Control of Calcium Entry Signals in B Cells

描述(由申请人提供)：B细胞中的钙信号在调节B细胞抗原受体(BCR)和激活B细胞反应之间的偶联过程中起着至关重要的作用。钙离子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细胞钙离子进入信号是如何控制的；(B)以B细胞为模型系统，了解钙离子进入的分子组成。这项工作结合了分子、遗传和生理方法，使用了鸡DT40B细胞系，通过靶向基因缺失，提供了一个系统来剖析BCR诱导的钙信号通路。利用这一系统，研究试图回答以下具体问题： 1.B细胞受体复合体的组成成分是如何控制钙内流信号的？BCR复合体是一个有组织的接头/效应器蛋白质阵列，用于转导BCR结合信号。需要检验的假设是，钙离子进入信号是由B细胞受体复合体的下游效应蛋白和接头蛋白控制的。研究集中在三类BCR偶联分子：(A)PLC-Gamma2，(B)BCR偶联接头蛋白BLNK和Bam32，以及(C)BCR偶联酪氨酸激酶。这项工作使用了一系列克隆的DT40B细胞系，其中每个BCR偶联效应/接头蛋白的基因都被删除了，并试图通过测量定义内源性钙进入途径的一系列钙信号参数来检查这些蛋白的作用。 2.色氨酸蛋白家族的成员能否在B细胞中发挥钙离子内流的调节作用？色氨酸通道广泛表达于包括B细胞在内的细胞中，但其生理激活尚不清楚。需要检验的假设是，Trp通道可以偶联到B细胞受体复合体的下游效应器和接头蛋白，并受其控制。使用DT40模型细胞系统，这些研究包括受控表达和抑制表达系统的组合，利用这些系统可以监测激活Trp通道的偶联过程。这些研究考察了TRPC3和TRPC4通道如何被两种关键信号蛋白-InsP3受体和磷脂酶C-伽马酶激活并与其分子相互作用的细节。 这些研究提供了一种新的BCR诱导的质膜偶联机制来控制B细胞钙信号，这是一个潜在的重要药理靶点。BCR诱导的钙信号是B细胞发育命运的主要决定因素，也就是说，它们是否增殖，是否保持无能，或者是否由于自我认识的结果，它们经历了凋亡的消除。这种钙离子进入调控过程的定义提供了一个关键的靶点，通过它可以改变B细胞的功能和发育，从而有可能控制主要的免疫疾病，包括原发的B细胞缺陷和淋巴增生性疾病。

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.