Mutational and biochemical analysis of Fgd2 and Fgd3

描述（由申请人提供）：这是一份修订后的R01提案，旨在解决两种新型白细胞限制性信号分子的生物化学和功能。Fgd2和Fgd3是Ras同源GTPases （Rho）的鸟嘌呤核苷酸交换因子（GEF）。它们与已知的Cdc42 gef具有显著的序列一致性，并且是gef的Dbl同源家族的成员。Fgd亚家族包括6个成员，其特点是包含在其他系统中促进磷脂结合和囊泡靶向的FYVE结构域。rhogef在受体信号传导、囊泡运输、细胞骨架调节、迁移和细胞生长调节等方面发挥着重要作用。在我们的工作之前，Fgd2没有被表征为一种蛋白质。Fgd2在B细胞和巨噬细胞中高水平表达。在B细胞中，Fgd2的表达受抗原受体信号的调控，并在记忆B细胞中表达。在某些情况下，Fgd2似乎与一个不同的家族成员Fgd3相互调节。我们最近产生了Fgd2的条件敲除。本文通过以下三个具体目标来阐述Fgd2和Fgd3的作用：1)在基因敲除和过表达的背景下，描述Fgd2的生物学功能。2)表征Fgd2及相关分子Fgd3的生化特性和特异性。将进行结构/功能分析，以确定单个基序的作用，产生突变形式，并评估Fgd2翻译后修饰和相互作用蛋白。3)生成并分析Fgd3缺陷小鼠，探讨Fgd3可能补偿Fgd2功能的问题。这些研究的长期目标是了解B细胞的发育和抗体反应是如何在分子水平上被调节的。

DESCRIPTION (provided by applicant): This is a revised R01 proposal to address the biochemistry and function of two novel, leukocyte restricted signaling molecules. Fgd2 and Fgd3 are guanine nucleotide exchange factors (GEF) for the Ras homology (Rho) GTPases. They have significant sequence identity with known Cdc42 GEFs and are members of the Dbl homology family of GEFs. The Fgd subfamily includes 6 members and is distinguished by inclusion of FYVE domains that in other systems promote phospholipid binding and vesicular targeting. RhoGEFs play a number of crucial roles in receptor signaling, vesicle trafficking, cytoskeletal regulation, migration and the regulation of cell growth. Prior to our work, Fgd2 had not been characterized as a protein. Fgd2 is expressed at high levels in B cells and in macrophages. In B cells, Fgd2 expression is regulated by antigen receptor signaling and is expressed in memory B cells. In some contexts Fgd2 appears to be regulated reciprocally with a distinct family member, Fgd3. We have recently generated a conditional knockout of Fgd2. In this proposal the roles of Fgd2 and Fgd3 are addressed through the following three Specific Aims. 1) To characterize the biological functions of Fgd2 in the contexts of genetic knockouts and overexpression. 2) To characterize biochemical properties and specificities of Fgd2 and the related molecule Fgd3. A structure/function analysis will be carried out to determine the roles of individual motifs, to generate mutant forms, and to assess Fgd2 posttranslational modifications and interacting proteins. 3) To generate and analyze Fgd3 deficient mice and to address the issue of possible compensation of Fgd2 function by Fgd3. The long term goal of these studies is to understand how B cell development and antibody responses are regulated at the molecular level.