Control of Calcium Entry Signals in B Cells

描述（由申请方提供）：B细胞中的Ca 2+信号在介导B细胞抗原受体（BCR）和B细胞应答激活之间的偶联中至关重要。Ca 2+进入信号的大小和持续时间是B细胞对抗原诱导的BCR交联的发育反应的主要决定因素。该项目有两个基本目标：（a）研究B细胞Ca 2+内流信号是如何控制的，以及（B）使用B细胞作为模型系统来理解Ca 2+内流的分子组成。这项工作需要使用鸡DT 40 B细胞系的分子、遗传和生理方法的组合，该细胞系通过靶向基因缺失提供了一个系统来剖析BCR诱导的Ca信号通路。利用这一系统，这些研究试图回答以下具体问题： 1. B细胞受体复合物的成分如何控制Ca 2+进入信号？BCR复合物是衔接子/效应子蛋白质的有序阵列，用于阻断BCR结合信号。有待检验的假设是Ca 2+进入信号受B细胞受体复合物的下游效应蛋白和接头蛋白的控制。研究集中于三类BCR偶联分子：（a）PLC-γ 2，（B）BCR偶联衔接蛋白BLNK和Bam 32，以及（c）BCR偶联酪氨酸激酶。这项工作使用了一系列的克隆DT 40 B细胞系，其中每个BCR偶联效应/衔接蛋白的基因已被删除，并试图检查这些蛋白质的作用，通过测量光谱的Ca 2+信号参数定义的内源性Ca 2+进入途径。 2. TRP蛋白家族成员能否在B细胞中作为Ca 2+进入介质发挥作用？TRP通道在细胞中广泛表达，包括B细胞，但其生理激活尚未解决。待检验的假设是TRP通道可偶联至B细胞受体复合物的下游效应子和衔接子蛋白并受其控制。使用DT 40模型细胞系统，该研究包括控制表达和抑制表达系统的组合，其中可以监测激活TRP通道的偶联过程。这些研究详细研究了TRPC 3和TRPC 4通道如何被两种关键信号蛋白-InsP 3受体和磷脂酶C-γ酶激活并与其分子相互作用。 这些研究提供了一种新的BCR诱导的质膜偶联机制，以控制B细胞Ca 2+信号，一个潜在的重要药理学目标的信息。BCR诱导的Ca 2+信号是B细胞发育命运的主要决定因素，即它们是否增殖，它们是否保持无反应性，或者作为自我识别的结果，它们是否通过凋亡而被消除。这种Ca 2+进入控制过程的定义提供了一个关键目标，通过该目标可以改变B细胞的功能和发育，从而提供控制主要免疫性疾病（包括原发性B细胞缺陷和淋巴增生性疾病）的潜力。

DESCRIPTION (provided by applicant): Ca2+ signals in B cells are crucial in mediating the coupling between the B cell antigen receptor (BCR) and activation of B cell responses. The size and duration of Ca2+ entry signals is a primary determinant of the developmental response of B cells to antigen-induced crosslinking of the BCR. The project has two fundamental objectives: (a) to study how B cell Ca2+ entry signals are controlled, and (b) to use B cells as a model system to understand the molecular components of Ca2+ entry. The work entails a combination of molecular, genetic, and physiological approaches using the chicken DT40 B cell line which, through targeted gene deletion, provides a system to dissect the BCR-induced Ca -signaling pathway. Using this system, the studies seek to answer the following specific questions: 1. How do components of the B cell receptor complex control Ca 2+ entry signals? The BCR complex is an organized array of adaptor/effector proteins serving to transduce the BCR-binding signal. The hypothesis to be tested is that Ca 2+ entry signals are controlled by the downstream effector and adaptor proteins of the B cell receptor complex. Studies focus on three classes of BCR-coupled molecules: (a) PLC-gamma2, (b) the BCR-coupled adaptor proteins BLNK and Bam32, and (c) BCR-coupled tyrosine kinases. The work uses a series of clonal DT40 B cell lines in which genes for each BCR-coupled effector/adaptor protein have been deleted, and seeks to examine the role of these proteins by measuring a spectrum of Ca2+ signaling parameters defining the endogenous Ca 2+ entry pathways. 2. Can members of the TRP family of proteins function as Ca2+ entry mediators in B cells? TRP channels are widely expressed in cells, including B cells, but their physiological activation is unresolved. The hypothesis to be tested is that TRP channels can couple to and be controlled by the downstream effector and adaptor proteins of the B cell receptor complex. Using the DT40 model cell system, the studies comprise a combination of controlled expression and repressed expression systems with which the coupling processes that activate TRP channels can be monitored. The studies examine details of how the TRPC3 and TRPC4 channels are activated by and molecularly interact with two key signaling proteins - the InsP3 receptor and the phospholipase C-gamma enzyme. The studies provide information on a novel BCR-induced coupling mechanism in the plasma membrane to control B cell Ca2+ signals, a potentially important pharmacological target. BCR-induced Ca 2+ signals are the primary determinants of the developmental fate of B cells, that is, whether they proliferate, whether they remain anergic, or whether as a result of self-recognition they undergo elimination by apoptosis. Definition of this Ca 2+ entry control process provides a key target through which B cell function and development can be modified providing the potential to control major immunological diseases including primary B cell deficiencies and lymphoproliferative disorders.