Analysis of Arg-Gly-Asp contact sites in the av subunit of integrin avb3 employing single amino acid mutations

Abstract number: P0745

Honda^* S., Tomiyama^* Y., Kashiwagi^* H., Kiyoi^* T., Kato^* H., Kosugi^* S., Shiraga^* M., Kurata† Y., Matsuzawa^* Y.

†Osaka Uiv Hosp, Department of Blood Transfusion, Japan ^*Osaka Uiv, Department of Int Medical and Mol Sci, Japan;

Integrin avb3 is mainly expressed on endothelial cells, vascular smooth muscle cells, osteoclasts, and certain invasive tumors (melanoma cells, prostate carcinoma cells), and it is thought to contribute to vascular remodeling, bone adsorption, and tumor progression. We have investigated the ligand-binding sites in the av subunit of avb3, and demonstrated that Tyr178 in the 2–3 loops of blade 3 of b-propeller domain (W3 4–1 loop) is critically required for ligand binding. More recently, the extracellular segment of integrin avb3 was solved by X-ray crystallography in the form complexed with a cyclic Arg-Gly-Asp (RGD) ligand. The crystal structure analysis revealed that the Arg side chain of the cyclic RGD ligand is held in a groove by making salt bridges to Asp150 and Asp218, and sandwiched between Tyr178 and Ala215 by forming hydrophobic contacts at walls of the groove. However, functional significance of these residues remains fully determined. To evaluate the relative functional significance of each av residue that binds RGD-containing ligands, we have introduced single amino acid mutations into the RGD-contact loops of av (Arg143-Phe154, Gly172-Gly181, Asn206-Leu222) defined by crystallography. The ligand-binding function of mutant avb3 was determined by the amounts of WOW-1 Fab binding, a monovalent and RGD-containing ligand-mimetic anti-avb3 antibody. All mutant avb3 transiently transfected into 293T cells were well expressed on the cell surface (70–130% of wild-type avb3) in flow cytometry, employing the mAb LM609 specific for avb3 complex. The binding of WOW-1 Fab were examined in the presence of 0.5 mM MnCl₂ because WOW-1 Fab recognizes preferentially activated avb3. Mutation of Asp218 to Ala (Asp218Ala) in av-abolished WOW-1 Fab binding, as well as that of Tyr178Ala previously described, and Asp219Ala at the residue adjacent to Asp218 inhibited moderately at levels similar to Trp179Ala (40–58% of wild type). The single mutation of Asp150Ala also reduced it by approximately 40 compared with wild type. Other mutations in av did not significantly affect WOW-1 Fab binding. These results suggest that Tyr178 and Asp218 of RGD-contact residues in av critically participate in the interaction between avb3 and its RGD-containing ligands.