STRUCTURE-FUNCTION ANALYSIS OF THE INTEGRIN BETA-1-SUBUNIT BY EPITOPE MAPPING

Monoclonal antibodies (mAbs) have been produced against the chicken beta1 subunit that affect integrin functions, including ligand binding, alpha 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 beta1 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 beta1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta1 subunit by homologous regions of the mouse beta1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta1 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 beta1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta 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 beta1 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 alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta1 subunit.