TOPOGRAPHY OF LIGAND-INDUCED BINDING-SITES, INCLUDING A NOVEL CATION SENSITIVE EPITOPE (AP5) AT THE AMINO-TERMINUS, OF THE HUMAN INTEGRIN BETA(3) SUBUNIT

Changes in ligand binding ability of the integrin alpha(IIb)beta(3) ca n be monitored by the concomitant expression of ligand-inducible bindi ng sites (LIES). A new LIES, the hexapeptide sequence GPNICT (residues 1-6) at the amino terminus of beta(3) recognized by the murine monocl onal antibody (mAb) AP5, is sensitive both to the binding of ligand an d to micromolar differences in divalent cation levels. Calcium or magn esium can completely inhibit the binding of AP5 to alpha(IIb)beta(3), on platelets, with ID50 values of 80 and 1500 mu M, respectively. The inhibitory effect of calcium plus magnesium is cumulative. In the pres ence of 1 mM calcium plus 1 mat magnesium, the peptide RGDW overcomes this inhibition and induces maximal binding of AP5. Maximal AP5 bindin g is also induced by a molar excess of EDTA. The unique location of th e AP5 LIES was determined by comparing the binding of LIBS specific mA b to recombinant human Xenopus beta(3) chimeras produced in a baculovi rus expression system. AP5 defines one region at the amino terminus be ta(3)1-6. A second region, defined by mAb D3GP3, is probably located w ithin beta(3)422-490, confirming the finding of Kouns ct al. (Kouns, W . C., Newman, P. J., Puckett, H. J., Miller, A. A., Wall, C, D., Fox, C. F., Seyer, J. M., and Jennings, L. K. (1991) Blood 78, 3215-3223). The third region, encompassing at most residues 490-690, and perhaps m ore precisely located within 602-690 (Du X., Gu, M., Weise, J. W., Nag aswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H. (1993) J . Biol. Chem. 268, 23087-23092), is recognized by the four mAb, anti-L IBS2, anti LIBSS, ant-LIBS6, and P41. Since its exposure is uniquely r egulated by both divalent cations and ligand, the amino terminus of be ta(3), may be involved in control of ligand binding by divalent cation mobilization.