Regulation of insulin-like growth factors by the ER chaperone GRP94

描述（由申请人提供）：胰岛素样生长因子（IGFs）介导胚胎发育、细胞生长信号传导和代谢应激反应过程中的分化决定。我们发现这些重要生长因子的产生依赖于内质网伴侣葡萄糖调节蛋白94 （GRP94）的活性，而GRP94对它们的生物合成至关重要。GRP94与其他分子伴侣的不同之处在于它只对少数客户蛋白有选择性，对晚期折叠中间体有偏好，并且没有任何已知的辅助因子。所有这些方面都表明GRP94具有独特的作用模式。本项目将利用依赖GRP94- igf轴生长的细胞来阐明GRP94的神秘作用周期。在我们的第一个目标中，我们将使用一种新的基于细胞的检测方法来筛选不支持IGF-II产生的GRP94突变体，从而确定对这种伴侣蛋白功能至关重要的氨基酸。在Aim 2中，我们将确定这些突变体在一般机制方面是否存在缺陷，例如ATP结合和水解或钙结合，或者突变是否定义了客户端结合域。我们还将测试GRP94突变体支持除IGF以外的其他客户端的能力，以发现是否存在特定于客户端的突变体。我们的第三个目标是通过利用一组胰岛素样蛋白之间的结构相似性和差异性来了解GRP94如何识别IGF。这些研究将告知GRP94的客户选择性，目前尚不清楚。最后，我们将利用IGF缺乏的临床结果来寻找功能重要的人类GRP94变异。根据我们的发现，至少有一种这样的变异会影响IGF的产生，我们将对IGF缺陷患者群体进行基因分型和测序，以寻找可能解释低IGF产生的等位基因GRP94变异。总之，这些结果将导致对GRP94如何陪伴客户蛋白的新理解。

DESCRIPTION (provided by applicant): Insulin-like growth factors (IGFs) mediate differentiation decisions during embryonic development, cell growth signaling and responses to metabolic stress. We found that the production of these important growth factors is dependent on the activity of the endoplasmic reticulum chaperone glucose regulate protein 94 (GRP94), which is essential for their biosynthesis. GRP94 is different from other molecular chaperones in its selectivity towards only a small number of client proteins, preference for late folding intermediates and the absence of any known co-factors. All these aspects suggest a unique mode of action for GRP94. This project will elucidate the enigmatic action cycle of GRP94 by taking advantage of cells whose growth depends on the GRP94-IGF axis. In our first aim we will use a novel cell-based assay to screen for mutants of GRP94 that do not support IGF-II production and thus define amino acids that are essential for this chaperone function. In Aim 2, we will determine whether these mutants are defective in general mechanistic aspects, such as ATP binding and hydrolysis or calcium binding, or whether the mutations define a client binding domain. We will also test the ability of GRP94 mutants to support clients other than IGF, to discover whether there are client-specific mutants. Our third aim is to understand how GRP94 recognizes IGF, by exploiting the structural similarities and differences among a set of insulin-like proteins. These studies will inform on the client selectivity of GRP94, which is currently not understood. Finally, we will take advantage of the clinical consequences of IGF deficiency to search for functionally important human GRP94 variants. Following on our finding that at least one such variant affects IGF production, we will genotype and sequence IGF-deficient patient populations to seek allelic GRP94 variants that may explain low IGF production. Together, these results will lead to a new understanding of how GRP94 chaperones client proteins. PUBLIC HEALTH RELEVANCE: This project aims to decipher how GRP94, representing the family of HSP90 chaperones, selects its client proteins and affects their proper production. The work will use a novel cell-based assay, with insulin-like growth factor as the client, to determine the functionality and characterize biochemically both natural and site-directed mutations in GRP94.