title: Quantification of integrin receptor agonism by fluorescence lifetime imaging.
abstract: 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 beta1 integrin and paxillin with alpha4 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.
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guys Campus, London, SE1 1UL, UK. maddy.parsons@kcl.ac.uk
authors.forename: Maddy
authors.initials: M
authors.lastname: Parsons
authors.forename: Anthea J
authors.initials: AJ
authors.lastname: Messent
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Humphries
authors.forename: Nicholas O
authors.initials: NO
authors.lastname: Deakin
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000235:genetics / Q000378:metabolism; D016023:Integrins / Q000819:agonists* / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016608:Talin / Q000378:metabolism; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D019012:Integrin beta1; D016023:Integrins; D008024:Ligands; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D016608:Talin; D000097573:Red Fluorescent Protein