Role of USP Ligand Binding Pocket During Development

USP 配体结合袋在开发过程中的作用

• 批准号: 6964831
• 负责人: GRACE M JONES
• 金额: $ 30万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6964831
• 关键词: Aedes; Drosophilidae; ecdysone; genetically modified animals; histogenesis; hormone binding protein; hormone receptor; hormone regulation /control mechanism; invertebrate endocrinology; invertebrate hormone; ligands; metamorphosis; molting; nuclear receptors; receptor binding; receptor expression; tissue /cell culture

DESCRIPTION (provided by applicant): A major unresolved question in insect endocrinology is whether the nuclear receptor ultraspiracle (USP) has an active, ligand-binding function or, alternatively, is a non-liganded, even passive, heterodimer partner. Our previous NIH-funded protein biochemical studies emphasized screening of many point mutants to the ligand-binding domain of the Drosophila USP. The specific aims were to identify optimal mutant USPs for future testing of an in vivo USP ligand binding function of 2 transgenic fly assays. Our studies, supported by recent studies by others, were successful in demonstrating in cultured cell assays that the USP of Drosophila (flies) and Aedes (mosquitoes) can transduce transcriptional signaling by methyl epoxyfarnesoate, a natural juvenile hormone (JH) in flies and mosquitoes. The studies also show that this intracellular JH signaling requires a surface region on USP corresponding to a ligand-activated co-activator binding surface of the vertebrate counterpart, RXR. Using the cultured cell functional assay, we were successful in preparing point mutants to the USP ligand-binding pocket and co-activator binding surface that act as dominant negatives to block USP-dependent JH signaling in the cultured cells. On this basis, the recent NIH study section panel summary directed that our next proposal move past the protein biochemical studies by using "the mutants already in hand rather than looking for more mutants" and to now orient the studies "into a direct biological context". Therefore, in this new proposal, we take these Drosophila USP mutants (and corresponding Aedes USP mutants) will be used in the transgenic fly assays in order to ask the following questions: 1) Is the USP ligand-binding pocket needed for USP function in JH/ecdysone-driven larval-to-larval molting? 2) Does the USP ligand-binding pocket transduce JH effects on pupal-adult metamorphosis? These in vivo studies are the next logical step, building upon protein biochemical and cultured-cell assays, and are insect endocirinology's first direct in vivo tests of a ligand-binding function for any USP. The outcome will have broad implications for insect endocrinology, enabling specific tests on the Aedes USP, the results of which will yield new insights on hormonal signaling in the technically more difficult mosquito.

描述（由申请人提供）：昆虫内分泌学中一个主要的未解决的问题是核受体超吸体（USP）是否具有主动的配体结合功能，或者是一个非配体的，甚至是被动的异二聚体伴侣。我们之前的nih资助的蛋白质生化研究强调筛选果蝇USP的配体结合结构域的许多点突变体。具体目的是确定最佳突变体USP，以便将来测试2种转基因苍蝇的体内USP配体结合功能。我们的研究得到了其他人最近研究的支持，成功地在培养细胞试验中证明了果蝇（苍蝇）和伊蚊（蚊子）的USP可以通过苍蝇和蚊子的天然幼体激素（JH）环氧法尼酸甲酯转导转录信号。研究还表明，这种细胞内JH信号需要USP上的一个表面区域，该表面区域对应于脊椎动物对应物RXR的配体激活的共激活物结合表面。利用培养细胞功能试验，我们成功地制备了USP配体结合袋和共激活剂结合表面的点突变体，这些突变体作为显性阴性体，阻断了培养细胞中USP依赖性JH信号传导。在此基础上，最近的NIH研究小组总结指示我们的下一个建议超越蛋白质生化研究