Structural and Functional Analyses of the Full-length Insulin Receptor (IR) and Type 1 Insulin-like Growth Factor Receptor (IGF1R) in the Liganded Active State

受体酪氨酸激酶（RTK）代表细胞表面受体家族。两种胰岛素 胰岛素样生长因子受体（IR）和1型胰岛素样生长因子受体（IGF 1 R）是II型RTK， 在控制葡萄糖代谢、细胞生长、增殖、分化和 迁移异常的IR或IGF 1 R信号传导导致许多人类疾病，包括 糖尿病和癌症，这使它们成为有前途的治疗靶点。尽管功能 重要性和与疾病的密切联系，确切的配体诱导的激活机制 IR和IGF 1 R的结构仍然是未知的，由于缺乏全长的高分辨率结构， 受体/配体复合物。该项目的目标是揭示配体（胰岛素和IGF 1， 分别）与这两种不同类型的受体结合，以及配体结合如何导致 受体激活尽管IR和IGF 1 R两者具有高序列同一性和结构同源性， 相似的是，它们已经显示出在配体结合特性方面表现出很大的多样性， 这表明了不同的激活机制。同时，我们的初步结构工作 显示完全配体化的IR二聚体在两种不同类型的胰岛素上与4个胰岛素分子结合。 位点-众所周知的位点1和我们新发现的位点2;然而，只有一个IGF 1分子 结合不对称的IGF 1 R二聚体，这意味着IGF 1与IGF 1 R的结合涉及负性结合。 协同性我们将结合联合收割机冷冻电子显微镜（cryo-EM），生物物理，生物化学和 基于细胞的方法来理解位点1和2胰岛素结合对IR激活的作用， 解释了IGF 1与IGF 1 R结合中负协同效应的起源，并揭示了IGF 1与IGF 1 R结合的负协同效应。 IR和IGF 1 R的跨膜结构域（TM）在受体活化中的功能。目标1将是 着重于活性状态下全长IR的功能和结构分析。这些研究 将揭示胰岛素与新IR位点2之间的详细结合模式，并确定胰岛素与新IR位点2之间的结合模式。 每种类型的胰岛素结合的关键作用。此外，我们将测试是否有不同的IR 许多结合的胰岛素表现出不同的活性水平，并触发不同的下游 发信号。目的2将集中在生物化学和全长IGF 1 R的结构分析， 活跃状态我们将确定负合作约束的结构要求， IGF 1对IGF 1 R的作用，并解释IGF 1 R的负协同作用的功能重要性 activation.目标3将侧重于直接可视化的TM-TM互动，在积极的 IR和IGF 1 R的状态，以了解为什么TM在IR和IGF 1 R的活性状态下二聚化 对受体激活很重要。

Receptor tyrosine kinases (RTKs) represent a family of cell surface receptors. Both of insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF1R) are type II RTKs that play essential roles in controlling glucose metabolism, cellular growth, proliferation, differentiation, and migration. Aberrant IR or IGF1R signaling causes a number of human diseases, including diabetes and cancers, which makes them promising therapeutic targets. Despite the functional importance and strong connections with diseases, the exact ligand induced activation mechanism of IR and IGF1R are still unknown, due to the lack of high resolution structures of full-length receptors/ligands complex. The goal of this project is to reveal how ligands (insulin and IGF1, respectively) binds to these two different types of receptors and how ligand binding leads to the receptor activation. Although both the IR and IGF1R share high sequence identity and structural similarity, they have been shown to exhibit great diversification in the ligand binding properties, which is indicative of diverse activation mechanism. Consistently, our preliminary structural work showed that the fully liganded IR dimer is bound by 4 insulin molecules at two distinct types of sites—the well-known site 1 and our newly discovered site 2; whereas, only one IGF1 molecule binds the asymmetric IGF1R dimer, meaning that the binding of IGF1 to IGF1R involves negative cooperativity. We will combine cryo-electron microscopy (cryo-EM), biophysical, biochemical and cellular based approaches to understand the roles of site 1 and 2 insulin binding for IR activation, explain the origin of the negative cooperativity in the binding of IGF1 to IGF1R, and reveal the function of transmembrane domains (TM) of IR and IGF1R in receptor activation. Aim 1 will be focused on the functional and structural analyses of full-length IR in the active state. These studies will reveal the detailed binding mode between insulin and the novel IR site 2, and identify the critical role of each type of insulin binding. In addition, we will test whether IRs with different number of insulins bound exhibit different levels of activities and trigger distinct downstream signaling. Aim 2 will be focused on biochemical and structural analyses of full-length IGF1R in the active state. We will identify the structural requirements for the negative cooperative binding of IGF1 to IGF1R, and explain the functional importance of the negative cooperative for IGF1R activation. Aim 3 will be focused on the direct visualization of the TM-TM interaction in the active state of IR and IGF1R to understand why the TM dimerization in the active state of IR and IGF1R is important for receptor activation.