Mapping the integrin α(v)β₃-ligand interface by photoaffinity cross- linking

Integrins are cell surface adhesion molecules involved in mediating cell-extracellular matrix interactions. High-resolution structural data are not available for these heterodimeric receptors. Previous cross-linking studies of integrins aimed at elucidating the nature of the receptor-ligand interface have been limited to identification of relatively large binding domains. To create reagents for 'photoaffinity scanning' of the RGD-binding site of human integrin α(v)β₃, new conformationally constrained ligands were designed. These photoreactive ligands are based on cyclo Ac-[Cys-Asn- Dmt-Arg-Gly-Asp-Cys]-OH, which displays an affinity of 50 nM for α(v)β₃. This molecular Scaffold was modified at the C-terminus by a benzophenone- containing amino acid residue, L-4-benzoylphenylalanine (Bpa). At the N- terminus, a molecular tag was introduced in the form of radioactive iodine or biotin. The newly designed tagged photoreactive RGD-containing ligands display an affinity of 0.5-0.7 μM for α(v)β₃, and cross-link efficiently and specifically to the receptor. A 100 kDa band corresponding to the β₃ subunit-ligand conjugate was detected as the major cross-linking product. Cross-linking was dependent upon the presence of Ca²⁺ and Mg²⁺ ions, and was competitively inhibited by a nonphotoreactive ligand. Enzymatic and chemical digestions of the radiolabeled photoconjugate enabled identification of a 20-amino acid fragment between positions 99 and 118 in the β₃ chain of the integrin as the contact domain for ligand at a site adjacent to the C- terminal portion of the RGD triad.