Monoclonal antibody 9EG7 defines a novel beta 1 integrin epitope induced by soluble ligand and manganese, but inhibited by calcium.
The monoclonal antibody 9EG7 has been previously found to recognize an epitope induced by manganese on the integrin beta 1 chain (Lenter, M., Uhlig, H., Hamann, A., Jeno, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 9051-9055). Here we show that treatment of beta 1 integrins with manganese or soluble integrin ligands (e.g. fibronectin and RGD peptide) induced the 9EG7 epitope. This epitope was also induced upon EGTA treatment to remove calcium, and the addition of calcium inhibited 9EG7 epitope induction by manganese or by ligand. Further emphasizing the importance of the 9EG7 epitope, the 9EG7 antibody itself stimulated adhesion mediated by multiple beta 1 integrins, and conversely, ligands for alpha 2 beta 1, alpha 3 beta 1, alpha 4 beta 1, and alpha 5 beta 1 all stimulated 9EG7 expression. Together these results support a model whereby (i) calcium inhibits beta 1 integrin function because it prevents the appearance of a conformation favorable to ligand binding and (ii) manganese enhances beta 1 integrin function because it induces the same favorable conformation that is induced by adding ligand, or removing calcium. Notably, other beta 1-stimulating agents (magnesium and mAb TS2/16) did not induce 9EG7 expression unless ligand was also present. Thus, although 9EG7 may reliable detect the ligand-bound conformation of beta 1 integrins, its expression does not always correlate with integrin "activation". Finally, mouse/chicken beta 1 chimeric molecules were used to map the 9EG7 epitope to beta 1 residues 495-602 within the cysteine-rich region, and antibody cross-blocking studies showed that the 9EG7 epitope is distinct from all previously defined human beta 1 epitopes.
Bazzoni
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Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Shih
D T
DT
Buck
C A
CA
Hemler
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002448:Cell Adhesion / Q000187:drug effects; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D018604:Epitope Mapping; D000939:Epitopes / Q000187:drug effects*; D005353:Fibronectins / Q000494:pharmacology; D006801:Humans; D019012:Integrin beta1 / Q000187:drug effects / Q000276:immunology*; D008024:Ligands; D008345:Manganese / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005353:Fibronectins; D019012:Integrin beta1; D008024:Ligands; D009842:Oligopeptides; D008345:Manganese; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium