Structure/Function Analysis of the Integrin β 1 Subunit by Epitope Mapping

• Published in: The Journal of cell biology Vol. 122; no. 6; pp. 1361 - 1371
• Main Authors: Shih, Daw-Tsun, Edelman, Jonathan M., Horwitz, Alan F., Grunwald, Gerald B., Buck, Clayton A.
• Format: Journal Article
• Language: English
• Published: United States Rockefeller University Press 01-09-1993; The Rockefeller University Press
• Subjects: 3T3 Cells; Amino acids; Animals; Antibodies; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - immunology; Base Sequence; Cell Adhesion; Cells; Chickens; Chimera; Complementary DNA; Cysteine - analysis; DNA - analysis; DNA - genetics; Epitopes; Epitopes - chemistry; Epitopes - genetics; Epitopes - immunology; Integrins; Integrins - genetics; Integrins - immunology; Integrins - physiology; Ligands; Mice; Molecular Sequence Data; Peptide Mapping; Sequence Homology, Nucleic Acid
• ISSN: 0021-9525; 1540-8140
• DOI: 10.1083/jcb.122.6.1361

Monoclonal antibodies (mAbs) have been produced against the chicken β 1 subunit that affect integrin functions, including ligand binding, α subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric β 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel of anti-chicken β 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken β 1 subunit by homologous regions of the mouse β 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse β 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken β 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the β 1 subunit. This region is homologous to a portion of the ligand-binding domain of the β 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce α/β subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of β 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the α subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the β 1 subunit.