Role for Vav Family Proteins in T Lymphocyte Activation

描述（由申请人提供）：Vav家族蛋白被认为作为分子衔接子和鸟嘌呤-核苷酸交换因子发挥作用，协调淋巴细胞中抗原受体下游的信号传导。以前的努力，以阐明Vav功能的淋巴细胞发育和活化在体内受到阻碍的冗余之间的Vav家族，其中包括3个高度同源的蛋白质。作为我们对这一提议的初步研究的一部分，我们首次产生并表征了缺乏所有3种Vav蛋白的小鼠，这使我们能够证明整个Vav家族的基本功能，并最终确定淋巴谱系中单个同种型之间的功能冗余限制。 然而，Vav功能的确切机制仍然难以捉摸。为了便于Vav功能的生化分析，在目的1中，我们提出使用经工程化以表达鼠α/β TCR、3.L2和鼠CD 4的Vav缺陷型抗原特异性Jurkat T细胞。由于3.L2 TCR-配体是共价连接至分级效力的抗原肽的可溶性I-Ek MHC分子，因此该系统允许抗原活化的微妙性（通过改变的肽配体），而且当使用转化的组织培养细胞系时，生物化学分析的便利性是可能的。使用J.3.L2系统，我们将确定大量Vav突变体在源自TCR的多个信号传导通路中的作用。这些Vav突变体的动机的结构特征，隐含地纳入几个不同的假设Vav机制。 在目的2中，我们提出使用一种新的Vav空造血干细胞互补（Vav nulI-HSCC）检测开发的突变体Vav蛋白在T细胞发育和活化的影响的快速分析。使用该测定，我们将确定体内T淋巴细胞中Vav蛋白功能的结构基础。基于这些实验的结果，将使用Vav外显子置换（敲入）将选择的Vav突变体引入鼠生殖系中。 在目标3中，我们将通过进行复杂的分析来研究围绕体内Vav功能调节的主要未回答的问题，这将建立在通过目标1和2中建立的方法检查的大量Vav突变体的基础上。

DESCRIPTION (provided by applicant): The Vav family proteins are thought to function as molecular adaptors and guanine-nucleotide exchange factors orchestrating signaling downstream of antigen receptors in lymphocytes. Previous efforts to elucidate Vav function in lymphocyte development and activation in vivo have been hampered by the redundancy among individual members of the Vav family, which comprises 3 highly homologous proteins. As a part of our preliminary studies for this proposal we generated and characterized for the first time mice lacking all 3 Vav proteins which allowed us to demonstrate the essential function of the entire Vav family as well as to conclusively define the limits of functional redundancy among the individual isoforms in the lymphoid lineage. However, the exact mechanism of Vav function remains elusive. To facilitate biochemical analyses of Vav function, in Aim 1 we propose to use Vav-deficient antigen-specific Jurkat T cells engineered to express a murine alpha/beta TCR, the 3.L2, and a murine CD4. Since 3.L2 TCR-ligands are soluble I-Ek MHC molecules covalently linked to antigenic peptides of graded potency, this system allows both the subtlety of antigen activation (by altered peptide ligands) but also the convenience of biochemical analysis possible when using a transformed tissue culture cell line. Using J.3.L2 system we will determine the effects of a large panel of Vav mutants in multiple signaling pathways emanating from the TCR. These Vav mutants were motivated by structural features which implicitly incorporate several distinct hypotheses about Vav mechanism. In Aim 2, we propose to use a novel Vav null-hematopoietic stem cell complementation (Vav nulI-HSCC)assay developed for rapid analyses of the effects of mutant Vav proteins in T cell development and activation. Using this assay, we will determine the structural basis for Vav protein function in T lymphocytes in vivo. Based on results of these experiments, selected Vav mutants will be introduced into murine germ line using Vav exon-replacement (knock-in). In Aim 3 we will examine the major unanswered questions surrounding the regulation of Vav function in vivo by carrying out complex analyses, which will build on the strength of a large panel of Vav mutants examined by approaches established in Aims 1 and 2.