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First published online January 23, 2008
doi: 10.1242/10.1242/jcs.018440

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Quantification of integrin receptor agonism by fluorescence lifetime imaging

Maddy Parsons^1,*,, Anthea J. Messent^2,*,, Jonathan D. Humphries², Nicholas O. Deakin² and Martin J. Humphries^2,

¹ Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guys Campus, London, SE1 1UL, UK
² Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

Authors for correspondence (e-mails: maddy.parsons{at}kcl.ac.uk; martin.humphries{at}manchester.ac.uk)

Accepted 4 October 2007

Summary

Both spatiotemporal analyses of adhesion signalling and the development of pharmacological inhibitors of integrin receptors currently suffer from the lack of an assay to measure integrin-effector binding and the response of these interactions to antagonists. Indeed, anti-integrin compounds have failed in the clinic because of secondary side effects resulting from agonistic activity. Here, we have expressed integrin-GFP and effector-mRFP pairs in living cells and quantified their association using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Association of talin with β1 integrin and paxillin with 4 integrin was dependent on both the ligand and receptor activation state, and was sensitive to inhibition with small molecule RGD and LDV mimetics, respectively. An adaptation of the assay revealed the agonistic activity of these small molecules, thus demonstrating that these compounds may induce secondary effects in vivo via integrin activation. This study provides insight into the dependence of the activity of small molecule anti-integrin compounds upon receptor conformation, and provides a novel quantitative assay for the validation of potential integrin antagonists.

Key words: FRET/FLIM, Integrin, Integrin activation, Antagonist, Microscopy

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