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Structural interlock between ligand-binding site and stalk-like region of beta1 integrin revealed by a monoclonal
1Department of Biological Sciences, Tokyo Institute of Technology, Midori-ku, Yokohama. jtakagi@bio.titech.ac.jp
Insights
A new antibody, AG89, reveals how integrin activation involves structural changes. This antibody binds to a specific site on beta1 integrin, showing a physical link between activation and ligand binding for better inside-out signaling understanding.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Integrin activation and ligand binding are regulated by inside-out signaling.
- The dynamic structural changes of integrins during activation are not fully understood.
Purpose of the Study:
- To characterize a novel monoclonal antibody, AG89, that recognizes a conformation-dependent epitope on human beta1 integrin.
- To investigate the structural changes of beta1 integrin during activation and ligand binding.
Main Methods:
- Production and detailed characterization of the monoclonal antibody AG89.
- Assessing AG89 binding to beta1 integrin under various activation conditions (activating antibodies, Mn2+, soluble ligands).
- Epitope mapping using interspecies chimeric beta1 integrins.
Main Results:
- AG89 reacts with the human integrin beta1 chain irrespective of the alpha subunit.
- AG89 binding increases upon integrin activation and is maximal when the ligand-binding pocket is occupied.
- The epitope for AG89 is located within residues 426-587, a cysteine-rich repeat structure.
- AG89 binding itself can activate resting beta1 integrin, suggesting a physical link between epitope exposure and ligand-binding pocket opening.
Conclusions:
- The AG89 epitope is exposed during beta1 integrin activation and ligation.
- Integrin signaling may be mediated by direct physical transduction of conformational information along the molecule.
- This study provides insights into the structural mechanisms of integrin inside-out signaling.
Abstract:
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.