Monoclonal Antibody 9EG7 Defines a Novel Integrin Epitope Induced by Soluble Ligand and Manganese, but Inhibited by Calcium

doi:10.1074/jbc.270.43.25570

Monoclonal Antibody 9EG7 Defines a Novel Integrin Epitope Induced by Soluble Ligand and Manganese, but Inhibited by Calcium (*)

From the (1)Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and the
(2) Wistar Institute, Philadelphia, Pennsylvania 19104

^§ To whom correspondence should be addressed:
Dana-Farber Cancer Institute, Rm. M613, 44 Binney St., Boston, MA 02115.
Tel.: 617-632-3410; Fax: 617-632-2662.

Abstract

The monoclonal antibody 9EG7 has been previously found to recognize an epitope induced by manganese on the integrin β 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 β 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 β integrins, and conversely, ligands for αβ, αβ, αβ, and αβ all stimulated 9EG7 expression. Together these results support a model whereby (i) calcium inhibits β integrin function because it prevents the appearance of a conformation favorable to ligand binding and (ii) manganese enhances β integrin function because it induces the same favorable conformation that is induced by adding ligand, or removing calcium. Notably, other β-stimulating agents (magnesium and mAb TS2/16) did not induce 9EG7 expression unless ligand was also present. Thus, although 9EG7 may reliably detect the ligand-bound conformation of β integrins, its expression does not always correlate with integrin “activation.” Finally, mouse/chicken β chimeric molecules were used to map the 9EG7 epitope to β 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 β epitopes.

Footnotes

↵* This work was supported by National Institutes of Health Grants GM38903 (to M. E. H.) and CA19144 and HLB47670 (to C. A. B.) and by a fellowship from the Sanofi Foundation for Thrombosis Research, Paris, France (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¹ The abbreviations used are:

mAb

monoclonal antibody

BCECF-AM

2′,6′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester

CLIBS

cation- and ligand-influenced binding site

hBSA

heat-denatured BSA

LIBS

ligand-induced binding site

PMA

phorbol 12-myristate 13-acetate

TBS

Tris-buffered saline

aa

amino acid(s)

BSA

bovine serum albumin.
²C. Pujades, J. Teixido, and M. E. Hemler, submitted for publication.
³C. Pujades, S. K. Craeft, R. Alon, A. Masumoto, L. Burkly, T. A. Springer, L. B. Chen, R. R. Lobb, M. Hemler, submitted for publication.
- Received August 4, 1995.