Architectural Basis of Leptin Transmembrane Signaling

描述（由申请人提供）：瘦素（L）及其受体（L-R）是调节能量稳态和体重的关键参与者。瘦素和L-R的细胞外部分之间的复合物形成导致Janus激酶2（JAK 2）的激活，JAK 2组成性地结合在受体的细胞内区域上。下丘脑核中瘦素刺激的JAK 2信号传导减少食物摄入并刺激能量消耗，而瘦素或L-R的功能缺陷导致病态肥胖、高血糖症、胰岛素敏感性降低和高脂血症。尽管瘦素系统对体重和其他生理反应有重要影响，但对L/L-R复合物的结构及其与JAK 2的关系知之甚少。这种缺乏洞察力的原因之一是L-R和JAK 2都具有相对较长且灵活的多结构域排列，这对于大规模纯化和X射线晶体学的实施都非常具有挑战性。本发明旨在通过应用单粒子低温电子显微镜（cryo-EM）来表征全长L/L-R和JAK 2的全复合物，克服这些限制，以解决L-R信号传导的结构先决条件。单粒子EM已经成为一个非常强大的工具，动态蛋白质组装的特点在相对较小的浓度，而不需要结晶。我们预计，单粒子EM技术在这个系统上的应用将揭示的L/L-R组件和JAK 2独立的，复杂的体系结构。考虑到这种膜定位信号复合物在肥胖、能量代谢和心脏病中的潜在重要性，所获得的结构结果将具有非常广泛的生物医学意义。考虑到目前缺乏任何受体/JAK复合物的结构信息，我们的研究将提供一般的架构框架，了解细胞外配体结合细胞因子受体如何导致细胞内JAK激活。

DESCRIPTION (provided by applicant): Leptin (L) and its receptor (L-R) are key players in the regulation of energy homeostasis and body weight. Complex formation between leptin and the extracellular portion of L-R results in the activation of Janus kinase 2 (JAK2) that is constitutively bound on the intracellular regions of the receptor. Leptin-instigated JAK2 signaling in hypothalamic nuclei reduces food intake and stimulates energy expenditure, while functional defects in either leptin or L-R result in morbid obesity, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. Despite the crucial impact of the leptin system on body weight and other physiological responses, little is known about the structure of the L/L-R complex and its association with JAK2. One of the reasons for this lack of insight is that both L-R and JAK2 have a relatively long and flexible multi-domain arrangement that has proved to be very challenging for both large-scale purification and implementation of X-ray crystallography. The present proposal aims to overcome these limitations in addressing the architectural prerequisites of L-R signaling by applying single- particle cryo-electron microscopy (cryo-EM) to characterize the holo-complex of full-length L/L-R and JAK2. Single-particle EM has emerged as a very powerful tool for the characterization of dynamic protein assemblies in relatively small concentrations and without the need for crystallization. We anticipate that the application of single-particle EM techniques on this system will reveal the architecture of the L/L-R assembly and JAK2 independently, and in complex. Given the underlying importance of this membrane-localized signaling complex in obesity, energy metabolism, and heart disease, the structural results obtained will be of very broad biomedical interest. Considering the current lack of structural information on any receptor/JAK complex, our studies will provide the general architectural framework for understanding how extracellular ligand binding on cytokine receptors results in intracellular JAK activation.