Exploiting a novel molecular toolkit to explore cell type specific adenosine receptor pharmacology and regulation at endogenous levels of expression.

细胞间的通讯过程是所有生命的重要组成部分，控制着生物体的内部运作，使它们能够做出反应、适应和生存。G蛋白偶联受体（gpcr）是一大类位于细胞表面的相关蛋白。这些受体可以检测细胞外的分子并激活细胞内的细胞信号通路。近年来，越来越清楚的是，GPCR药理学（药物与GPCR相互作用的机制）依赖于GPCR所在的局部膜环境，并且越来越多的证据表明，它们的功能可以通过与低聚物复合物内邻近蛋白质（包括其他GPCR）的相互作用来调节。这可能为开发针对特定细胞类型或亚细胞结构域的gpcr的药物提供了新的机会，并提供了一种避免靶上副作用的方法（即药物与正确的受体结合但在错误的位置）。腺苷是一种重要的内源性核苷，通过四种不同的gpcr （A1AR, A2AAR, A2BAR, A3AR）在局部释放。腺苷受体分布广泛，与肾脏、呼吸系统和心血管疾病以及癌症有关。在缺氧、炎症和缺血等代谢应激条件下，腺苷的局部生成显著增强，腺苷受体表达改变，从而保护细胞、组织和器官免受感染和炎症时的附带损伤。有越来越多的证据表明，在同型二聚体和异质复合物中存在几种腺苷受体。在我们之前的项目批准（MR/N020081/1）中，我们证明了腺苷受体的所有组合都可以产生异质复合物。其他人也报道了A2AAR和A2BAR之间的复合物。在纹状体中检测到A2AAR-D2多巴胺受体异构体，并被认为是帕金森病的可行靶点。最后，有证据表明a1ar - β -肾上腺素受体和a1ar - β -肾上腺素受体异构体改变了配体结合和信号特性。然而，到目前为止，大多数研究都是在模型细胞系中使用重组过表达受体，使用生物发光共振能量转移（BRET）等方法来探测寡聚化。然而，这种实验可能会被过表达混淆，过表达会增加gpcr形成二聚体的倾向。因此，迫切需要开发新的方法来监测受体表达水平，并了解在内源性受体表达水平下，寡聚化对天然细胞中GPCR功能的影响，以及它们形成稳定和短暂复合物的倾向。我们最近开发了高度敏感的方法来量化受体表达水平、它们的药理学和蛋白质-蛋白质相互作用，这些方法在天然细胞中发现的低水平受体表达时特别有效。这些方法包括荧光配体技术、内源性gpcr的配体定向共价标记、结合NanoBiT技术的CRISPR/Cas9基因组编辑和单分子生物物理方法在单个活细胞和更复杂的生理相关的离体血管和心脏制备中的应用。该计划的目的是研究来自心血管和免疫系统的天然细胞中受体表达水平和受体寡聚化是如何调节的，这些细胞内源性表达在健康和疾病中起关键作用的腺苷受体。我们的目标是研究这些相互作用在人内皮细胞、平滑肌细胞、成纤维细胞、巨噬细胞和干细胞来源的心肌细胞中的作用。我们还旨在确定这些相互作用如何受到缺氧和炎症介质的影响。

The process of cell to cell communication is a vital and integral part of all life and controls the inner workings of organisms allowing them to respond, adapt and survive. G protein-coupled receptors (GPCRs) are a large group of related proteins that are located on the surface of cells. These receptors can detect molecules outside of the cell and activate cell signalling pathways inside cells. It has become clear, in recent years, that GPCR pharmacology (the mechanisms by which drugs interact with them) is dependent on the local membrane environment within which the GPCR resides, and there is growing evidence that their function can be regulated by interactions with neighbouring proteins (including other GPCRs) within oligomeric complexes. This may provide novel opportunities to develop drugs which are targeted at GPCRs in a particular cell type or subcellular domain and provide a way to circumvent on-target side effects (i.e. where the drug binds to the right receptor but in the wrong location). Adenosine is an important endogenous nucleoside that is released locally and acts via four different GPCRs (A1AR, A2AAR, A2BAR, A3AR). Adenosine receptors have a widespread distribution and have been implicated in renal, respiratory and cardiovascular diseases, as well as cancer. Local generation of adenosine is markedly enhanced, and adenosine receptor expression changed, under metabolic stress e.g. during hypoxia, inflammation and ischaemia and this protects cells, tissues and organs from collateral damage during infection and inflammation. There is increasing evidence for the presence of several adenosine receptors in homodimer and heteromeric complexes. In our previous programme grant (MR/N020081/1), we showed that heteromeric complexes can be produced for all combinations of adenosine receptor. Others have also reported complexes between A2AAR and A2BAR. A2AAR-D2 dopamine receptor heteromers have been detected in striatum and have been proposed to be a viable target for Parkinson's disease. Finally, there is evidence for A1AR-beta1-adrenoceptor and A1AR-beta2-adrenoceptor heteromers which have altered ligand binding and signaling characteristics. To date, however, the majority of studies have used recombinantly overexpressed receptors in model cell lines to probe for oligomerisation using methods such as bioluminescence resonance energy transfer (BRET). However, such experiments can be confounded by overexpression which can increase the propensity of GPCRs to form dimers. There is therefore an urgent need to develop new approaches to monitor receptor expression levels and to understand the effect of oligomerisation on GPCR function in native cells at endogenous levels of receptor expression and their propensity to form stable and transient complexes. We have recently developed highly sensitive methods to quantify receptor expression levels, their pharmacology and protein-protein interactions that are particularly effective at the low levels of receptor expression found in native cells. These approaches include fluorescent ligand technologies, ligand-directed covalent labelling of endogenous GPCRs, CRISPR/Cas9 genome editing in combination with NanoBiT technologies and single molecule biophysical approaches in single living cells and more complex physiologically relevant ex vivo isolated blood vessels and heart preparations. The aim of this programme is study how receptor expression levels and receptor oligomerisation are regulated in native cells from the cardiovascular and immune systems, which endogenously express adenosine receptors that play key roles in health and disease. We aim to investigate these interactions in human endothelial cells, smooth muscle cells, fibroblasts, macrophages and stem cell-derived cardiomyocytes. We also aim to determine how these interactions are affected by hypoxia and inflammatory mediators.