Architectural Basis of Leptin Transmembrane Signaling

描述(申请人提供)：瘦素(L)及其受体(L-R)是调节能量平衡和体重的关键分子。瘦素和L-R的胞外部分形成复合体，导致Janus kinase2(JAK2)的激活，JAK2结构性地结合在受体的胞内区域。瘦素在下丘脑核团中激活JAK2信号，减少食物摄入量，刺激能量消耗，而瘦素或L-R的功能缺陷会导致病态肥胖、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复合体的结构及其与JAK2的关系知之甚少。缺乏洞察力的原因之一是，L-R和JAK2都有一个相对较长和灵活的多域排列，事实证明，这对大规模提纯和X射线结晶学的实施都非常具有挑战性。本提案旨在克服这些限制，通过应用单粒子冷冻电子显微镜(CRYO-EM)来表征全长L/L-R和JAK2的全息复合体，从而解决L-R信号的结构先决条件。单粒子EM已经成为一种非常强大的工具，可以在相对较小的浓度下表征动态蛋白质组装，而不需要结晶。我们预计，单粒子EM技术在该系统上的应用将独立地、复杂地揭示L/L-R组件和JAK2的结构。鉴于这种膜定位的信号复合体在肥胖、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兴趣。考虑到目前缺乏关于任何受体/JAK复合体的结构信息，我们的研究将为理解细胞外配体与细胞因子受体结合如何导致细胞内JAK激活提供总体架构框架。 与公共健康相关：瘦素信号是调节哺乳动物能量平衡和体重的关键。瘦素与其受体的胞外结合导致Janus kinase2(JAK2)的胞内激活，进而调节许多生理上至关重要的信号级联反应。我们建议应用分子电子显微镜技术来表征瘦素、其受体和JAK2之间的信号复合体的结构。对这一结构的阐明将提供一个通过瘦素参与细胞外受体激活JAK2的机制。由于缺乏瘦素信号与严重的病理有关，如肥胖和糖尿病，这一知识最终将被用于设计基于靶向瘦素受体复合体的治疗策略。

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. PUBLIC HEALTH RELEVANCE: Leptin signaling is key to the regulation of mammalian energy homeostasis and body weight. Extracellular binding of leptin to its receptor results in the intracellular activation of Janus kinase 2 (JAK2) which, in turns, regulates a number of physiologically crucial signaling cascades. We propose to apply molecular electron microscopy techniques to characterize the structure of the signaling complex between leptin, its receptor, and JAK2. The elucidation of this architecture will provide a mechanistic understanding of JAK2 activation via extracellular receptor engagement by leptin. Since deficient leptin signaling is associated with severe pathologies, such as obesity and diabetes, this knowledge will be ultimately used for the design of therapeutic strategies that are based on targeting the leptin receptor complex.