Structural Studies of Metabotropic GABA Receptors

描述（由申请人提供）：代谢性GABAB受体介导哺乳动物大脑中的抑制性神经传递，是其功能的核心。GABAB受体是调节Ca2+和K+通道活性，抑制腺苷酸环化酶功能的g蛋白偶联受体（gpcr）。GABAB受体的功能障碍与包括痉挛、癫痫和疼痛在内的许多疾病的发生有关。因此，阐明GABAB受体的结构和功能将有助于设计有价值的治疗剂。与大多数gpcr不同，GABAB受体是GBR1和GBR2亚基的异二聚体。这些受体的反式激活需要异源二聚化，其中GBR1负责配体结合，GBR2参与g蛋白偶联。本提案的第一部分旨在了解GBR1和GBR2外畴在激活过程中的作用。我们建议用生化方法解决单个亚基的结构和研究它们的相互作用。第二个目的是揭示配体结合和异源二聚体二聚化的结构基础，并确定受体-配体相互作用的亲和力和特异性。我们的第三个目标是了解细胞内螺旋结构域异二聚化的分子基础及其在受体组装和运输中的作用。研究的最后一部分是利用全长受体了解跨膜信号转导机制。我们建议使用全细胞膜片钳方法对野生型和突变型受体进行功能研究。影响配体结合、受体异二聚化和受体激活的突变将基于现有的结构信息和同源模型进行设计。结合对GABA受体的结构和功能分析，将有助于我们进一步了解GABA作用的分子基础以及GPCR异源二聚体特有的激活机制。公共卫生相关性：代谢性GABA受体功能障碍导致人类神经系统疾病。我们正在研究GABA受体不同组分的结构模型，以了解它们在受体功能中的作用。所得到的原子结构可能有助于新型治疗剂的设计。

DESCRIPTION (provided by applicant): Metabotropic GABAB receptors mediate inhibitory neurotransmission in the mammalian brain and are central to its function. GABAB receptors are G-protein coupled receptors (GPCRs) that regulate the activity of Ca2+ and K+ channels, and inhibit the function of adenylyl cyclase. Malfunction of GABAB receptors has been implicated in the development of a number of diseases including spasticity, epilepsy and pain. Therefore, elucidating the structure and function of GABAB receptors would assist the design of valuable therapeutic agents. Unlike most GPCRs, GABAB receptors function as a heterodimeric assembly of GBR1 and GBR2 subunits. Heterodimerization is required for the trans-activation of these receptors, where GBR1 is responsible for ligand-binding and GBR2 is involved in G-protein coupling. The first part of this proposal is aimed at understanding the role of GBR1 and GBR2 ectodomains in the activation process. We propose to solve the structures of the individual subunits and study their interactions using biochemical methods. The second aim is to reveal the structural basis of ligand-binding and dimerization of an ectodomain heterodimer, and determine the affinity and specificity of receptor-ligand interactions. Our third aim is to understand the molecular basis of heterodimerization by the intracellular coiled-coil domains and their role in receptor assembly and trafficking. The last part of the proposed research is focused on understanding transmembrane signal transduction mechanism using full-length receptors. We propose to carry out functional studies of wild-type and mutant receptors using whole cell patch clamp methodology. Mutations that affect ligand binding, receptor heterodimerization, and receptor activation will be designed based on the available structural information and homology models. A combination of structural and functional analysis of GABAB receptors will advance our understanding of the molecular basis of GABA action and the activation mechanisms particular to GPCR heterodimers. PUBLIC HEALTH RELEVANCE: Malfunction of metabotropic GABA receptors leads to neurological disorders in human. We are pursuing structural models for different components of GABA receptors in order to understand their roles in receptor function. The resulting atomic structures may assist the design of novel therapeutic agents.