Membrane Microdomains And B- Cell Signaling
膜微域和 B 细胞信号传导
基本信息
- 批准号:7196688
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:B cell receptorB lymphocyteCD19 moleculeantibody formationantigen receptorsapoptosisbiological signal transductioncell growth regulationcell membraneconfocal scanning microscopyflow cytometryfluorescence resonance energy transfergenetically modified animalsimmunoprecipitationintermolecular interactionlaboratory mouseleukopoiesismembrane lipidsmembrane structureposttranslational modificationsprotein localizationreceptor bindingreceptor expressiontissue /cell culturetransfectionwestern blottings
项目摘要
B cell antibody responses are triggered by the binding of antigen to the clonally distributed B cell antigen receptors (BCRs). Over the last several years a great deal has been learned about the biochemistry of the complex signal cascades triggered by BCR antigen engagement. Signaling is initiated by phosphorylation of the BCR by a membrane associated member of the Src family kinase, Lyn. At present, the initiating event in B cell activation that brings the antigen bound BCR into contact with Lyn is not known. It now appears that cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, serve as a platform for BCR signaling. Lipid rafts can be isolated from cells based on their relative insolubility in certain nonionic detergents at 4 degrees Celsius. Using detergent insolubility to isolate rafts we learned that in resting cells the BCR is excluded from rafts that concentrate Lyn but upon multivalent antigen binding, the BCR oligomerizes and associates with rafts where it is phosphorylated by Lyn and signaling is initiated. The translocation of the BCR into rafts does not require two of the earliest events in BCR signaling, namely the phosphorylation of the BCR by Lyn or association of the BCR with the actin cytoskeleton. However, the failure of the BCR to signal or to associate with the actin cytoskeleton results in only weak and transient association of the BCR with lipid rafts. The initiation of signaling in the rafts is followed by raft clustering and ultimately by the formation of a highly organized structure termed an immunological synapse from which BCR signaling may be prolonged. Over the last year we have applied the new technology of FRET imaging that allows us to view the interactions of proteins and lipids in living cells, to better characterize the earliest events in antigen-driven BCR signaling, namely, the oligomerization of the receptor and its association with lipid rafts. Using quantitative FRET imaging we showed that the BCR is a monomer of the surface of resting cells and that multivalent antigen binding resulted in the simultaneous phosphorylation of the receptor?s cytoplasmic domains, a change in the BCR?s cytoplasmic domains from a clustered to an open form and the rapid yet transient association with lipids that compose lipid rafts. These events precede the activation of downstream signaling events and require the continuous activity of Src-family kinases but not the binding of Syk. Thus, the initiation of BCR signaling is a remarkably dynamic process accompanied by reversible conformation changes and raft lipid associations induced by Src-family kinase activity. Efforts are under way to introduce appropriate FRET donor and acceptor pairs into transgenic mice to allow us to image signal events in normal B cell subsets.
An exciting theme that emerged from our studies of the relationship of the BCR with rafts is one in which BCR raft association is regulated by a variety of factors that control the outcome of the B cell's encounter with antigen including the developmental state of the B cell, the engagement of coreceptors and viral infection. Determining how these factors influence BCR/raft association should add fundamentally to our understanding of how rafts function. Over the last year we have made progress in defining the mechanisms by which coreceptors function to regulate the association of the BCR with lipid rafts and as a consequence regulate signaling. The B cell coreceptors CD19/CD21 when coligated to the BCR through the binding of complement tagged antigens prolongs BCR residency in and signaling from rafts. We determined that the ability of the CD19/CD21 complex to function in rafts was dependent on a tetraspanin CD81 that is a component of the CD19/CD21 complex. Thus, in B cells from CD81-deficient mice and B cells expressing chimeric CD19 receptors that fail to associate with CD81, the CD19/CD21 complex when coligated to the BCR failed to stabilize the BCR in rafts. Many proteins that associate with lipid rafts do so by virtue of their acylation in particular by their palmitoylation, a reversible acylation event. We determined that CD81 becomes palmitoylated in the lipid rafts following crosslinking of the BCR and the CD19/CD21 complex. Palmitoylation appeared essential for the function of CD81 as blocking palmitoylation using the inhibitor 2-bromopalmitate blocked the ability of the CD19/CD21 complex to stabilize the BCR in lipid rafts when coligated to the BCR. Studies are in progress to determine the nature of the palmitoylating enzyme and the cysteines in the cytoplasmic domains of CD81 that are targets of palmitoylating enzymes.
Progress was also made in determining how the FcgammaRIIB, a potent negative regulator of BCR signaling when coligated to the BCR, signals for apoptosis when crosslinked to itself. We learned that the FcgammaRIIB when crosslinked to itself becomes associated with lipid rafts and signals for apoptosis by a mechanism dependent on c-Abl but independent of both the phosphatase SHIP and the FcgammaRIIB?s ITIM motifs that are required for FcgammaRIIB?s inhibition of BCR signaling. Although the signaling pathways following homo-versus hetero-aggregation are distinct there appears to be a feedback mechanism by which once signaling is initiated in one pathway signaling is shut down in the opposing pathway. Studies are in progress to define the role of c-Abl and SHIP in this feedback mechanism.
B细胞抗体应答是由抗原与克隆分布的B细胞抗原受体(BCR)的结合触发的。在过去的几年中,已经了解了大量关于BCR抗原接合触发的复杂信号级联的生物化学。信号传导由Src家族激酶的膜相关成员林恩对BCR的磷酸化启动。目前,使抗原结合的BCR与林恩接触的B细胞活化中的起始事件尚不清楚。现在看来,胆固醇和鞘脂丰富的膜微区,称为脂筏,作为一个平台的BCR信号。脂筏可以基于其在4摄氏度下在某些非离子去污剂中的相对不溶性而从细胞中分离。使用去污剂不溶性分离筏,我们了解到,在静息细胞中,BCR被排除在浓缩林恩的筏之外,但在多价抗原结合时,BCR寡聚化并与筏缔合,在筏中BCR被林恩磷酸化并启动信号传导。BCR向筏的转移不需要BCR信号传导中最早的两个事件,即BCR被林恩磷酸化或BCR与肌动蛋白细胞骨架的结合。然而,BCR的信号或与肌动蛋白细胞骨架的失败导致只有微弱的和短暂的协会的BCR与脂筏。筏中信号的起始之后是筏聚集,并最终形成被称为免疫突触的高度组织化的结构,BCR信号可以从该免疫突触延长。在过去的一年中,我们应用了FRET成像的新技术,使我们能够查看活细胞中蛋白质和脂质的相互作用,以更好地表征抗原驱动的BCR信号传导中的最早事件,即受体的寡聚化及其与脂筏的关联。使用定量FRET成像,我们表明,BCR是一个单体的表面静息细胞和多价抗原结合导致同时磷酸化的受体?s胞质结构域,BCR的变化?的细胞质结构域从一个集群到开放的形式和快速而短暂的协会与脂质组成脂筏。这些事件先于下游信号传导事件的激活,并且需要Src家族激酶的持续活性,但不需要Syk的结合。因此,BCR信号的启动是一个非常动态的过程,伴随着可逆的构象变化和筏脂协会诱导的Src家族激酶活性。目前正在努力将合适的FRET供体和受体对引入转基因小鼠,以使我们能够在正常B细胞亚群中成像信号事件。
从我们对BCR与筏的关系的研究中出现的一个令人兴奋的主题是,BCR筏的结合受到多种因素的调节,这些因素控制B细胞与抗原相遇的结果,包括B细胞的发育状态、辅助受体的参与和病毒感染。确定这些因素如何影响BCR/筏协会应该从根本上增加我们对筏功能的理解。在过去的一年里,我们已经在确定辅助受体的功能,以调节与脂筏的BCR协会,并作为一个结果,调节信号转导的机制方面取得了进展。当B细胞共受体CD 19/CD 21通过补体标记抗原的结合与BCR共连接时,BCR驻留在筏中并从筏发出信号。我们确定了CD 19/CD 21复合物在筏中发挥作用的能力依赖于四跨膜蛋白CD 81,四跨膜蛋白CD 81是CD 19/CD 21复合物的一个组分。因此,在来自CD 81缺陷小鼠的B细胞和表达不能与CD 81结合的嵌合CD 19受体的B细胞中,当与BCR共连接时,CD 19/CD 21复合物不能稳定筏中的BCR。许多与脂筏结合的蛋白质通过其酰化,特别是通过其棕榈酰化(可逆的酰化事件)而这样做。我们确定,在BCR和CD 19/CD 21复合物交联后,CD 81在脂筏中变为棕榈酰化。棕榈酰化似乎对CD 81的功能至关重要,因为使用抑制剂2-溴棕榈酸酯阻断棕榈酰化阻断了CD 19/CD 21复合物在与BCR共连接时稳定脂筏中BCR的能力。研究正在进行中,以确定棕榈酰化酶的性质和作为棕榈酰化酶靶点的CD 81细胞质结构域中的半胱氨酸。
在确定Fc γ RIIB(当与BCR共连接时,BCR信号传导的有效负调节剂)当与自身交联时如何发出细胞凋亡信号方面也取得了进展。我们了解到,FcgammaRIIB交联时,成为与脂筏和信号细胞凋亡的机制依赖于c-Abl,但独立的磷酸酶SHIP和FcgammaRIIB?s FcgammaRIIB所需的ITIM基序?抑制BCR信号传导。虽然同源与异源聚集后的信号传导途径是不同的,但似乎存在反馈机制,一旦在一个途径中启动信号传导,则在相反的途径中关闭信号传导。研究正在进行中,以确定c-Abl和SHIP在这种反馈机制中的作用。
项目成果
期刊论文数量(0)
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科研奖励数量(0)
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Susan Pierce其他文献
Susan Pierce的其他文献
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{{ truncateString('Susan Pierce', 18)}}的其他基金
Intracellular Trafficking Of The B cell Antigen Receptor
B 细胞抗原受体的细胞内运输
- 批准号:
6521528 - 财政年份:
- 资助金额:
-- - 项目类别:
The Mechanism of Co-Receptor Regulation of B-cell Activation
B 细胞激活的共受体调节机制
- 批准号:
8555905 - 财政年份:
- 资助金额:
-- - 项目类别:
B cell Receptor Dysregulation in Cancer and Autoimmune Disease
癌症和自身免疫性疾病中的 B 细胞受体失调
- 批准号:
8745551 - 财政年份:
- 资助金额:
-- - 项目类别:
The Mechanism of Co-Receptor Regulation of B-cell Activation
B 细胞激活的共受体调节机制
- 批准号:
8745432 - 财政年份:
- 资助金额:
-- - 项目类别:
Intracellular Trafficking and Signaling Of The B-cell Antigen Receptor
B 细胞抗原受体的细胞内运输和信号转导
- 批准号:
8745390 - 财政年份:
- 资助金额:
-- - 项目类别:
The Mechanism of Co-Receptor Regulation of B-cell Activation
B 细胞激活的共受体调节机制
- 批准号:
9566642 - 财政年份:
- 资助金额:
-- - 项目类别:
B cell Receptor Dysregulation in Cancer and Autoimmune Disease
癌症和自身免疫性疾病中的 B 细胞受体失调
- 批准号:
8157106 - 财政年份:
- 资助金额:
-- - 项目类别:
The Mechanism of Co-Receptor Regulation of B-cell Activation
B 细胞激活的共受体调节机制
- 批准号:
8156981 - 财政年份:
- 资助金额:
-- - 项目类别:
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