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

背景GPCRs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，GPCRs经历了配体特异性的结构重排，促进了不同信号通路的细胞内转导的关联和激活。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了细胞生理、酶活性、离子通道状态和基因表达水平。因此，GPCRs作为外部环境的传感器，允许细胞外配体调节细胞内的过程。尽管最初认为GPCRs只作为单体发挥作用，但越来越多的证据表明，一些GPCRs采用具有新的功能和药理特性的同聚和异构体组装。虽然GPCR同聚体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、变构和化学计量仍然不清楚。GPCR异构体的高分辨率结构从未被报道过，它将促进异构化缺陷受体的产生，以去除它们对体内功能的特定贡献。更关键的是，结构数据可以用于合理设计偏向或偏离异构体特定信号的药物。此外，计算方法可以用来加强我们对其他相关异构体的结构和功能的理解。目的本项目旨在利用分子、计算、生物物理、细胞和超分辨显微镜技术研究腺苷A2A-多巴胺D2异构体复合体的结构和功能。从药理学的角度来看，了解A2A和D2受体的同构体和异构体信号模式之间的差异可能会导致在治疗帕金森氏病、成瘾和精神分裂症等多巴胺能疾病方面的新策略。方法本项目的目标是获得足够稳定的A2A-D2齐聚物，用于结构研究。为了提高稳定性，突变方法将被用来产生交联型突变体、构象稳定突变体和截短形式。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。此外，还将评估配体和稳定蛋白质等其他工具的可行性。A2A-D2异构体的稳定和功能形式将被提纯，生物物理/结构分析将旨在阐明原-原异构体相互作用的性质和异构体功能。结果该项目预期的结果是稳定A2A-D2异构体中的原核糖体之间的相互作用，以便提纯高质量的蛋白质，并获得足够的数量，用于高分辨结构测定。这些信息将有助于药物的合理设计，以改变独特的低聚物特定结果。

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.