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 （JAK2）的激活，JAK2组成性地结合在受体的细胞内区域。瘦素引发的下丘脑核JAK2信号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、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，但人们对L/L- r复合物的结构及其与JAK2的关系知之甚少。这种缺乏洞察力的原因之一是，L-R和JAK2都具有相对较长且灵活的多畴排列，这对于大规模纯化和x射线晶体学的实施都是非常具有挑战性的。本提案旨在通过应用单粒子低温电子显微镜（cryo-EM）表征全长L/L- r和JAK2的全息配合物，克服这些限制，解决L- r信号的结构先决条件。单颗粒电子显微镜已经成为一种非常强大的工具，可以在相对较小的浓度下，在不需要结晶的情况下，表征动态蛋白质组装。我们期望单粒子EM技术在该系统上的应用将揭示L/L- r组件和JAK2的独立和复杂的结构。鉴于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，我们的研究将为理解细胞外配体结合细胞因子受体如何导致细胞内JAK激活提供一般的结构框架。

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.