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

doi:10.1074/jbc.270.20.11947

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

From the (1) From The Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis of the Departments of Molecular and Experimental Medicine and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, the Blood Research Institute of the Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233, and The Second Department of Internal Medicine, Osaka University Medical School, Osaka 565, Japan

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Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, 10666 N. Torrey Pines Rd., Maildrop SBR13, La Jolla, CA 92037.
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Abstract

Changes in ligand binding ability of the integrin αβ₃ 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 αβ₃ on platelets, with ID 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.

Footnotes

↵* This study was supported by National Heart, Lung, and Blood Institute Grants HL-46979 (to T. J. K.) and HL-31950 (to Z. M. R.). This is manuscript number 8196-MEM from The Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¹ The abbreviations used are:

LIBS

ligand-inducible binding sites

mAb

monoclonal antibody

FITC

fluorescein isothiocyanate

HPLC

high performance liquid chromatography

PRP

platelet-rich plasma

PGE₁

prostaglandin E₁

bp

base pair(s)

ELISA

enzyme-linked immunosorbant assay

MFI

mean fluorescence intensity.
↵²A. J. Pelletier, manuscript in preparation.