Structural biology of shared cytokine signaling receptors

描述（由申请人提供）：i型四螺旋束细胞因子通过对细胞生长、分化和增殖的影响介导广泛的生理过程。总的来说，通过多效性和独特的活动，它们有助于免疫系统、造血系统和神经系统的稳态。大多数造血和免疫调节细胞因子通过一小群共享的跨膜信号受体起作用。两种最广泛使用的共享受体是gp130和共同γ链（3c），它们通过细胞外区域与它们的细胞因子配体结合，导致细胞内JAK/STAT信号级联的激活。Gp130是11种细胞因子（IL-6、IL-11、纤毛神经营养因子、抑癌素、白血病抑制因子等）的共享受体，3c是6种细胞因子（IL-2、-4、-7、- 9、-15、-21）的共享受体。在该奖项的前期，我们阐明了gp130/IL-6和3c/IL-2两个典型受体复合物家族的结构结构。这些三元和四元配合物代表了每个共享受体家族中所有配体所使用的受体-配体复合物的组装蓝图。在本研究中，我们将从细胞外配体识别、异寡聚细胞表面复合物的组装、跨膜信号传导以及JAK和STAT的细胞内激活等方面对这些共享受体的结构生物学进行深入研究。为此，我们将：1-测定可溶性细胞外多聚体受体-配体复合物的x射线晶体结构，2-测定细胞因子-受体复合物逐步形成的协同性和组装能量，3-纯化与细胞因子和细胞内接头结合的整个全长受体复合物作为膜蛋白并通过电镜成像，4-重构由JAK和STAT组成的细胞内信号复合物。5-用核磁共振来补充晶体学研究，以询问信号传导过程中的潜在变构。在这种方式下，通过采取多学科策略，我们建议通过激活细胞内信号级联，从配体的初始接合中获得共享信号受体的完整分子快照。项目描述：被称为细胞因子的生长因子及其受体对免疫、造血和神经系统的正常功能至关重要，而生长因子网络的功能障碍是许多人类疾病（如自身免疫和癌症）的基础。我们建议使用生物化学和结构生物学技术来可视化生长因子与其细胞受体结合的三维形状，并了解这种结合事件如何在细胞膜上传递。这些研究将使我们深入了解基本的受体信号机制，并促进细胞因子受体系统的治疗目的的操纵。

DESCRIPTION (provided by applicant): Type-I four-helix bundle cytokines mediate a wide range of physiological processes through their effects on cell growth, differentiation, and proliferation. Collectively, through both pleiotropic and unique activities, they contribute to the homeostasis of the immune, hematopoietic, and nervous systems. The majority of hematopoietic and immune regulatory cytokines act through a small group of shared transmembrane signaling receptors. The two most widely used shared receptors are gp130, and the common gamma chain (3c), which engage their cytokine ligands through their extracellular regions, resulting in activation of intracellular JAK/STAT signaling cascades. Gp130 is the shared receptor for eleven cytokines (IL-6, IL-11, Ciliary Neurotrophic Factor, Oncostatin, Leukemia Inhibitory Factor, and others), and 3c is the shared receptor for 6 cytokines (IL-2, -4, -7,- 9, -15, -21). During the prior term of this award, we elucidated structural architectures of the two paradigmatic receptor complexes in each family, gp130/IL-6 and 3c/IL-2. These ternary and quaternary complexes represent blueprints for the assembly of receptor-ligand complexes used by all ligands in each shared receptor family. In this proposal, we escalate our investigations into the structural biology of these shared receptors in regards to extracellular ligand recognition, assembly of hetero-oligomeric cell surface complexes, transmembrane signaling, and intracellular activation of JAK and STAT. In order to do this, we are: 1- determining x-ray crystal structures of soluble extracellular multimeric receptor-ligand complexes, 2- measuring the cooperativity and assembly energetics of stepwise cytokine-receptor complex formation, 3- purifying the entire full-length receptor complexes, as membrane proteins, bound to both cytokine and intracellular adaptors and imaging them by electron microscopy, 4- reconstituting the intracellular signaling complexes consisting of JAK and STAT, and 5- complementing the crystallographic studies with NMR to interrogate potential allostery during signaling. In this fashion, by taking a multi-disciplinary strategy we propose to obtain a complete molecular snapshot of a shared signaling receptor from the initial engagement of ligand through the activation of intracellular signaling cascades. PROJECT NARRATIVE: Growth factors termed cytokines, and their receptors, are essential to the normal functioning of the immune, hematopoietic, and nervous systems, and dysfunction of the growth factor network underlies numerous human disease conditions such as autoimmunity and cancer. We propose to use the techniques of biochemistry and structural biology to visualize the three-dimensional shapes of growth factors bound to their cellular receptors, and understand how this binding event is communicated across the cell membrane. These studies will give us insight into basic receptor signaling mechanisms, as well as facilitate the manipulation of the cytokine receptor system for therapeutic purposes.