Structural characterisation of A2A adenosine- D2 dopamine receptor heteromer

A2A 腺苷-D2 多巴胺受体异聚体的结构表征

• 批准号: 2453250
• 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: 2020
• 资助国家: 英国
• 起止时间: 2020 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2453250
• 关键词: Structural; characterisation; A2A; adenosine; D2

G-protein coupled receptors (GPCRs) are the largest family of protein receptors and it is now widelyaccepted that many can not only function as monomers but also as oligomers with distinct biologicalfunctions (Maurice et al., 2011). However, there is a lack of structural information about these oligomers,particularly at the interface between protomers. A2A adenosine receptor (A2AR) and D2 dopamine receptor(D2R) are Class A GPCRs which are co- expressed in striatopallidal GABA neurons in the central nervoussystem (Fuxe et al., 2010; Schiffmann et al., 1991). A2AR allosterically inhibits D2R signalling in the brain,thus A2AR-D2R heteromers have been implicated in disordered dopamine signalling such as in Parkinson's,schizophrenia and cocaine addiction (Ferre et al., 1991; Fuxe et al., 2010). Evidence to suggest theexistence of an A2AR-D2R heteromers was found through coimmunoprecipitation, BRET (Bioluminescenceresonance energy transfer) and FRET (florescence resonance energy transfer) analyses (Canals et al.,2003; Hillion et al., 2002). Since then, studies to investigate the interface and overall structure of theheteromer have been carried out (Borroto-Escuela et al., 2018). in However, the precise molecular basisof the interactions between protomers remain unclear. The characterisation of receptor interface along witha high-resolution structure of the A2AR-D2R heteromer would allow the development of novel drugs to targetthe receptors in a heteromer- specific manner. Furthermore, the structure of this heteromer may allow agreater understanding of the nature of oligomer formation and regulation and other similar Class A GPCRoligomers.Since GPCRs are membrane proteins, they are notoriously difficult to isolate for structural studies due totheir dynamic flexibility (Zhao and Wu, 2012). Therefore, methods to engineer the GPCR to increase itsstability are common and widely used. This is even more relevant in the case of the receptor heterodimers,however methods to stabilise these complexes are less well developed.Therefore, the overall aim of the project is to stabilise the A2AR and D2R heteromer in mammalian systemsfor high resolution structural analysis. This optimally stable heteromer will be obtained by:1. Applying mutagenesis to introduce cross-linkages2. Screening of conformationally restricted mutants3. Use of nanobodiesWhile these strategies aim to obtain optimal constructs for structural studies, these experiments alone willprovide vital information on the precise molecular interactions between protomers.Site directed mutagenesis of A2AR and D2R will be carried out to try to establish cysteine cross-linkingbetween the two receptors. Our collaborator used molecular modelling to predict the heteromer interfaceand identify the optimal residues to create cross-linkages (Francesca Fanelli, unpublished). This hasidentified 4 candidate residue pairs for mutagenesis to cysteine. These cysteine mutant pairings will beassessed individually using BRET to determine their effect on interprotomer affinity and proximity. Mutantpairs which have successfully cross-linked and result in an increased affinity at the interface between A2AR and D2R will be combined to create a more stable heteromer. This will not only increase heteromer stability for structural analysis but will give further evidence for the predicted receptor interface, which is currently poorly understood. Previous work has also identified nanobodies which are effective in stabilising the A2A homomer (Thomas Diaz, unpublished). This will be applied to the cysteine mutated A2A-D2 heteromer to investigate if these further alter associations by BRET analysis. These strategies for stabilisation along with the use of thermostabilised A2A and D2 mutants will be used for isolation, purification and structural studies with the heteromer.

G蛋白偶联受体（GPCR）是最大的蛋白质受体家族，现在已被广泛吸现的是，许多人不仅可以充当单体，而且可以作为具有不同生物学功能的低聚物（Maurice等，2011）。但是，缺乏有关这些低聚物的结构信息，尤其是在造物质之间的界面上。 A2a腺苷受体（A2AR）和D2多巴胺受体（D2R）是A类GPCR，在中枢神经系统中共同表达在纹状体GABA神经元中（Fuxe等，2010; Schiffmann等，1991）。 A2AR变构抑制大脑中的D2R信号传导，因此A2AR-D2R杂体与无序的多巴胺信号传导有关，例如帕金森氏症，精神分裂症和可卡因成瘾（Ferre等，1991; Fuxe等，2010）。通过共免疫沉淀，BRET（Biomuminescencenceresonance Energy转移）和FRET（Florescentence Resonance Energy Transners）分析（Canals等，2003; Hillion等，2002），发现A2AR-D2R异构体的存在的证据是通过共免疫沉淀的存在。从那时起，已经进行了研究界面的界面和整体结构的研究（Borroto-Escuela等，2018）。然而，在原始人之间的相互作用的精确分子基础尚不清楚。受体界面的表征以及A2AR-D2R异构体的高分辨率结构的表征将允许新型药物的发展以异构体的方式靶向受体。此外，该异构体的结构可以使Agreater了解低聚物形成和调节的性质以及其他类似的A gpcroligomers。由于GPCR是膜蛋白，因此，由于Totheir Dynamigibility和Wu，Zhao和Wu，2012年），众所周知，它们很难分离出结构性研究。因此，对GPCR提高稳定性的设计方法是普遍的，并且广泛使用。在受体异二聚体的情况下，这甚至更为相关，但是稳定这些复合物的方法的发展较不那么发达。因此，该项目的总体目的是稳定哺乳动物系统中的A2AR和D2R异构体，用于高分辨率高分辨率结构分析。此最佳稳定异构体将通过：1获得。应用诱变引入交叉链接2。筛选构象受限突变体3。这些策略旨在获得结构研究的最佳构建体的使用，仅这些实验将提供有关原始物体之间精确分子相互作用的重要信息。位于A2AR和D2R的位置诱变将耗尽以试图建立两个受体之间的囊性交叉链接。我们的合作者使用分子建模来预测异构体的互动识别最佳残基以创建交联（Francesca Fanelli，未发表）。这已经识别了4个候选残留物，用于诱变到半胱氨酸。这些半胱氨酸突变配对将使用BRET单独评估，以确定它们对脑型物的亲和力和接近性的影响。成功交联并导致A2AR和D2R之间界面上的亲和力增加的Mutantpairs将被组合起来，以创建更稳定的异构体。这不仅将提高异构体的结构分析稳定性，而且还将为预测的受体界面提供进一步的证据，目前知之甚少。先前的工作还确定了有效稳定A2A同类体（Thomas Diaz，未发表）的纳米词。这将应用于半胱氨酸突变的A2A-D2异构体，以调查这些是否通过BRET分析进一步改变关联。这些稳定策略以及使用恒温的A2A和D2突变体的使用策略将用于与异构体进行分离，纯化和结构研究。