Intervesicle cross-linking with integrin alpha(IIb)beta(3) and cyclic-RGD-lipopeptide. A model of cell-adhesion processes

We report the synthesis of a new integrin alpha(IIb)beta(3)-specific cyclic hexapeptide that contains an Arg-Gly-Asp (RGD) sequence and is coupled to a dimyristoylthioglyceryl anchor. We demonstrate that this ligand is useful to study specific integrin binding to membrane surfaces. With the help of biotinylated analogues of the peptide, a spacer of optimal length between t he peptide and lipid moieties was searched for by evaluating the binding st rength with an enzyme-coupled immunosorbent assay (ELISA) and by surface pl asmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the lig ands, which consisted either of E-aminohexanoic acid (epsilon Ahx) or of ep silon Ahx with two additional glycine units. Best results were obtained wit h c[Arg-GlyAsp-D-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constant s of K-D = 0.158 mu M from ELISA and K-D = 1.1 mu M from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-D-Phe-Lys([dimyristoyl-3-thioglyce ryl-succinimido-propanoyl]Ahx-Gly- Gly)-Gly], was used as a membrane-anchor ed integrin ligand. It is shown by fluorescence microscopy and cryo electro n microscopy that integrin reconstituted into phospholipid vesicles binds t o vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allo ws establishment of new model systems for systematic studies of the self-or ganization of integrin clusters and focal adhesion complexes.