Directing luteinising hormone receptor activity in vivo: A convergent approach to study GPCR molecular complexes

我们对生物系统的科学认识的发展突出了这些系统的复杂性和相互联系的性质。当生物系统的整合失败时，就会导致疾病。因此，为了促进我们对这些基本机制的理解，来自不同领域的科学家正在共同努力解决这些生物学挑战，从而开发出针对一系列疾病的更好的治疗方法。该项目将把细胞生物学与生物物理学、人工智能、化学、计算建模和生理学结合起来，称为融合或跨学科的方法，以解决一个基本问题：激素如何作用于女性卵巢——一个由不同相互联系的细胞组成的复杂器官。卵巢是女性生殖的中枢。在卵巢内，每个卵子都被包裹在一组高度组织的细胞中，这些细胞被称为卵泡。卵泡内不同类型的细胞对激素信号做出反应，与卵子沟通，以确保每个月释放一个成熟的卵子进行受精。黄体生成素（LH）是一种关键的激素，它能使卵子发育、释放，并在卵子受精的早期提供对怀孕至关重要的激素。LH通过与细胞表面的特异性受体LHR结合来协调这些功能。LHR是一个名为G蛋白偶联受体（gpcr）的受体大家族的一部分，有800多种不同类型的受体对光、气味、味觉、大脑中的化学递质和各种不同的激素做出反应。因此，gpcr是一种受欢迎的药物靶标，然而，对更特异性、副作用更少、活性更长时间的新药的需求很高。这种发展需要深入了解复杂生物系统的分子机制，以高度可控的方式控制受体活性。受体改变它们之间交流方式的一个重要方式是相互联系。我们之前由bbsrc资助的研究已经剖析了LHR信号是如何通过与自身（同质体）和另一种重要的生殖激素受体——促卵泡激素（FSHR）作为异质体的关联而改变的。我们通过使用一种称为超分辨率成像的显微镜技术（一种称为光活化染料定位显微镜（PD-PALM）的技术）可视化了LHR同质体和LHR/FSHR异质体，该技术提供了对细胞表面单个受体成像的能力。我们的工作表明，LHR受体以单体和大小不等的异构体存在。改变这些受体-受体关联的模式可以改变细胞内产生的信号的类型、持续时间和强度。一个突出而重要的问题仍然是LHR复合物的组织如何有助于LHR在卵泡中的多种功能。我们将使用我们的PD-PALM单分子显微镜技术和机器学习技术来创建一个新的自动化平台，以可视化不同卵泡细胞类型和保留与卵子通信的“开放”卵泡中的LHR分子复合物（具有信号机制的受体）。将卵泡细胞的PD-PALM图像与计算模拟相结合，将揭示LHR如何相互作用，并采用新的化学、小分子和抗体工具来破坏或操纵这些相互作用，以通过生物物理、生化和遗传方法了解它们在调节多种卵巢功能中的作用。我们预计，在未来，所产生的信息可以直接应用于改善患有多囊卵巢综合征、激素依赖性癌症、不孕症、卵巢早衰等疾病的女性的生活质量，并可能应用于其他涉及gpcr的疾病。

Developments in our scientific understanding of biological systems highlight the complexity and interconnected nature of such systems. When integration of biological systems fails, disease can result. Thus, to advance both our understanding of these fundamental mechanisms, scientists from different fields are working together to address these biological challenges, and in turn develop better treatments for a range of diseases. This project will integrate cell biology with biophysics, artificial intelligence, chemistry, computational modeling and physiology, termed a convergent or trans-disciplinary approach, to problem-solve the fundamental question: how do hormones act on the female ovary- a complex organ of distinct interconnected cells. The ovary is a hub of female reproduction. Within the ovary, each egg is encapsulated in a highly organized group of cells, termed the follicle. Different cell types within the follicle respond to hormonal cues, communicating with the egg to ensure a single mature egg is released each month for fertilisation. A key hormone that develops the egg, causes its release and provides the hormones critical in early pregnancy if the egg is fertilized, is luteinising hormone (LH). LH coordinates these functions by binding to its specific receptor on the surface of the cell, the LHR. LHR is part of a large family of receptors called G protein-coupled receptors (GPCRs) with more than 800 different types that respond to light, smells, taste, chemical transmitters in the brain and a variety of different hormones. Thus, GPCRs are a popular drug target, however, there is high demand for new drugs that are more specific, have fewer side effects, and that are active for longer. Such developments require an in-depth understanding of the molecular mechanisms from complex biological systems to control receptor activity in a highly controlled manner. One important way receptors can modify how they communicate is by associating with each other. Our previous BBSRC-funded studies have dissected how LHR signalling is altered via its association with itself (homomers) and another important reproductive hormone receptor, the follicle stimulating hormone (FSHR) as heteromers. We have visualised LHR homomers and LHR/FSHR heteromers by employing a form of microscopy called super resolution imaging- a technique called photoactivated dye localisation microscopy (PD-PALM), which provides the ability to image single receptors on the surface of the cell. Our work has revealed that LHR receptors exist as monomers and a range of size of homomers/heteromers. Altering the pattern of these receptor-receptor association can change the type, duration and magnitude of signals generated inside cells. An outstanding and important question that remains is how the organization of LHR complexes contributes to LHR's multiple functions in the follicle. We will use our single molecule microscopy technique of PD-PALM with machine learning technology to create a novel automated platform to visualize LHR molecular complexes (receptor with it's signaling machinery) in the different follicle cell types and 'open' follicles that retains the communication with the egg. Combining PD-PALM images from follicle cells with computational simulations will unpick how LHR engages with each other, and employ novel chemical, small molecule and antibody tools to disrupt or manipulate these interactions to understand their role in regulating multiple ovarian functions via biophysical, biochemical and genetic methods. We anticipate that in the future, the information generated can be directly applied to improve the quality of life of women with conditions such as polycystic ovarian syndrome, hormone-dependent cancer, infertility, premature ovarian failure, and potentially applied to other diseases that involve GPCRs.