Revealing Molecular Bases of Signal Transduction through the Drosophila Insulin Receptor: cryoEM and Functional Studies.

我们有机会解决我们对胰岛素与胰岛素受体的生理结合以及由此产生的分子信号信号的理解。胰岛素和胰岛素样生长因子1和2 （IGF1/2）是关键的人类蛋白质激素，具有共同的起源和类似的三维组织。尽管结构相似，但它们调节着广泛的生理事件，胰岛素负责广泛的代谢控制和衰老，而IGF1/2是生长因子，也参与调节寿命和生长。胰岛素和igf通过结合其高度同源的约450 kDa的同型二聚体酪氨酸激酶受体（RTKs）：胰岛素受体（hIR）和IGF-1R来发挥其活性。这些激素之间密切的分子和生理联系隐藏在胰岛素- igf信号轴（IIS）背后，负责生长、寿命、糖尿病、癌症和神经变性的稳态调节和病理。IIS是动物王国中最保守的信号轴之一，显示了息肉与人类相似、同源的激素、受体和信号节点的共性。近年来，研究人员描述了几种低温电镜结构的全长hIR。然而，他们显示了各种可能的，令人困惑的，不同的胰岛素：hIR化学计量（1到4个胰岛素/hIR），并且受体TK部分的结构变化也未被揭示，因为它的流动性和被ir稳定脂质遮挡。因此，我们开始研究与黑腹果蝇（Drosophila melanogaster, Dm）高度同源的hIR/IGF-1R仅在昆虫中发挥作用的IR （dmIR）。提出的研究结果的主要好处还来自dmIR 60kDa/亚基tk结构域扩展（CTD），其部分同源于IRS1 - hIR第一下游磷酸化蛋白底物。因此，它提供了一个独特的机会，将IR的工作扩展到IR:IRS1的相互作用，及其随后与PI3K的p85/SH2结构域的关联，PI3K是基于IR的信号转导的下一个关键介质。也可以研究dmIR与CHICO蛋白（人类IRS1-4的Dm同源物）的复合物，因为似乎dmIR以两种不同的方式结合下游PI3K效应物：一种是通过CTD - dmIR-内部irs1样模块，另一种是通过dmIR近膜段的CHICO对接位点。本提案的主要目的是解决我们对胰岛素与胰岛素受体的生理结合的理解，并定义该复合物的分子信号特征。我们将利用果蝇胰岛素受体（dmIR），利用其与人类胰岛素受体（hIR）的高度同源性，并利用其与hIR的第一细胞内底物强烈对应的大细胞内延伸。因此，dmIR为冷冻电镜和功能研究提供了一个独特的机会，可以研究整个激素：受体：效应分子信号机制，这里称为IR信号体。目的:1。提供对生理胰岛素的独立见解：IR化学计量学和IR外结构域（ecd-dmIR）的激活构象（已经开展的冷冻电镜工作）。阐明全长IR从无激素状态到激活状态的结构转变（低温电镜研究）为了深入了解IR与其下游信号伙伴的第一个细胞内成分相互作用的性质和激活（dmIR及其PI3K和CHICO信号伙伴片段的低温em）。通过对观察到的1-3个关键蛋白：蛋白界面进行突变的转基因和基因替代蝇，并对下游复合物进行串联亲和纯化，对结构发现进行功能验证。通过将1-4的发现转化为人类基于ir的IIS系统，概述基于ir的信号体中的分子组装和信号传导步骤。

We have the opportunity to resolve our understanding of the physiological binding of insulin to the insulin receptor and the resultant molecular signalling signatures of this complex. Insulin and Insulin-like Growth Factors 1 and 2 (IGF1/2) are the key human protein hormones with a common origin and similar 3-D organisation. Despite similar structures they regulate a wide spectrum of physiological events, with insulin being responsible for broad metabolic control and aging, while IGF1/2 are growth factors involved also in regulation of life span and growth. Insulin and IGFs exert their activities by binding to their highly homologous ca. ~450 kDa homo-dimeric tyrosine-kinase receptors (RTKs): Insulin Receptor (hIR) and IGF-1R, respectively. The close molecular and physiological links between these hormones lie behind the insulin-IGF signalling axis (IIS) responsible for the homeostatic regulations and pathologies in growth, life span, diabetes, cancer and neurodegeneration. IIS is one of the most conserved signalling axis in the animal kingdom showing commonality of similar, orthologues hormones, receptors and signalling nodes from polyps to humans. Recently, several cryoEM structures of the full length hIR were described. However, they showed a variety of possible, and puzzling, different insulin:hIR stoichiometries (one to four insulins/hIR), and the structural changes in the TK part of the receptor were also not revealed due to its mobility and occlusion by the IR-stabilising lipids. Hence we initiated work on highly homologous to hIR/IGF-1R insect Drosophila melanogaster (Dm) only IR (dmIR) which takes their roles in insects. The key benefits of the proposed research results also from dmIR 60kDa/subunit TK-domain extension (CTD) that is in part homologous to IRS1 - the hIR first downstream phosphorylation protein substrate. Therefore it presents a unique opportunity to expand IR work into IR:IRS1 interaction, and its subsequent association with p85/SH2 domain of PI3K: the next key mediator of IR-based signal transduction. The dmIR complex with CHICO protein (Dm orthologue of human IRS1-4) can be studied as well, as it seems that dmIR binds the downstream PI3K effector in two different ways: one by the CTD - dmIR- internal IRS1-like module, and the other one through the CHICO docking sites in the dmIR juxtamembrane-segment.The main Aim of this proposal is to resolve our understanding of the physiological binding of insulin to the insulin receptor and define the molecular signalling signatures of this complex. We will use here Drosophila insulin receptor (dmIR), exploit its high homology to the human IR (hIR), and exploit its large intracellular extension which corresponds strongly to the first intracellular substrate of the hIR. Therefore dmIR presents an unique opportunity for the cryoEM and functional studies to study the whole of the hormone:receptor:effector molecular signalling machinery referred to here as the IR signalosome. Objectives:1. To provide an independent insight into physiological insulin:IR stoichiometry and the activated conformation of the IR ectodomain (ecd-dmIR)(already advanced cryoEM work).2. To elucidate the structural transitions of the full-length IR from its hormone-free to activated state (cryoEM studies)3. To provide an insight into the nature of the IR interaction with, and activation of the first intra-cellular components of its-downstream signalling partners (cryoEM of dmIR with fragments of its PI3K and CHICO signalling partners).4. To undertake the functional validation of the structural findings through making transgenic and gene replacement flies with mutations on the observed in 1-3 key protein:protein interfaces, and tandem-affinity purification of the downstream complexes.5. To outline the molecular assembly and signalling steps in the IR-based signalosome by translating findings of 1-4 to the human hIR-based IIS system.