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Exploring the selectivity and consequences of GPCR homo and hetero- dimerisation/oligomerisation using RASSLs

ReferenceBB/E006302/1
Principal Investigator / Supervisor Professor Graeme Milligan
Co-Investigators /
Co-Supervisors
Institution University of Glasgow
DepartmentInstitute of Biomedical & Life Sciences
Funding typeResearch
Value (£) 416,349
StatusCompleted
TypeResearch Grant
Start date 17/04/2007
End date 16/04/2010
Duration36 months

Abstract

Many, if not all, rhodopsin-like, class A G protein- coupled receptors appear to exist as dimers or higher-order oligomers. A series of questions remain. Among the most demanding to investigate are whether GPCR monomers remain physically associated throughout their life-history, whether the binding of a single agonist molecule to a GPCR dimer is sufficient to initiate signal transduction, internalisation from the cell surface and desensitisation, the extent and selectivity of hetero-dimerisation/oligomerisation between co-expressed pairs of GPCRs and whether GPCRs are generally restricted to dimers or routinely form more complex oligomeric structures. A major limitation to examining the contribution of each element of homo-dimers of aminergic GPCRs is that each binds the same natural ligand. Mutation of GPCRs such that they lose affinity for the natural ligand but gain affinity for a specific synthetic molecule offers means to overcome this problem. We will employ such Receptors Activated Solely by Synthetic Ligands (RASSLs) of muscarinic acetylcholine receptors for this purpose. We have been developing improved and novel means to image the presence of oligomeric protein complexes in single living cells via sequential 3 colour fluorescence resonance energy transfer and will continue to develop this system to monitor muscarinic receptor oligomers. We have also employed bi-molecular fluorescence complementation as a means to identify receptor dimers and will use this approach to examine if muscarinic receptors exist as multi-polypeptide complexes throughout their life history. These studies will develop and utilise a series of technically demanding imaging approaches to address many of the key unanswered questions about GPCR 'dimerisation'.

Summary

G protein-coupled receptors (GPCRs) are the largest family of proteins that transmit signals from the outside of cells to the inside. They are also the targets for a wide range of small molecule medicines developed and produced by the pharmaceutical industry. It has become increasingly clear that GPCRs can exist as dimers or higher-order oligomers and this appears to be integral to their function. Because a wide range of different GPCRs are expressed by individual cells, then it is probable that as well as dimers or oligomers consisting of two of more copies of the same GPCR, 'hetero-dimers' that consist of pairs of different GPCRs are present in cells. It is likely that these will function and be regulated in a distinct manner to homo-dimers consisting of two identical GPCRs. It is difficult to examine different roles of the two elements of a homo-dimer because they bind and respond to the same ligand and this is also true for hetero-dimers between pairs of highly related GPCRs. However, mutagenesis of GPCRs can produce forms of GPCRs generally called Receptors Activated Solely by Synthetic Ligands (RASSLs). I will use RASSLs for the group of GPCRs that respond to acetylcholine to examine these questions. I will also refine and further develop methods we have developed that allow us to predict which GPCRs are able to interact to form hetero-dimers and which are not. Finally, I also plan to develop novel techniques to 'see' whether GPCRs in living cells are organised only as dimers or may form larger, oligomeric structures.
Committee Closed Committee - Biochemistry & Cell Biology (BCB)
Research TopicsStructural Biology, Technology and Methods Development
Research PriorityX – Research Priority information not available
Research Initiative X - not in an Initiative
Funding SchemeX – not Funded via a specific Funding Scheme
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