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这种复合体的分子信号特征的理解。胰岛素和胰岛素样生长因子1和2(IGF1/2)是人类关键的蛋白质荷尔蒙，具有共同的来源和相似的三维结构。尽管它们的结构相似，但它们调节着广泛的生理事件，其中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IGFS分别通过与其高度同源的约450 kDa同源二聚体酪氨酸激酶受体(RTKs)：胰岛素受体(HIR)和IGF-1R结合来发挥作用。这些激素之间密切的分子和生理联系位于胰岛素-胰岛素样生长因子信号轴(IIS)后面，IIS负责生长、寿命、糖尿病、癌症和神经退化的动态平衡调节和病理。IIS是动物界中最保守的信号轴之一，显示了从息肉到人类的相似同源激素、受体和信号节点的共性。最近，人们描述了几种全长HIR的低温电子显微镜结构。然而，他们显示了各种可能的和令人费解的不同的胰岛素：HIR化学计量学(1到4个胰岛素/HIR)，并且由于受体的流动性和被IR稳定的脂类遮挡，受体TK部分的结构变化也没有被揭示。为此，我们开展了与昆虫hir/IGF-1R高度同源的研究工作，发现了在昆虫中发挥作用的只有果蝇的IR(DmIR)。拟议研究结果的主要好处还来自dmIR 60 kDa/亚单位TK结构域扩展(CTD)，它部分与HIR第一个下游磷酸化蛋白底物IRS1同源。因此，这为将IR的工作扩展到IR提供了一个独特的机会：IRS1相互作用，以及它与PI3K的P85/SH2结构域的后续关联：PI3K是基于IR的信号转导的下一个关键中介。DmIR与CHICO蛋白(人IRS1-4的DM同源)的复合体也可以被研究，因为dmIR似乎以两种不同的方式结合下游的PI3K效应器：一种是通过CTD-dmIR内部的IRS1样模块，另一种是通过dmIR近膜片段上的CHICO对接位点。本研究的主要目的是解决我们对胰岛素与胰岛素受体生理结合的理解，并确定这种复合体的分子信号特征。我们将在这里使用果蝇胰岛素受体(DmIR)，利用它与人胰岛素受体(HIR)的高度同源性，并利用其巨大的细胞内延伸，这与hIR的第一个细胞内底物强烈对应。因此，dmIR为冷冻EM和功能研究提供了一个独特的机会来研究整个激素：受体：效应器分子信号机制，这里称为IR信号体。目的：1.提供对生理性胰岛素的独立认识：IR化学计量学和IR胞外结构域的激活构象(ECD-dmIR)(已有的高级低温EM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EM研究)3.深入了解IR与其下游信号伙伴(dmIR及其PI3K和ChICO信号伙伴片段的低温EM)相互作用和激活的性质。通过对观察到的1-3个关键蛋白的突变进行转基因和基因置换，对结构发现进行功能验证，并对下游复合体进行串联亲和纯化。通过将1-4的发现转化为基于人HIR的IIS系统，概述基于IR的信号体中的分子组装和信号传递步骤。

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.