The inhibitory anti-beta1 integrin monoclonal antibody 13 recognizes an epitope that is attenuated by ligand occupancy. Evidence for allosteric inhibition of integrin function.
Integrin-ligand binding causes conformational changes in the integrin, as evidenced by the increased expression of epitopes known as ligand-induced binding sites. Some monoclonal antibodies (mAbs) that recognize ligand-induced binding sites stimulate ligand binding, possibly by stabilizing the ligand-occupied conformation of the integrin. Here we have investigated the effect of ligand recognition by alpha5beta1 on the binding of a mAb that inhibits beta1 integrin function (mAb 13). Ligand (fibronectin fragment or GRGDS peptide) decreased the binding of mAb 13 to alpha5beta1. Analysis of this inhibition showed that at high ligand concentrations, approximately 50% of the total integrin bound mAb 13 with >50-fold lower affinity than in the absence of ligand. The concentration of ligand required for half-maximal inhibition of antibody binding was independent of antibody concentration, suggesting that ligand acts as an allosteric inhibitor of mAb 13 binding. Hence, ligand and mAb 13 did not appear to compete directly for binding to alpha5beta1. The stimulatory anti-beta1 mAb 9EG7 was found to increase the maximum level of ligand binding approximately 2-fold, indicating that up to 50% of the total integrin could not bind ligand without 9EG7 stimulation. Analysis of mAb 13 binding in the presence of 9EG7 and ligand (i.e. maximal ligand occupancy) demonstrated that essentially all of the integrin bound mAb 13 with very low or zero affinity. Our results demonstrate that mAb 13 recognizes an epitope that is dramatically attenuated in the ligand-occupied form of alpha5beta1. Hence, since mAb 13 preferentially recognizes the unoccupied conformation of the integrin, the antibody may inhibit ligand binding by stabilizing the unoccupied state of alpha5beta1. In addition, we present evidence that the binding of mAb 13 to ligand-occupied alpha5beta1 may also induce a conformational change in the integrin, resulting in the displacement of ligand.
Mould
A P
AP
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
Akiyama
S K
SK
Humphries
M J
MJ
D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000937:Antigen-Antibody Reactions; D001667:Binding, Competitive; D000939:Epitopes; D006801:Humans; D019012:Integrin beta1 / Q000276:immunology*; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D051381:Rats; D017469:Receptors, Fibronectin / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019012:Integrin beta1; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D017469:Receptors, Fibronectin; C047981:arginyl-glycyl-aspartic acid