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Volume 272, Number 7, Issue of February 14, 1997 pp. 4103-4107
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular Determinants of Arg-Gly-Asp Ligand Specificity for beta 3 Integrins

(Received for publication, September 4, 1996, and in revised form, November 19, 1996)

Thomas J. Kunicki , Douglas S. Annis and Brunhilde Felding-Habermann

From the Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Department of Molecular and Experimental Medicine, and the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

The Arg-Tyr-Asp (RYD) and Arg-Gly-Asp (RGD) sequences within the third complementarity-determining region of the heavy chain (H3) of murine recombinant Fab molecules OPG2 and AP7, respectively, are responsible for their specific binding to the platelet integrin alpha IIbbeta 3. In this study, we evaluated the influence of divalent cation composition and single amino acid substitutions at key positions within H3 on the selectivity of these Fab molecules for integrin alpha IIbbeta 3 versus the vitronectin receptor alpha Vbeta 3. The parent Fab molecule OPG2 (H3 sequence, HPFYRYDGGN) binds selectively to alpha IIbbeta 3 and not at all to any other RGD-cognitive integrin, particularly alpha Vbeta 3, under any divalent cation conditions. The binding of the AP7 Fab molecule (HPFYRGDGGN) to alpha IIbbeta 3 is not affected by the relative composition of calcium, magnesium or manganese. However, AP7 binding to alpha Vbeta 3, either expressed by M21 cells or as the purified integrin, is supported by manganese and inhibited by calcium. If the flanking asparagine 108 residue within the AP7 H3 loop is replaced by alanine (HPFYRGDGGA), the resulting Fab molecule AP7.4 binds selectively to alpha Vbeta 3 in a cation-dependent manner, but does not bind at all to alpha IIbbeta 3 under any conditions. AP7.4 binding to alpha Vbeta 3 is supported by manganese, completely inhibited by calcium, and largely unaffected by magnesium. This behavior mimics that of the adhesive protein, osteopontin, another ligand that binds preferentially to alpha Vbeta 3. Despite these differences in specificity for alpha IIbbeta 3 and alpha Vbeta 3, AP7 and AP7.4 remain selective for the beta 3 integrins and do not bind to cell lines that express the RGD-cognitive integrins alpha Vbeta 5 or alpha 5beta 1. These results confirm that subtle changes in the amino acid composition immediately flanking the RGD or RYD motifs can have a profound effect on beta 3 integrin specificity, most likely because they influence the juxtaposition of the arginine and aspartate side chains within the extended RGD loop sequence.


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