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Adenosine A1-receptor binding and signalling in membrane microdomains of single living cells

Reference	BB/D521581/1
Principal Investigator / Supervisor	Professor Stephen Hill
Co-Investigators / Co-Supervisors	Dr Stephen Briddon, Professor Barrie Kellam
Institution	University of Nottingham
Department	Sch of Biomedical Sciences
Funding type	Research
Value (£)	295,119
Status	Completed
Type	Research Grant
Start date	01/12/2005
End date	30/11/2008
Duration	36 months

Abstract

The adenosine-A1 receptor is a member of the G-protein coupled receptors (GPCR) superfamily, and is responsible for many of the physiological actions of adenosine in the control of respiration, the cardiovascular system, and in the central nervous system. In the cell membrane, the adenosine A1-receptor is thought to exist within organised signalling domains such as caveolae and receptor activation is accompanied by redistribution among these domains. It is not known, however, whether or how the pharmacology of the receptor differs between the different compartments or when associated with different signalling or partner molecules. Current methodology (e.g. radioligand binding) does not afford such information. The aim of this project is to use the technique of fluorescence correlation spectroscopy (FCS) in conjunction with novel fluorescent A1-receptor ligands to investigate receptor pharmacology in different membrane domains at the single cell and receptor level. FCS uses a diffraction-limited laser beam to create a very small detection volume (less than 0.5fl). Diffusion of fluorescent species through this volume results in fluctuations in the fluorescent signal detected. Statistical analysis of these fluctuations yields both the diffusion rate of the flurosphore (related to its mass) and its concentration. As FCS is a non-invasive technique, positioning of the detection volume on a cell membrane allows fast-moving free ligand and slow moving bound ligand to be distinguished and quantified. This yields all the necessary information for pharmacological analysis in a small (0.1 micrometre squared) area of membrane. A further extension of this technique is cross-correlation or two-colour FCS, which uses co-localised blue and red laser lines to detect the co-diffusion of two spectrally different but interacting species. We have previously synthesised red fluorescent antagonist and agonist ligands for the A1-receptor and showed that FCS can quantify their binding to the receptor in the membranes of A1-expressing cells. These studies have shown important differences in the way the agonist- and antagonist-receptor complexes diffuse. Initially, we will extend these studies to investigate the nature of these differences, by doing more extensive kinetic analysis of the to the native and GFP-tagged A1-receptors (using cross-correlation FCS between the red ligand and green receptor). Using known fluorescent markers for different membrane domains (e.g. caveolae, cytoskeleton), we will then investigate the domains within which the receptor and the bound ligand are found using confocal imaging. Subsequently, FCS measurements will be localised to these domains, using the appropriate label. A full comparison of ligand pharmacology between different receptor subpopulations will be carried out. In instances where the domains are highly mobile, these measurements will be confirmed by cross-correlation analysis of ligand and domain marker. Further to this, the A1-receptor interacts with a number of scaffolding and signalling proteins, such as adenosine deaminase and caveolin. Cross-correlation analysis between GFP labelled G-protein alpha subunits, GFP-caveolin or antibody-tagged adensine deaminase, will be used to investigate the pharmacology of the A1-receptor when associated with each of these proteins. In particular ligand effects on receptor G-protein interaction between different alpha subunits (alpha 1/2/3, alphas and alphaq) will be investigated. We will then extend the study to simultaneously measure ligand binding and functional response within these domains using a relationship between receptor occupancy and functional response within different compartments.

Summary

Adenosine is a molecule that binds to specialised docking sites on the outside of cells called adenosine receptors. If a cell is stimulated, adenosine is released and this binds to the receptors of neighbouring cells and hence stimulates them. This is important in many physiological responses including breathing control, sleeping and protecting the heart or brain from damage caused by lack of oxygen (for example in a heart attack or stroke). It is now clear that there are several different types of adenosine receptors (of which the adenosine A1-receptor is one example) and secondly that these receptors are localised in very tiny and highly specialised regions of the cell membrane called microdomains. These microdomains contain a collection of different molecules that are involved in telling the cell how to respond to drugs or hormones. It is quite possible that the adenosine receptors in the different microdomains can be recognised by drugs differently. The aim of this proposal is to use highly sophisticated laser-based microscopy to study the way that drugs bind to A1-receptors in these small membrane microdomains in living cells. This is achieved by using a drug molecule that has a fluorescent label attached to it. The fluorescent drug can then be followed as it binds to the adenosine receptor in real time at the single molecule level. On its own, the small fluorescent drug molecule moves quickly through a laser beam and gives off light (photons). When the drug binds to a receptor, it becomes much bigger and heavier and so moves much more slowly and gives off a different pattern of light. By analysing the time that each fluorescent molecule is present within the laser be4am, we can count the number of free drug molecules and the number of receptor-bound drug molecules that are present. The relationship between these two numbers gives important information on how well the drug is binding at a particular concentration of the drug.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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