Mapping the integrin alpha(v)beta(3)-ligand interface by photoaffinity cross-linking

Integrins are cell surface adhesion molecules involved in mediating cell-ex tracellular matrix interactions. High-resolution structural data are not av ailable for these heterodimeric receptors. Previous cross-linking studies o f integrins aimed at elucidating the nature of the receptor-ligand interfac e have been limited to identification of relatively large binding domains. To create reagents for "photoaffinity scanning"' of the RGD-binding site of human integrin alpha(v)beta(3), new conformationally constrained ligands w ere designed. These photoreactive ligands are based, on cycle Ac-[Cys-Asn-D mt-Arg-Gly-Asp-Cys]-OH, which displays an affinity of 50 nM for alpha(v)bet a(3), This molecular scaffold was modified at the C-terminus by a benzophen one-containing amino acid residue, L-4-benzoylphenylalanine (Bpa). At the N -terminus, a molecular lag 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 mu M for alpha(v)beta(3), and cross-link eff iciently and specifically to the receptor. A 100 kDa band corresponding to the beta(3) subunit-ligand conjugate was detected as the major cross-linkin g product. Cross-linking was dependent upon the presence of Ca2+ and Mg2+ i ons, and was competitively inhibited by a nonphotoreactive ligand. Enzymati c and chemical digestions of the radiolabeled photoconjugate enabled identi fication of a 20-amino acid fragment between positions 99 and 118 in the be ta(3) chain of the integrin as the contact domain for ligand at a site adja cent to the C-terminal portion of the RGD triad.