BIOLOGICAL FUNCTION OF B CELL RECEPTOR DESTABILIZATION

描述（改编自申请人的摘要）： B细胞抗原受体（BCR）有助于免疫应答， 导致增殖或分化。免疫反应失调 由于癌蛋白转化或不能区分自身与外来 蛋白质可导致淋巴增生性疾病和自身免疫性疾病。的 候选人的兴趣是定义“沉默”自我的分子机制， 特异性B淋巴细胞。 Vilen博士的近期职业目标是进一步 描述了她最近观察到的B细胞抗原 受体导致BCR复合物的不稳定，如由 降低免疫共沉淀mIg和Ig-α/Ig-β的能力。第一 具体目的是定义BCR复合物完整性的变化 在抗原诱导的脱敏之后。 这将有助于评估 BCR失稳是否代表mIG与IG的物理解离- α和β免疫球蛋白，或生化不稳定。 一种方法是 采用化学交联剂来估计mIg和 受体不稳定细胞中的Ig-α/Ig-β。 第二种方法将 采用荧光显微镜来解决，如果个别成分的 不稳定的受体在聚集时独立地分离。 在第二个具体目标中，申请人将检验BCR 不稳定发生在细胞表面，并与B时间相关 细胞无反应性和受体脱敏，表明BCR 去稳定化可能是脱敏表型的原因。 这将 通过创建一组嵌合受体来评估， 不稳定。 这些受体将含有Ig-α或IG-α。 融合至mIg胞外结构域和MHC I类的β胞质尾 跨膜结构域 嵌合受体将被稳定地转染到 B细胞系，并测定嵌合受体的能力， 变得麻木。 申请人的长期目标是继续确定分子基础 B细胞受体脱敏作为无反应性的模型， 导致BCR不稳定的分子机制。 BCR的时间 不稳定和激酶激活的需要表明， mIg或Ig-α/Ig-β上的磷酸化事件可能是导致 大会及其他人 因此，她未来的计划，超出了工作的持续时间， 本申请中所述的方法，包括建立一种新的磷酸化， 事件与受体不稳定同时发生，确定 靶向位点，并证实其在受体不稳定中的作用， 一种突变受体 未来的其他研究包括评估 通过产生“基因敲入”小鼠在免疫应答中的BCR不稳定 携带阻止BCR不稳定的突变。 最后 申请人感兴趣的是确定mIG和IG- 当从细胞表面去除时，α/Ig-β随之而来。

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.