Membrane Microdomains And B- Cell Signaling

B细胞抗体反应是由抗原与克隆分布的B细胞抗原受体(BCR)结合而触发的。在过去的几年里，关于BCR抗原结合引发的复杂信号级联的生物化学已经有了很大的了解。信号转导是通过膜相关的Src家族成员Lyn对BCR的磷酸化而启动的。目前，B细胞活化的起始事件使抗原结合的BCR与LYN接触尚不清楚。现在看来，富含胆固醇和鞘磷脂的膜微域，称为脂筏，是BCR信号传递的平台。根据脂筏在4摄氏度的某些非离子洗涤剂中的相对不溶性，可以将脂筏从细胞中分离出来。使用洗涤剂的不溶性来分离RAFT，我们了解到在静息细胞中，BCR被排除在浓缩Lyn的RAFT之外，但在多价抗原结合时，BCR寡聚并与RAFT结合，在那里它被Lyn磷酸化并启动信号传递。BCR的移位到RAFT中不需要BCR信号中最早的两个事件，即LYN对BCR的磷酸化或BCR与肌动蛋白细胞骨架的结合。然而，BCR不能发出信号或与肌动蛋白细胞骨架结合，只会导致BCR与脂筏的微弱和短暂的结合。在RAFT中信号的起始之后是RAFT聚集，并最终形成一个被称为免疫突触的高度组织化的结构，BCR信号可能从这个突触延长。在过去的一年里，我们应用了FRET成像的新技术，使我们能够观察活细胞中蛋白质和脂类的相互作用，以更好地表征抗原驱动的BCR信号中的早期事件，即受体的寡聚化及其与脂筏的关联。FRET定量成像显示bcr是静息细胞表面的单体，多价抗原结合导致S受体胞质结构域同时磷酸化，bcr、S胞质结构域由簇状变为开放，并与组成脂筏的脂类迅速而短暂地结合。这些事件先于下游信号事件的激活，需要Src家族激酶的持续活性，而不是Syk的结合。因此，BCR信号的启动是一个显著的动态过程，伴随着可逆的构象变化和由Src家族激酶活性诱导的RAFT脂质结合。目前正在努力将合适的FRET供体和受体对引入转基因小鼠，以使我们能够成像正常B细胞亚群中的信号事件。 从我们对BCR和RAFT关系的研究中出现了一个令人兴奋的主题，即BCR RAFT关联受到多种因素的调节，这些因素控制着B细胞与抗原相遇的结果，包括B细胞的发育状态、辅助受体的结合和病毒感染。确定这些因素如何影响BCR/RAFT关联应该从根本上增加我们对RAFT功能的理解。在过去的一年里，我们在确定辅助受体调节bcr与脂筏的关联并因此调节信号的机制方面取得了进展。当B细胞辅助受体CD19/CD21通过补体标记抗原与BCR结合时，可延长BCR在RAFT中的停留时间和发出的信号。我们确定CD19/CD21复合体在RAFT中发挥作用的能力依赖于CD19/CD21复合体的一种成分--Tetraspanin CD81。因此，在CD81缺陷小鼠的B细胞和表达嵌合CD19受体但未能与CD81结合的B细胞中，CD19/CD21复合体与BCR结合时不能稳定筏子中的BCR。许多与脂筏相关的蛋白质都是通过它们的酰化作用，特别是棕榈酰化作用来做到这一点的，这是一个可逆的酰化事件。我们确定CD81在BCR和CD19/CD21复合体交联后在脂筏中发生棕榈酰化。棕榈酰化对于CD81的功能似乎是必不可少的，因为使用阻断剂2-溴棕榈酸酯阻断了CD19/CD21复合体与BCR结合时稳定BCR的能力。研究正在进行中，以确定棕榈酰化酶的性质和CD81细胞质区域中的半胱氨酸，它们是棕榈酰化酶的靶标。 在确定FcGammaRIIB与BCR连接时如何发出细胞凋亡信号方面也取得了进展。FcGammaRIIB是BCR信号的有力负调控因子。我们了解到，当FcGammaRIIB与自身交联时，通过一种依赖于c-Abl的机制与脂筏和凋亡信号联系在一起，而不依赖于磷酸酶SHIP和FcGammaRIIB？S ITIM基序，这是FcGammaRIIB？S抑制bcr信号所必需的。虽然同种和异种聚集后的信号通路是不同的，但似乎有一种反馈机制，一旦在一条途径中启动信号，在相反的途径中信号就会被关闭。目前正在进行研究，以确定c-Abl和Ship在这一反馈机制中的作用。

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.