title: Disruption of integrin-fibronectin complexes by allosteric but not ligand-mimetic inhibitors.
abstract: Failure of Arg-Gly-Asp (RGD)-based inhibitors to reverse integrin-ligand binding has been reported, but the prevalence of this phenomenon among integrin heterodimers is currently unknown. In the present study we have investigated the interaction of four different RGD-binding integrins (α5β1, αVβ1, αVβ3 and αVβ6) with fibronectin (FN) using surface plasmon resonance. The ability of inhibitors to reverse ligand binding was assessed by their capacity to increase the dissociation rate of pre-formed integrin-FN complexes. For all four receptors we showed that RGD-based inhibitors (such as cilengitide) were completely unable to increase the dissociation rate. Formation of the non-reversible state occurred very rapidly and did not rely on the time-dependent formation of a high-affinity state of the integrin, or the integrin leg regions. In contrast with RGD-based inhibitors, Ca2+ (but not Mg2+) was able to greatly increase the dissociation rate of integrin-FN complexes, with a half-maximal response at ~0.4 mM Ca2+ for αVβ3-FN. The effect of Ca2+ was overcome by co-addition of Mn2+, but not Mg2+. A stimulatory anti-β1 monoclonal antibody (mAb) abrogated the effect of Ca2+ on α5β1-FN complexes; conversely, a function-blocking mAb mimicked the effect of Ca2+. These results imply that Ca2+ acts allosterically, probably through binding to the adjacent metal-ion-dependent adhesion site (ADMIDAS), and that the α1 helix in the β subunit I domain is the key element affected by allosteric modulators. The data suggest an explanation for the limited clinical efficacy of RGD-based integrin antagonists, and we propose that allosteric antagonists could prove to be of greater therapeutic benefit.
authors.affiliation: *Biomolecular Analysis Core Facility, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester M13 9PT, U.K.
authors.forename: A Paul
authors.initials: AP
authors.lastname: Mould
authors.affiliation: †Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
authors.forename: Susan E
authors.initials: SE
authors.lastname: Craig
authors.affiliation: †Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Byron
authors.affiliation: †Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
authors.affiliation: *Biomolecular Analysis Core Facility, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester M13 9PT, U.K.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Jowitt
mesh_terms: D000495:Allosteric Site / Q000187:drug effects; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D002478:Cells, Cultured; D005353:Fibronectins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D039081:Integrin alpha5beta1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D016023:Integrins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D046912:Multiprotein Complexes / Q000187:drug effects* / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D019030:Receptors, Vitronectin / Q000037:antagonists & inhibitors / Q000378:metabolism; D061987:Sf9 Cells; D012910:Snake Venoms / Q000494:pharmacology; D018411:Spodoptera
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D005353:Fibronectins; D039081:Integrin alpha5beta1; D039302:Integrin alphaVbeta3; D016023:Integrins; D008024:Ligands; D046912:Multiprotein Complexes; D009842:Oligopeptides; D010446:Peptide Fragments; D019030:Receptors, Vitronectin; D012910:Snake Venoms; C094340:integrin alphavbeta1; C073878:integrin alphavbeta6; C422910:Cilengitide; C047981:arginyl-glycyl-aspartic acid