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其对细胞生长、分化和增殖的作用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，造血和神经系统的稳态。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gp 130和共同的γ链（3c），其通过其细胞外区域接合其细胞因子配体，导致细胞内JAK/STAT信号级联的激活。Gp 130是11种细胞因子（IL-6、IL-11、睫状神经营养因子、制瘤素、白血病抑制因子等）的共有受体，3c是6种细胞因子（IL-2、-4、-7、-9、-15、-21）的共有受体。在该奖项的前期，我们阐明了每个家族中两种典型受体复合物的结构体系，gp 130/IL-6和3c/IL-2。这些三元和四元复合物代表了每个共享受体家族中所有配体所使用的受体-配体复合物组装的蓝图。在这个建议中，我们升级我们的调查到这些共享受体的结构生物学方面的胞外配体识别，装配异源寡聚细胞表面复合物，跨膜信号，和细胞内激活JAK和STAT。为了做到这一点，我们：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-测量逐步形成的精氨酸-受体复合物的协同性和组装能，3-纯化作为膜蛋白的整个全长受体复合物，其结合细胞因子和细胞内衔接子，并通过电子显微镜对其成像，4-重构由JAK和STAT组成的细胞内信号传导复合物，和5-用NMR补充晶体学研究以询问信号传导期间的潜在变构。以这种方式，通过采取多学科的策略，我们建议获得一个完整的分子快照的共享信号受体从配体的初始参与，通过激活细胞内信号级联。项目叙述：被称为细胞因子的生长因子及其受体对于免疫、造血和神经系统的正常功能是必需的，并且生长因子网络的功能障碍是许多人类疾病状况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通过细胞膜进行通信。这些研究将使我们深入了解基本的受体信号传导机制，以及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.