Use of fluorescence correlation spectroscopy to study GPCR oligomerisation and allosterism in membrane micro domains of single living cells.

The way in which cells communicate with each other and change cellular responses is an essential part of all life, and controls the inner workings of organs within the body allowing them to respond, adapt and survive. This communication between cells is largely based on chemical messenger molecules, which can be small (e.g. adenosine, adrenaline) or large (e.g. vascular endothelial growth factor, VEGF). These molecules work by binding to specific proteins (receptors) on the surface of their target cells that in turn activate signalling responses inside the cell. G Protein-Coupled Receptors (GPCRs) are the largest family of these cell surface proteins. They are major targets for drug discovery and over 30% of all prescribed drugs target these receptors.

Recently we have discovered much more about the physical structure of GPCRs using x-ray crystallography. This has led to a better understanding of how the structure of these proteins changes when stimulated by agonist molecules that act at the same site (orthosteric) as the natural hormone or neurotransmitter. However, over the last decade it has become clear that drugs can also bind to an additional site, called the allosteric site, which is in a separate location on the GPCR protein. These drugs cause a different change in protein structure that can alter how well a hormone or neurotransmitter binds to the orthosteric binding site and activates its receptor. As well as small molecule allosteric drugs, neighbouring cellular proteins (including other GPCRs) can also bind to GPCRs and act as allosteric modulators to enhance or inhibit the binding and/or function of the natural ligand. This means that how well a drug binds or activates a GPCR can depend on where that receptor is in the cell and what other cellular proteins are present in that location. This can also change the signals stimulated by the receptor (leading to something called biased signalling).

Traditional ways of measuring the way ligands bind to receptors (their pharmacology) require large numbers of cells to achieve a measurable response. During our current MRC programme grant we have developed new highly sensitive imaging approaches (based on a technique called fluorescence correlation spectroscopy or FCS) to study the pharmacology of GPCRs in very small areas of the membrane of single living cells. We have focused on two receptors for the hormone adenosine - the A1 and A3 receptors. We are the only group in the UK (and one of few worldwide) to have applied FCS to look at the interaction of GPCRs with both drugs and other cellular signaling proteins. The aim of this renewal is to extend this work to address key questions about the molecular pharmacology of GPCRs. This will use FCS to look at GPCRs in complex with other proteins (receptors and signaling proteins) in specific areas of living cell membranes. In particular, we will take advantage of the exquisite sensitivity of FCS to detect GPCRs at the low expression levels normally found in native cells. Our major emphasis will be on receptors for adenosine and adrenaline (beta-adrenoceptors) that are important for the cardiovascular system.

Specific questions we will try and answer include: (a) How many receptors of each type do the signaling complexes contain? (b) What impact do changes in how these complexes are made up have on how each of the constituent receptors binds its ligand? (c) Does this vary between neighbouring cells and membrane locations? (d) To what extent does binding of a ligand to one receptor in the complex affect binding of ligands to the others? Can this knowledge be exploited to target drugs to complexes with a specific composition? (e) Can these complexes and their functional interactions be demonstrated in native cells from the cardiovascular system?

The techniques of fluorescence correlation spectroscopy (FCS) and photon counting histogram analysis (PCH) will be used to monitor the number, diffusional charactersitics and brightness of ligand-bound (using fluorescent agonists and antagonist ligands) and free (tagged using GFP, Halotag or SNAP tag technologies) receptors in small defined regions of the plasma membrane (~0.2 square microns) in multiple areas of a single living cell. This will provide insight into the ligand-binding characteristics, composition and molecular size of oligomeric structures containing cell surface receptors such as G protein-coupled receptors (GPCRs). Stoichiometry of GPCR homo- and hetero-dimer complexes will be investigated using antibody cross-linking and subsequent FCS/PCH analysis. This will be confirmed in parallel experiments using raster image correlation spectroscopy (RICS), number and brightness analysis and two-colour super resolution microscopy (dSTORM; Zeiss Elyra).

Kinetic studies in single cells and in multiple areas of the plasma membrane (FCS, PCH) will evaluate the kinetics of ligand-receptor binding interactions and the extent of cooperativity (negative or positive) across dimer interfaces. This work will also provide important information on ligand binding residence times and the amino acid residues involved in conveying allosteric influences across oligomeric interfaces. Bioluminescence resonance enegy transfer (BRET) between a fluorescent ligand and the novel bioluminescent protein NanoLuc fused to the N-terminus of the GPCR under study will allow alternative strategies to investigate allostery. Intramolecular fluorescence resonance energy transfer (FRET) approaches will be used to look at conformation changes induced by agonists in different GPCRs. BRET and FRET strategies will also be used to study signalling bias from particular oligomeric configurations. The techniques above will be applied to native cells from the cardiovascular system.

There are two major impact areas of the proposed research and technologies developed during this proposed programme of research:
The first is in terms of new and detailed quantitative molecular pharmacology knowledge gained regarding allosterism arising from cooperative interactions occurring across oligomeric interfaces within discrete membrane domains of living cells. These interactions can lead to changes in ligand binding affinity, agonist efficacy and signalling bias. It has become clear that the pharmacology of a cell surface G protein-coupled receptor can be markedly changed by its local environment. This is largely a consequence of interactions with neighbouring proteins in specific membrane microdomains. This can lead to cell type or microdomain specific pharmacology and signalling, and may explain why drugs developed through high throughput screening campaigns often lack efficacy in the clinic. The drugs are effectively targeted at the right receptor but in the wrong oligomeric context and location for therapeutic benefit in the disease in question. Our work therefore has direct relevance to the academic community, the pharmaceutical industry and the biotechnology industry.

Ultimately, the healthcare sector may benefit as a consequence of new therapeutic opportunities arising out of the new knowledge gained. GPCRs are a major target for drug discovery. The research on the receptors targeted in this programme of work has wide implications for the cardiovascular system and the treatment of cancer. Our planned research therefore has the potential to provide significant long term impact in meeting future clinical need, delivering and improving therapies for a range of debilitating diseases and contributing to human health.

The second major impact will come from the technologies and methodologies developed and implemented to interrogate the molecular pharmacology of GPCRs. The application of exquisitely sensitive fluorescence correlation spectroscopy (FCS) measurements in small areas of the membrane together with novel BRET technologies for the evaluation ligand-binding and protein-protein interactions should revolutionize the study of cell surface receptors and have application for most drug discovery targets. Beneficiaries will include academics and drug discovery scientists in research institutes, major pharma, SMEs and the biotechnology industry.

The impact of the work will be initially communicated through publication of our research findings and technology advances, according to the research councils' open access policy. However, in parallel, steps will be taken to showcase the power of the technology in international scientific meetings. The project will also train research staff in multidisciplinary skills at the interface between molecular pharmacology, medicinal chemistry and imaging/biophysics.