|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
First published online January 23, 2008
doi: 10.1242/10.1242/jcs.018440
Short Report |



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
![]()
CiteULike
Complore
Connotea
Del.icio.us
Digg
Reddit
Technorati
Twitter What's this?
This article has been cited by other articles:
![]() |
D. J. Killock, M. Parsons, M. Zarrouk, S. M. Ameer-Beg, A. J. Ridley, D. O. Haskard, M. Zvelebil, and A. Ivetic In Vitro and in Vivo Characterization of Molecular Interactions between Calmodulin, Ezrin/Radixin/Moesin, and L-selectin J. Biol. Chem., March 27, 2009; 284(13): 8833 - 8845. [Abstract] [Full Text] [PDF] |
||||
![]() |
E. Puklin-Faucher and M. P. Sheetz The mechanical integrin cycle J. Cell Sci., January 15, 2009; 122(2): 179 - 186. [Abstract] [Full Text] [PDF] |
||||
![]() |
N. Watanabe, L. Bodin, M. Pandey, M. Krause, S. Coughlin, V. A. Boussiotis, M. H. Ginsberg, and S. J. Shattil Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin {alpha}IIb{beta}3 J. Cell Biol., October 22, 2008; 181(7): 1211 - 1222. [Abstract] [Full Text] [PDF] |
||||