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?