title: Dual functionality of the anti-beta1 integrin antibody, 12G10, exemplifies agonistic signalling from the ligand binding pocket of integrin adhesion receptors.
abstract: Although integrins are known to mediate connections between extracellular adhesion molecules and the intracellular actin cytoskeleton, the mechanisms that are responsible for coupling ligand binding to intracellular signaling, for generating diversity in signaling, and for determining the efficacy of integrin signaling in response to ligand engagement are largely unknown. By characterizing the class of anti-integrin monoclonal antibodies (mAbs) that stimulate integrin activation and ligand binding, we have identified integrin-ligand-mAb complexes that exhibit differential signaling properties. Specifically, addition of 12G10 mAb to cells adhering via integrin alpha4beta1 was found to trigger disruption of the actin cytoskeleton and prevent cell attachment and spreading, whereas mAb addition to cells adhering via alpha5beta1 stimulated all of these processes. In contrast, soluble ligand binding to either alpha4beta1 or alpha5beta1 was augmented or unaffected by 12G10. The regions of the integrin responsible for differential signaling were then mapped using chimeras. Surprisingly, a chimeric alpha5 integrin containing the beta-propeller domain from the ligand binding pocket of alpha4 exhibited the same signaling properties as the full-length alpha4 integrin, whereas exchanging or removing cytoplasmic domains had no effect. Thus the mAb 12G10 demonstrates dual functionality, inhibiting cell adhesion and spreading while augmenting soluble ligand binding, via a mechanism that is determined by the extracellular beta-propeller domain of the associating alpha-subunit. These findings therefore demonstrate a direct and variable agonistic link between the ligand binding pocket of integrins and the cell interior that is independent of the alpha cytoplasmic domains. We propose that either ligand-specific transmembrane conformational changes or ligand-specific differences in the kinetics of transmembrane domain separation underlie integrin agonism.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Humphries
authors.forename: Neil R
authors.initials: NR
authors.lastname: Schofield
authors.forename: Zohreh
authors.initials: Z
authors.lastname: Mostafavi-Pour
authors.forename: Linda J
authors.initials: LJ
authors.lastname: Green
authors.forename: Alistair N
authors.initials: AN
authors.lastname: Garratt
authors.forename: A Paul
authors.initials: AP
authors.lastname: Mould
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001665:Binding Sites; D019556:COS Cells; D002412:Cations; D002448:Cell Adhesion; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D039041:Integrin alpha4beta1 / Q000378:metabolism; D039081:Integrin alpha5beta1 / Q000378:metabolism; D019012:Integrin beta1 / Q000737:chemistry* / Q000378:metabolism; D016023:Integrins / Q000737:chemistry* / Q000378:metabolism; D020014:K562 Cells; D007700:Kinetics; D008024:Ligands; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D002412:Cations; D018076:DNA, Complementary; D039041:Integrin alpha4beta1; D039081:Integrin alpha5beta1; D019012:Integrin beta1; D016023:Integrins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D019010:Vascular Cell Adhesion Molecule-1; D004247:DNA