nvestigating functional and structural roles of G-protein coupled receptor oligomerization

研究 G 蛋白偶联受体寡聚化的功能和结构作用

• 批准号: 1949441
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1949441
• 关键词: nvestigating; functional; structural; roles; protein

BackgroundGPCRs are the largest family of receptor proteins in eukaryotes and play essential roles in signal transduction in all physiological systems. To relay extracellular information to the cell, GPCRs undergo ligand-specific structural rearrangements that promote association with, and activation of intracellular transducers of different signalling pathways. Through differential post-translational modifications and allosteric interactions, these intracellular signalling proteins alter cell physiology; enzymatic activities, ion channel states, and gene expression levels. Thus, GPCRs act as sensors of the external environment, allowing extracellular ligands to modulate intracellular processes.Though originally thought to function exclusively as monomers, increasing evidence has shown that some GPCRs adopt homomeric and heteromeric assemblies with novel functional and pharmacological properties. Though structural information is available for GPCR homomers, our understanding of the mechanisms driving heteromer formation, the interfaces involved, their stability, allostery, and stoichiometry remain unclear. A high-resolution structure of a GPCR heteromer has never been reported and would facilitate the generation of heteromerisation-deficient receptors to unpick their specific contributions to in vivofunctions. More critically, structural data could be used for rational design of drugs biased towards or away from heteromer-specific signalling. Furthermore, computational approaches could be employed to enhance our understanding of the structure and function of other, related heteromers. AimsThis project aims to use molecular, computational, biophysical, cellular and super-resolution microscopy approaches to investigate the structure and function of the Adenosine A2A -Dopamine D2 heteromer complex. From a pharmacological perspective, understanding the differences between homomeric and heteromeric signalling modes of A2A and D2 receptors in may lead to novel strategies in the treatment of dopaminergic disorders such as Parkinson's Disease, addiction, and schizophrenia. MethodsIn particular, this project will aim to obtain sufficiently stable A2A-D2 oligomers for structural studies. To enhance the stability, mutational approaches will be used to generate cross-linked mutants, conformationally-stabilised mutants, and truncated forms. Mutagenesis approaches will be rationalised by computational modelling, and assessed by functional studies and super-resolution microscopy. The viability of additional tools such as ligands and stabilising proteins will also be assessed. Stable and functional forms of the A2A-D2 heteromer will be purified and biophysical/structural analyses will aim to elucidate the nature of protomer-protomer interactions and heteromer function. Outcomes The outcomes expected from this project are to stabilise the inter-protomer interactions in the A2A-D2 heteromer, in order to purify high quality protein, and in sufficient quantity, for high-resolution structure determination. This information would facilitate the rational design of pharmaceuticals that modify unique, oligomer-specific outcomes.

dpcr是真核生物中最大的受体蛋白家族，在所有生理系统的信号转导中发挥着重要作用。为了将细胞外信息传递给细胞，gpcr进行配体特异性结构重排，促进与不同信号通路的细胞内转导器的关联和激活。通过不同的翻译后修饰和变构相互作用，这些细胞内信号蛋白改变细胞生理；酶活性，离子通道状态和基因表达水平。因此，gpcr作为外部环境的传感器，允许细胞外配体调节细胞内过程。虽然最初认为gpcr仅作为单体发挥作用，但越来越多的证据表明，一些gpcr采用具有新功能和药理特性的同质和异质组装。虽然GPCR同源物的结构信息是可用的，但我们对驱动异构体形成的机制、所涉及的界面、它们的稳定性、变构和化学计量学的理解仍然不清楚。GPCR异聚体的高分辨率结构从未被报道过，这将促进异聚缺陷受体的产生，以分离其对体内功能的特定贡献。更重要的是，结构数据可以用于合理设计偏向或远离异源特异性信号的药物。此外，计算方法可以用来提高我们对其他相关异构体的结构和功能的理解。本项目旨在利用分子、计算、生物物理、细胞和超分辨率显微镜等方法研究腺苷A2A -多巴胺D2异构体复合物的结构和功能。从药理学角度来看，了解A2A和D2受体的同质和异质信号传导模式的差异可能会为治疗多巴胺能疾病（如帕金森病、成瘾和精神分裂症）提供新的策略。方法本项目旨在获得足够稳定的A2A-D2低聚物，用于结构研究。为了提高稳定性，突变方法将用于产生交联突变体、构象稳定突变体和截断形式。诱变方法将通过计算模型合理化，并通过功能研究和超分辨率显微镜进行评估。还将评估其他工具（如配体和稳定蛋白）的可行性。A2A-D2异聚体的稳定和功能形式将被纯化，生物物理/结构分析将旨在阐明原聚体-原聚体相互作用和异聚体功能的性质。本项目预期的结果是稳定A2A-D2异聚体中原体间的相互作用，以纯化高质量的蛋白，并获得足够的量，用于高分辨率的结构测定。这一信息将有助于合理设计修改独特的低聚物特异性结果的药物。