An Allosteric Ca2+ Binding Site on the β3-Integrins That Regulates the Dissociation Rate for RGD Ligands

doi:10.1074/jbc.271.36.21745

An Allosteric Ca²⁺ Binding Site on the β3-Integrins That Regulates the Dissociation Rate for RGD Ligands*

From the ^‡ Program on Cell Adhesion, Cancer Research Center, The Burnham Institute, La Jolla, California 92037 and the
^¶ Department of Molecular Biology (MB11), The Scripps Research Institute, La Jolla, California 92037

** An Established Investigator of the American Heart Association under the sponsorship of Genentech. To whom correspondence should be addressed:
Program on Cell Adhesion, Cancer Research Center, The Burnham Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037.

Abstract

Here we use a model RGD-containing ligand to study how Ca²⁺ and Mg²⁺ regulate ligand binding to β3-integrins. Fab-9, an antibody that contains an optimized RGD loop in its antigen binding site (Barbas, C. F., Languino, L., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10003-10007), was used as the model ligand. Across a physiologic range of Mg²⁺, Fab-9 bound to both αvβ3 and αIIbβ3 with a monophasic binding isotherm. Across the same range of Ca²⁺, the binding of Fab-9 to the β3-integrins was biphasic. Low concentrations of Ca²⁺ (μM) promoted the binding of Fab-9. Higher concentrations of Ca²⁺ (mM) blocked Fab-9 binding. These data suggest that Ca²⁺ binds to two distinct classes of sites on the β3-integrins, with the low affinity Ca²⁺ binding site(s) being an inhibitory site. We designate this inhibitory site(s) as the I site. Further biochemical characterization showed that the I site has the following characteristics: 1) it is specific for Ca²⁺; 2) it is allosteric to the ligand binding site; 3) its occupation increases the dissociation rate between integrin and RGD ligand; and 4) occupation of the I site can induce cellular deadhesion.

Footnotes

↵^§ Supported initially by a postdoctoral grant from Monsanto/Searle and subsequently by a postdoctoral grant from the California Affiliate of the American Heart Association.
↵^∥ A Scholar of the American Foundation for AIDS Research and the recipient of an Investigator Award from the Cancer Research Institute.
↵* This work was supported by National Institutes of Health Grants CA 56483 and AR 42750 (to J. W. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Received February 23, 1996.
- Revision received May 1, 1996.