Structural interlock between ligand-binding site and stalk-like region of beta1 integrin revealed by a monoclonal antibody recognizing conformation-dependent epitope.
Integrin activation and sebsequent ligand binding to it are regulated by intracellular mechanisms called inside-out signaling, which are not fully understood and are accompanied by dynamic structural changes of the integrin molecule itself. A monoclonal antibody recognizing a conformation-dependent epitope on human beta1 integrin was produced and characterized in detail. This antibody, AG89, reacted with human integrin beta1 chain regardless of the alpha subunit. AG89 can recognize resting state beta1 integrin on the cells, but the reactivity is increased approximately 2-fold upon integrin activation by activating anti-beta1 antibodies and approximately 3-fold by Mn2+. Furthermore, occupation of the ligand-binding pocket by a soluble ligand (RGD peptide for alpha(v)beta1 and CS-1 peptide for alpha4beta1) resulted in maximum binding of AG89, indicating that the epitope for AG89 is exposed during the conformational changes of beta1 integrin upon activation/ligation. Epitope mapping by using interspecies chimeric beta1 revealed that the epitope for AG89 lies within residues 426-587, which corresponds to the cysteine-rich repeat structure located in the middle of the beta1 chain. The fact that binding of AG89 itself could activate the resting beta1 integrin indicates that exposure of the AG89 epitope in the membrane-proximal stalk-like domain and "opening" of the ligand-binding pocket at the outermost domain are physically linked. We propose that the integrin "signaling" is mediated by this direct physical transduction of conformational information along the integrin molecule.
Takagi
J
J
Department of Biological Sciences, Tokyo Institute of Technology, Midori-ku, Yokohama. jtakagi@bio.titech.ac.jp
Isobe
T
T
Takada
Y
Y
Saito
Y
Y
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D011233:Precipitin Tests; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000939:Epitopes; D016023:Integrins; D008024:Ligands; D016650:Fluorescein-5-isothiocyanate