Topography of Ligand-induced Binding Sites, Including a Novel Cation-sensitive Epitope (AP5) at the Amino Terminus, of the Human Integrin β3 Subunit

• Published in: The Journal of biological chemistry 1995-05, Vol.270 (20), p.11947-11954
• Main Authors: Honda, Shigenori, Tomiyama, Yoshiaki, Pelletier, Anthony J., Annis, Doug, Honda, Yumiko, Orchekowski, Randal, Ruggeri, Zaverio, Kunicki, Thomas J.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.20.11947

Changes in ligand binding ability of the integrin αIIbβ3 can be monitored by the concomitant expression of ligand-inducible binding sites (LIBS). A new LIBS, the hexapeptide sequence GPNICT (residues 1-6) at the amino terminus of β3 recognized by the murine monoclonal antibody (mAb) AP5, is sensitive both to the binding of ligand and to micromolar differences in divalent cation levels. Calcium or magnesium can completely inhibit the binding of AP5 to αIIbβ3 on platelets, with ID50 values of 80 and 1500 μM, respectively. The inhibitory effect of calcium plus magnesium is cumulative. In the presence of 1 mM calcium plus 1 mM magnesium, the peptide RGDW overcomes this inhibition and induces maximal binding of AP5. Maximal AP5 binding is also induced by a molar excess of EDTA. The unique location of the AP5 LIBS was determined by comparing the binding of LIBS-specific mAb to recombinant human-Xenopus β3 chimeras produced in a baculovirus expression system. AP5 defines one region at the amino terminus β31-6. A second region, defined by mAb D3GP3, is probably located within β3422-490, confirming the finding of Kouns et al. (Kouns, W. C., Newman, P. J., Puckett, K. 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 more precisely located within 602-690 (Du X., Gu, M., Weise, J. W., Nagaswami, C., Bennett, J. S., Bowditch, R., and Ginsberg, M. H.(1993) J. Biol. Chem. 268, 23087-23092), is recognized by the four mAb, anti-LIBS2, anti-LIBS3, ant-LIBS6, and P41. Since its exposure is uniquely regulated by both divalent cations and ligand, the amino terminus of β3 may be involved in control of ligand binding by divalent cation mobilization.