Defining the functional roles of the enigmatic G protein-coupled receptor GPR35

G蛋白偶联受体（gpcr）是细胞表面蛋白，在细胞对外部信号和信号作出反应中起着关键作用。它们通常被认为是通过激活一组或多组所谓的G蛋白来实现的。由于在控制对许多激素和神经递质的反应方面的这些关键作用，大量的gpcr是目前使用的药物的分子靶标，一般来说，gpcr被认为是“可处理的”，即激活或阻止其激活的分子可以被发现并开发为潜在的新药。然而，有许多gpcr特征不明确，它们在控制生理功能中的作用是未知或不确定的。在这种情况下，它们在较长期内成为新药靶标的潜力往往部分取决于在编码GPCR的基因已被消除（敲除）的小鼠身上产生的影响，或者在有这种证据的情况下，通过研究发现，在人类中编码GPCR的基因序列的变异与发生疾病的可能性有关。GPR35就是这样一种GPCR。已知它存在于结肠中，蛋白质序列的变化与包括溃疡性结肠炎在内的下肠道炎症性疾病的潜在发展有关。GPR35在调节炎症方面也可能有更普遍的作用。例如，一些能够激活GPR35的配体已经成为治疗气道/肺部炎症性疾病哮喘的有用药物。要更好地了解GPR35是否能成为新药的有用靶点，有两个主要挑战。首先，尽管有两种化学物质可以阻断人类GPR35的功能，但它们对大鼠或小鼠的GPR35都不起作用。这意味着在啮齿动物的生理和疾病模型中探索GPR35的功能是非常困难的，尽管这些模型对于建立一个可能值得在人类患者中进行测试的病例提供了至关重要的支持。其次，由于GPR35的信号转导机制不寻常且研究较少，因此尚不清楚GPR35产生的各种信号中哪一种可能最适合模拟或阻断以治疗疾病。在拟议的研究中，我们计划使用各种高度创新的方法来克服这些挑战。我们已经使用了一种叫做“基因编辑”的技术来开发一系列细胞系，在这些细胞系中，只有GPCR能产生的信号子集才会被真正诱导。这将使我们能够评估GPR35的不同激活剂是否可能在生理系统中引起不同的影响。第二个关键方法将涉及生产转基因（即转基因）小鼠。总的来说，老鼠和人类有一组非常相似的基因。然而，在某些情况下，可以激活或阻断人类受体的配体对小鼠受体没有相同的效果。这就是GPR35的情况。因此，我们计划通过用人类基因替换GPR35的小鼠基因来“人源化”小鼠。这将产生的小鼠对GPR35配体的反应将类似于我们在激活或阻断人类GPR35时所预期的反应，并将提供更清晰的图像，说明GPR35配体如何影响人类疾病治疗的发展。虽然申请的主要目的是开发方法，加深我们对GPR35的作用及其功能的理解，但所获得的结果将极大地影响未来关于该受体是否可能成为开发新药的新靶点的决定。

G protein-coupled receptors (GPCRs) are cell surface proteins that play key roles in allowing cells to respond to external cues and signals. They are routinely considered to do so by activating one or more of a group of so called G proteins. Because of these key roles in controlling responses to many hormones and neurotransmitters a substantial number of GPCRs are the molecular targets for currently employed medicines and, in general, GPCRs are considered 'tractable' i.e. that molecules that activate or block their activation can be found and developed as potential new medicines. However, there are a number of GPCRs that are poorly characterised and the roles they play in the control of physiological functions are unknown or uncertain. In such cases the potential for them to be targets for new medicines in the longer term is often assessed partially by the effects produced in mice in which the gene encoding the GPCR has been eliminated (knocked-out) or, where such evidence is available, in studies in which variations in the sequence of the gene encoding the GPCR in humans is linked to the potential to develop a disease. GPR35 is such a GPCR. It is known to be present in the colon and variations in sequence of the protein have been linked to the potential development of inflammatory diseases of the lower gut including Ulcerative Colitis. There may also be a more general role for GPR35 in the regulation of inflammation. For example, some ligands that are able to activate GPR35 have been useful medicines for the treatment of airway/lung inflammatory disease asthma. There are two major challenges to better understand if GPR35 could be a useful target for new medicines. Firstly, although there are two chemicals available that can block the function of the human form of GPR35, neither of them work at either the rat or mouse versions of GPR35. This means that it is very difficult to explore the function of GPR35 in rodent models of physiology and disease although such models are vital to provide support to build a case that this might be worth testing in human patients. Secondly, as the mechanisms of signal transduction by GPR35 are both unusual and poorly explored it is unclear which of the various signals generated by GPR35 might be most appropriate to mimic or block to treat disease. In the proposed studies we plan to use a variety of highly innovative approaches to overcome these challenges. We have used a technique called 'gene-editing' to develop a range of cell lines in which only subsets of signals that can be generated by a GPCR are actually induced. These will allow us to assess if different activators of GPR35 are likely to cause different effects in physiological systems. The second key approach will involve the production of transgenic (i.e. genetically modified) mice. Overall, mice and humans have a very similar set of genes. However, in some circumstances ligands that can activate or block a human receptor do not have the same effect at the mouse receptor. This is the case for GPR35. As such, we plan to 'humanise' mice, by replacing the mouse gene for GPR35 with the equivalent gene from humans. This will produce mice in which the responses to GPR35 ligands will be akin to those we would anticipate if we activated or blocked GPR35 in humans and will provide a much clearer picture of how GPR35 ligands might affect the development of treatment of diseases in humans. Although the key objectives of the application are to develop approaches that will deepen our understanding of the roles of GPR35 and how it functions, the results obtained will greatly influence future decisions on whether this receptor might become a new target for the development of novel medicines.