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

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.

Recent technological developments have illuminated how G protein-coupled receptors (GPCRs) are intricately organized from the subcellular to atomic level; exhibiting a multiplicity in conformational states and protein microenvironments. These developments also inform the study of GPCR homomers and heteromers; addressing fundamental mechanisms in how such complexes are allosterically modulated and their coupling to its signalling machinery. Our studies with a GPCR key in reproduction and pregnancy, the luteinsing hormone receptor (LHR), have integrated super-resolution imaging with modelling to demonstrate LHR forms an array of complexes, from monomer, dimer to a variety of low order oligomers, including heteromers with follicle-stimulating hormone receptor (FSHR). Reorganization of protomers alters signal activity and is likely to be mediated by multiple interfaces. Unpicking the physiological role of these complexes is challenging when applied to LHR in female reproduction, where its functions must be coordinated within a highly interconnected system of somatic and germ cells; the ovarian follicle. Our team of investigators and project partners will apply a trans-disciplinary approach to these fundamental mechanisms, by integrating automated super-resolution imaging with machine learning, computational modelling, chemistry, nanobody technology and biophysical sensors. Employing mouse models expressing HA-tagged LHR and FLAG-FSHR, we will image the organization of LHR complexes, with FSHR and its signal machinery, in follicular systems. Protomer reorganization during follicle maturation, and by receptor nanobodies and small molecules that induce distinct conformational and signalling states, will also be applied. Modelling of follicular LHR complexes will identify interfaces for chemical disruption. We will characterise the impact of disrupting or modifying highly defined receptor complexes on classical physiological roles of LH in both mouse and human systems.