Spatial Intensity Distribution Analysis: Studies of G Protein-Coupled Receptor Oligomerisation

Spatial intensity distribution analysis (SpIDA) is a recently developed approach for determining quaternary structure information on fluorophore-labelled proteins of interest in situ. It can be applied to live or fixed cells and native tissue. Using confocal images, SpIDA generates fluorescence inte...

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Bibliographic Details
Published in:Trends in pharmacological sciences (Regular ed.) 2018-02, Vol.39 (2), p.175-186
Main Authors: Pediani, John D., Ward, Richard J., Marsango, Sara, Milligan, Graeme
Format: Article
Language:English
Subjects:
Animals
dimerisation
G protein-coupled receptor
Humans
ligand regulation
Microscopy, Fluorescence - methods
Protein Multimerization
quaternary structure
Receptors, G-Protein-Coupled - chemistry
Receptors, G-Protein-Coupled - metabolism
Single Molecule Imaging - methods
SpIDA
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Summary:Spatial intensity distribution analysis (SpIDA) is a recently developed approach for determining quaternary structure information on fluorophore-labelled proteins of interest in situ. It can be applied to live or fixed cells and native tissue. Using confocal images, SpIDA generates fluorescence intensity histograms that are analysed by super-Poissonian distribution functions to obtain density and quantal brightness values of the fluorophore-labelled protein of interest. This allows both expression level and oligomerisation state of the protein to be determined. We describe the application of SpIDA to investigate the oligomeric state of G protein-coupled receptors (GPCRs) at steady state and following cellular challenge, and consider how SpIDA may be used to explore GPCR quaternary organisation in pathophysiology and to stratify medicines. GPCRs may exist and function as monomers: however, abundant evidence suggests they can form dimers/oligomers. This concept has implications for drug discovery as it may offer opportunities to modulate the effects of known pharmaceuticals or identify new drug therapies. A variety of approaches have been applied to this issue from traditional biochemical techniques, via resonance energy transfer approaches to recently developed image analysis-based techniques such as SpIDA. This uses mathematical analysis of confocal microscopy images to generate quantal brightness and density information for a fluorophore-tagged receptor. SpIDA can be applied to live or fixed cells and native tissue. SpIDA has been applied to GPCRs from each of the major subfamilies to explore their oligomerisation status at steady state and their regulation by receptor density and ligand binding.
ISSN:0165-6147
1873-3735
DOI:10.1016/j.tips.2017.09.001