Multi-colour single molecule fluorescence-based analysis of native G protein-coupled receptor organization

G-protein coupled receptors (GPCRs) are proteins present on the surfaces of most cells in humans and other animals. These proteins detect both light and molecules in solution, producing signals inside the cell that tell it that the particular substance has been detected. These signals form the basis of our sense of sight and taste as well as regulating our mood and behaviour. The GPCRs are the largest group of cellular receptors encoded by the human genome, and are the targets for more than half the drugs presently in clinical use. It is, therefore, of the utmost importance to properly understand how these molecules exert their effects. Many important studies have not been performed due to the lack of sufficiently sensitive methods to detect individual proteins on live cells. We have developed a fluorescence-based microscopic method, which we have shown to have the necessary sensitivity to analyse individual proteins on live cells. We intend to use this new method to address some of the key questions about the structure and behaviour of GPCRs. In particular, we will determine how the receptors are organised on the cell surface and how this changes when GPCRs detect molecules from solution. These questions are among the most contentious in the entire field of GPCR biology. This research has the potential to fundamentally change our thinking about how these molecules work.

G- protein coupled receptors (GPCRs) are the largest family of receptors on the surface of eukoryotic cells and the main target for clinical drugs. For this reason it is important to study the endogenous receptors, using methods applicable to living cells, and resolve the current controversy whether many GPCRs exist or function as monomers or dimers, how they are distributed on the cell surface and this changes on activation. We propose to use the single molecule methods we have recently developed, based on the detection of coincident fluorescence signal from individual labelled receptors or ligands (PNAS 104, 17662) to directly address these questions. We will single molecule florescence to probe unactivated GPRs, activated GPCRs, the interaction of GPCRs with the G protein and with membrane signalling compartments for both beta2AR and CCR5. These studies should provide new insights into the distribution and function of these important receptors.