The organisational structure of class A GPCRs: Implications for pharmacology, function and therapeutic regulation

A 类 GPCR 的组织结构：对药理学、功能和治疗调节的影响

• 批准号: MR/L023806/1
• 负责人: Graeme Milligan
• 金额: $ 242.75万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FL023806%2F1
• 关键词: organisational; structure; class; GPCRs; Implications

Proteins located at the surface of cells act as receptors for information provided by the presence of a bewildering number of hormones, neurotransmitters and various other cell modulators. In certain cases, such as the single transmembrane domain receptor-tyrosine kinases, it is well established that for binding of the messenger to be transmitted into a response inside the cell, the receptor has to dimerise, i.e. two copies of the receptor must come together into a complex and interact. The family of G protein-coupled receptors (GPCRs) are the targets for key hormones and neurotransmitters that control everything from heart rate to emotional responses. Given their importance in physiology and as the targets of a very broad group of medicines that alleviate major diseases, these proteins have been studied extensively for many years. Indeed, in a drive to both understand the molecular basis of action of medicines that function by binding to GPCRs and also to improve the next generation of such medicines, in recent years enormous progress has been made via crystallising certain GPCRs so that amazing detail of their structures can be observed. In a number of recent cases the receptor has crystallised as either a dimer (two copies) or a tetramer (containing four copies of the receptor) with well defined contacts between the individual copies of the receptor. Despite this, and clear evidence that GPCRs can and do exist as dimers or even, indeed, as multimers in living cells, it is also well established that such interactions are not absolutely required for their primary function. As such, key outstanding questions that so far lack answers include, what range of sizes of complexes of GPCRs exist, does this vary between different members of the family, does this vary in different cells and tissues in which the receptor is expressed and, if so, what are the consequences of this for function in both health and disease? However, as the moment there is little agreement on these topics. The first substantial component of the studies proposed is to address and answer each of these questions. These studies are designed to answer both fundamental questions about these receptors but also to explore the significiance of the answers for the effectiveness of various medicines and how such variation might be used to develop improved medicines.Integral to developing approaches to address many of these questions has been a series of recent efforts to better incorporate mathematics into analysis of biological processes and the appreciation that photographic images of cells expressing such receptors labelled with fluorescent markers often contain far more information that is usually abstracted from them. Thus, in preparation for this application we have developed ways in which detailed mathematical analysis can uncover hitherto unappreciated insights into the size and the shape of a receptor complex inside a single cell. In isolation, this would be interesting but insufficient. Therefore, the second major element of the work will be to use information from the crystal structure data to attempt to generate modified receptors that disassemble or that prevent the formation of such complexes and then use these modified forms of the receptor to assess the functional consequences. Such studies will take advantage of a wide array of approaches in pharmacology, biochemistry and cellular signalling assays that my team and I have built up and used over many years. The other major component of the proposal reflects that individual GPCRs do not only interact to form complexes with other copies of the same receptor. They can also interact with other members of the GPCR superfamily to generate heteromers. Such heteromers have been reported to display very distinct pharmacology and function than the corresponding homomers and we will address similar questions as above for such complexes incorporating GPCRs for the neurotransmitters dopamine and glutamate.

位于细胞表面的蛋白质充当信息的受体，这些信息是由大量激素、神经递质和各种其他细胞调节剂提供的。在某些情况下，例如单跨膜结构域受体-酪氨酸激酶，已经确定的是，为了将信使的结合传递到细胞内的应答中，受体必须二聚化，即受体的两个拷贝必须一起形成复合物并相互作用。G蛋白偶联受体（GPCR）家族是关键激素和神经递质的靶点，这些激素和神经递质控制着从心率到情绪反应的一切。鉴于它们在生理学中的重要性以及作为缓解重大疾病的非常广泛的药物组的靶点，这些蛋白质已被广泛研究多年。事实上，为了了解通过与GPCR结合发挥作用的药物的分子基础，以及改善下一代此类药物，近年来通过结晶某些GPCR取得了巨大进展，以便可以观察到其结构的惊人细节。在最近的一些情况下，受体结晶为二聚体（两个拷贝）或四聚体（含有四个受体拷贝），在受体的各个拷贝之间具有明确的接触。尽管如此，并且有明确的证据表明GPCR可以并且确实作为二聚体甚至实际上作为多聚体存在于活细胞中，但也已经确定，这种相互作用对于它们的主要功能并不是绝对必需的。因此，迄今为止缺乏答案的关键未决问题包括，存在GPCR复合物的大小范围，这在家族的不同成员之间是否不同，这在表达受体的不同细胞和组织中是否不同，如果是这样，这对健康和疾病的功能有什么影响？然而，目前在这些问题上几乎没有达成一致意见。拟议研究的第一个实质性组成部分是处理和回答这些问题。这些研究旨在回答关于这些受体的基本问题，同时也探索各种药物有效性答案的重要性，以及如何利用这些变化来开发改进的药物。最近的一系列努力，更好地将数学纳入生物过程的分析中，并赞赏摄影图像用荧光标记物标记的表达这种受体的细胞通常包含更多的信息，这些信息通常是从它们中提取的。因此，在准备这一应用时，我们已经开发出了一些方法，通过这些方法，详细的数学分析可以揭示迄今为止尚未得到重视的关于单个细胞内受体复合物的大小和形状的见解。孤立地看，这将是有趣的，但还不够。因此，这项工作的第二个主要内容将是使用来自晶体结构数据的信息，试图产生能够分解或阻止这种复合物形成的修饰受体，然后使用这些修饰形式的受体来评估功能后果。这些研究将利用我和我的团队多年来建立和使用的药理学、生物化学和细胞信号分析的各种方法。该提案的另一个主要组成部分反映了单个GPCR不仅与同一受体的其他副本相互作用形成复合物。它们还可以与GPCR超家族的其他成员相互作用以产生异聚体。据报道，这种异聚体显示出与相应的同聚体非常不同的药理学和功能，我们将解决与上述结合神经递质多巴胺和谷氨酸的GPCR的复合物类似的问题。

项目成果

Distinct Agonist Regulation of Muscarinic Acetylcholine M2-M3 Heteromers and Their Corresponding Homomers.

• DOI: 10.1074/jbc.m115.649079
• 发表时间: 2015-06-05
• 期刊: The Journal of biological chemistry
• 作者: Aslanoglou D;Alvarez-Curto E;Marsango S;Milligan G
• 通讯作者: Milligan G

Muscarinic receptor oligomerization.

• DOI: 10.1016/j.neuropharm.2017.11.023
• 发表时间: 2018-07-01
• 期刊: Neuropharmacology
• 影响因子: 4.7
• 作者: Marsango S;Ward RJ;Alvarez-Curto E;Milligan G
• 通讯作者: Milligan G

Analysis of Human Dopamine D3 Receptor Quaternary Structure.

• DOI: 10.1074/jbc.m114.630681
• 发表时间: 2015-06-12
• 期刊: The Journal of biological chemistry
• 作者: Marsango S;Caltabiano G;Pou C;Varela Liste MJ;Milligan G
• 通讯作者: Milligan G

Spatial Intensity Distribution Analysis: Studies of G Protein-Coupled Receptor Oligomerisation.

空间强度分布分析：G蛋白偶联受体低聚的研究。

• DOI: 10.1016/j.tips.2017.09.001
• 发表时间: 2018-03
• 期刊: Trends in pharmacological sciences
• 影响因子: 13.8
• 作者: Pediani JD;Ward RJ;Marsango S;Milligan G
• 通讯作者: Milligan G

Approaches to Characterize and Quantify Oligomerization of GPCRs.

GPCR 寡聚化的表征和定量方法。

• DOI: 10.1007/978-1-4939-2914-6_7
• 发表时间: 2015
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Marsango S