Structural characterisation of A2A adenosine- D2 dopamine receptor heteromer

G蛋白偶联受体(GPCRs)是最大的蛋白质受体家族，现在被广泛接受，许多受体不仅可以作为单体发挥功能，而且还可以作为具有不同生物学功能的寡聚体(Maurice等人，2011年)。然而，缺乏关于这些低聚物的结构信息，特别是在原聚体之间的界面上。A2A腺苷受体(A2AR)和D2多巴胺受体(D2R)是A类GPCRs，在中枢神经系统的纹状体皮质GABA神经元中共同表达(Fuxe等人，2010；Schiffmann等人，1991)。A2AR变构抑制大脑中的D2R信号，因此A2AR-D2R异构体与帕金森氏症、精神分裂症和可卡因成瘾等多巴胺信号的紊乱有关(Ferre等人，1991年；Fuxe等人，2010年)。通过免疫共沉淀、BRET(生物荧光共振能量转移)和FRET(荧光共振能量转移)分析发现了A2AR-D2R异构体存在的证据(Canals等人，2003年；Hillion等人，2002年)。此后，开展了研究异构体的界面和整体结构的研究(Borroto-Escuela等人，2018年)。然而，原核酸体之间相互作用的确切分子基础仍不清楚。受体界面的特征以及A2AR-D2R异构体的高分辨率结构将允许以异构体特异性的方式开发针对受体的新药。此外，这种异构体的结构可能使人们更好地理解寡聚体的形成和调节以及其他类似的A类gpcr的性质。由于gpcr是膜蛋白，众所周知，由于它们的动态灵活性，它们很难分离出来用于结构研究(赵和吴，2012)。因此，对GPCR进行改造以提高其稳定性的方法是常见和广泛使用的。这与受体异二聚体的情况更相关，然而稳定这些复合体的方法还不太成熟。因此，该项目的总体目标是稳定哺乳动物系统中的A2AR和D2R异构体，用于高分辨率结构分析。这种最稳定的异构体将通过以下步骤获得：1。应用诱变引入交联剂。筛选构象限制性突变体3。纳米体的使用虽然这些策略的目的是为结构研究获得最佳构建体，但这些实验本身将提供关于原核体之间精确分子相互作用的重要信息。将对A2AR和D2R进行定点突变，试图建立两个受体之间的半胱氨酸交联。我们的合作者使用分子建模来预测异构体界面，并确定创建交叉连接的最佳残基(Francesca Fanelli，未发表)。这已经鉴定了4个候选残基对半胱氨酸的诱变。这些半胱氨酸突变对将使用BRET进行单独评估，以确定它们对原核间亲和力和亲和力的影响。已成功交联并导致A2AR和D2R之间界面亲和力增加的突变对将结合在一起，创建更稳定的异构体。这不仅将增加结构分析的异构体稳定性，而且将为预测的受体界面提供进一步的证据，目前人们对此知之甚少。以前的工作也发现了有效稳定A2a同聚体的纳米体(Thomas Diaz，未发表)。这将应用于半胱氨酸突变的A2A-D2异构体，以通过Bret分析来研究这些异构体是否会进一步改变关联。这些稳定策略以及热稳定的A2A和D2突变体将用于异构体的分离、纯化和结构研究。

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.