains a large bank of diverse LRR cassettes, available for insertion into an incomplete germline VLR gene. Individual lymphocytes express a uniquely rearranged VLR gene in monoallelic fashion. Different evolutionary strategies were thus used to generate highly diverse lymphocyte receptors through rearrangement of LRR modules in agnathans (jawless fish) and of immunoglobulin gene segments in gnathostomes (jawed vertebrates).
Assuntos
Rearranjo Gênico/genética , Imunogenética , Lampreias/genética , Lampreias/imunologia , Linfócitos/metabolismo , Receptores de Antígenos/genética , Adaptação Fisiológica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Evolução Biológica , Genoma , Mutação em Linhagem Germinativa/genética , Glicosilfosfatidilinositóis , Imunidade/genética , Imunidade/imunologia , Linfócitos/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos/química , Receptores de Antígenos/imunologia , Sequências Repetitivas de Aminoácidos , Análise de Sequência de DNARESUMO
CD38 is a multifunctional cell surface receptor expressed on multiple cell lineages of hematopoietic origin with high levels of expression on human plasma cells. Previously, we isolated the monoclonal variable lymphocyte receptor B (VLRB) MM3 antibody from the evolutionarily distant sea lamprey, which recognized the CD38 ectoenzyme exclusively on human plasma cells in a manner that correlated with CD38 enzymatic activity. The plasma cell-specific binding of VLRB MM3 contrasts with the broad pattern of expression of CD38-determined conventional antibodies specific for this antigen. In an effort to facilitate the application of this unique reagent in combination with conventional antibody panels, we explored a strategy to generate VLRB MM3 tetramers. The resulting reagent maintained the threshold-based recognition of CD38. Increased sensitivity achieved with VLRB MM3 tetramers also showed preferential recognition of germinal center centroblasts over centrocytes. VLRB MM3 tetramers thus provided a unique and versatile single-step staining reagent for the detection of human CD38 that is readily incorporated into multi-color flow cytometry panels.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Citometria de Fluxo/métodos , Linfócitos/imunologia , Humanos , Modelos MolecularesRESUMO
Memory B cells and plasma cells are antigen-experienced cells tasked with the maintenance of humoral protection. Despite these prominent functions, definitive cell surface markers have not been identified for these cells. We report here the isolation and characterization of the monoclonal variable lymphocyte receptor B (VLRB) N8 antibody from the evolutionarily distant sea lamprey that specifically recognizes memory B cells and plasma cells in humans. Unexpectedly, we determined that VLRB N8 recognizes the human leukocyte antigen-I (HLA-I) antigen in a tyrosine sulfation-dependent manner. Furthermore, we observed increased binding of VLRB N8 to memory B cells in individuals with autoimmune disorders multiple sclerosis and systemic lupus erythematosus. Our study indicates that lamprey VLR antibodies uniquely recognize a memory B cell- and plasma cell-specific posttranslational modification of HLA-I, the expression of which is up-regulated during B cell activation.
Assuntos
Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Memória Imunológica/imunologia , Plasmócitos/imunologia , Receptores de Antígenos/imunologia , Tirosina/análogos & derivados , Animais , Anticorpos Monoclonais/sangue , Linfócitos B/metabolismo , Células Cultivadas , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Lampreias/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Plasmócitos/metabolismo , Receptores de Antígenos/metabolismo , Tirosina/químicaRESUMO
Although GTPases of the Ras family have been implicated in many aspects of the regulation of cells, little is known about the roles of individual family members. Here, we analyzed the mechanisms of activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by two types of external stimuli, growth factors and ligation of the antigen receptors of B or T lymphocytes (BCRs and TCRs). The growth factors interleukin-3, colony-stimulating factor 1, and epidermal growth factor all preferentially activated M-Ras and K-Ras 4B over H-Ras or N-Ras. Preferential activation of M-Ras and K-Ras 4B depended on the presence of their polybasic carboxy termini, which directed them into high-buoyant-density membrane domains where the activated receptors, adapters, and mSos were also present. In contrast, ligation of the BCR or TCR resulted in activation of H-Ras, N-Ras, and K-Ras 4B, but not M-Ras. This pattern of activation was not influenced by localization of the Ras proteins to membrane domains. Activation of H-Ras, N-Ras, and K-Ras 4B instead depended on the presence of phospholipase C-gamma and RasGRP. Thus, the molecular mechanisms leading to activation of Ras proteins vary with the stimulus and can be influenced by either colocalization with activated receptors or differential sensitivity to the exchange factors activated by a stimulus.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Genes ras , Substâncias de Crescimento/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Proteína Adaptadora GRB2 , Humanos , Interleucina-3/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Microdomínios da Membrana/metabolismo , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Ésteres de Forbol/metabolismo , Fosfolipase C gama , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína SOS1/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fosfolipases Tipo C/metabolismo , Proteínas rasRESUMO
Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.