BIOLOGICAL FUNCTION OF B CELL RECEPTOR DESTABILIZATION

描述(改编自申请人的摘要)：由 B细胞抗原受体(BCR)有助于正常情况下的免疫反应 导致增殖或分化。放松对免疫反应的管制 由于癌蛋白转化或不能区分自己和外来者 蛋白质可导致淋巴增生性疾病和自身免疫性疾病。这个 候选人的兴趣是定义分子机制，使自我“沉默” 特异性B淋巴细胞。S博士近期的职业目标是进一步 描述了她最近观察到的B细胞抗原连接 受体导致bcr复合体的不稳定，如 免疫共沉淀Mig和Ig-α/Ig-β的能力减弱。第一 具体目标是定义BCR复合体完整性的变化 在抗原诱导脱敏之后。这将允许评估 BCR失稳是否代表了Mig和Ig的物理解离- 阿尔法和Ig-β，或生化不稳定。其中一种方法将是 Employeed使用化学交联剂来估计MiG和 受体失稳细胞中的Ig-α/Ig-β。第二种方法将 使用荧光显微镜来研究是否有单个组件 不稳定的受体在聚集时独立分离。 在第二个具体目标中，申请人将检验BCR的假设 不稳定发生在细胞表面，并在时间上与B 细胞无反应和受体脱敏，提示bcr 不稳定可能是不敏感表型的原因。这将是 通过创建一组不敏感的嵌合受体进行评估 造成不稳定。这些受体将含有Ig-α或Ig- 与MiG胞外区和MHC I类分子融合的β细胞质尾巴 跨膜结构域。嵌合受体将稳定地转染到 B细胞系，嵌合受体将被检测其能力 变得麻木了。 申请者的长期目标是继续定义分子基础 通过定义B细胞受体脱敏作为无能的模型 BCR失稳的分子机制(S)。BCR的时机 不稳定和对激酶激活的要求表明， Mig或Ig-α/Ig-β上的磷酸化事件可能负责 这件事。因此，她未来的计划，超出了工作的持续时间 在本申请中描述的，涉及确定一种新的磷酸化 事件与受体失稳同时发生，识别 靶点，并确认其在受体失稳中的作用 一种突变的受体。未来的其他研究包括评估 BCR通过产生“敲入”小鼠而使免疫反应不稳定 携带防止bcr不稳定的突变。最后， 申请人有兴趣定义MiG和Ig-的内吞途径。 当从细胞表面移除时，α/Ig-β紧随其后。

DESCRIPTION (adapted from the applicant's abstract): Signals transduced by the B cell antigen receptor (BCR) contribute to an immune response that normally leads to proliferation or differentiation. Deregulation of the immune response due to oncoprotein transformation or failure to discriminate self from foreign proteins can lead to lymphoproliferative disorders and autoimmune disease. The candidate's interests are to define molecular mechanisms that "silence" self- specific B lymphocytes. Dr. Vilen s immediate career goals are to further characterize her recent observation that ligation of the B cell antigen receptor leads to destabilization of the BCR complex, as evidenced by the diminished ability to co-immunoprecipitate mIg and Ig-alpha/Ig-beta. The first Specific Aim is to define changes in the integrity of the BCR complex following antigen-induced desensitization. This will allow assessment as to whether BCR destabilization represents a physical dissociation of mIg from Ig- alpha and Ig-beta, or a biochemical destabilization. One method that will be employed uses chemical crosslinkers to estimate the distance between mIg and Ig-alpha/Ig-beta in receptor destabilized cells. A second approach will employ fluorescence microscopy to address if the individual components of destabilized receptors segregate independently upon aggregation. In the second Specific Aim, the applicant will test the hypothesis that BCR destabilization occurs on the cell surface and is temporally correlated with B cell unresponsiveness and receptor desensitization, suggesting that BCR destabilization may be responsible for the desensitized phenotype. This will be assessed by creating a panel of chimeric receptors that are not susceptible to destabilization. These receptors will contain either the Ig-alpha or Ig- beta cytoplasmic tail fused to the mIg extracellular domain and an MHC class I transmembrane domain. The chimeric receptors will be stably transfected into a B cell line, and the chimeric receptors will be assayed for their ability to be desensitized. The applicant's long range goals are to continue to define the molecular basis of B cell receptor desensitization as a model for anergy by defining the molecular mechanism(s) responsible for BCR destabilization. The timing of BCR destabilization and the requirement for kinase activation suggest that a phosphorylation event on either mIg or Ig-alpha/Ig-beta may be responsible for this event. Therefore, her future plans, beyond the duration of the work described in this application, involve establishing if a novel phosphorylation event occurs coincident with receptor destabilization, identifying the targeted site, and confirming its role in receptor destabilization by creating a mutant receptor. Additional future studies include assessing the role of BCR destabilization in an immune response by generating a "knock-in" mouse harboring a mutation that prevents BCR destabilization. Finally, the applicant is interested in defining the endocytic pathways that mIg and Ig- alpha/Ig-beta follow when removed from the cell surface.