Intervesicle Cross-Linking with Integrin αIIbβ3and Cyclic-RGD-Lipopeptide. A Model of Cell-Adhesion Processes

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Abstract
We report the synthesis of a new integrin αIIbβ3-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 the peptide and lipid moieties was searched for by evaluating the binding strength with an enzyme-coupled immunosorbent assay (ELISA) and by surface plasmon resonance (SPR). It was found to be strongly dependent on the length of the spacer introduced between the biotin and peptide moieties of the ligands, which consisted either of ε-aminohexanoic acid (εAhx) or of εAhx with two additional glycine units. Best results were obtained with c[Arg-Gly-Asp-d-Phe-Lys(Biot-Ahx-Gly-Gly)-Gly-] with dissociation constants of KD = 0.158 μM from ELISA and KD = 1.1 μM from SPR measurements. The analogous lipopeptide, c[Arg-Gly-Asp-d-Phe-Lys([dimyristoyl-3-thioglyceryl-succinimido-propanoyl]Ahx-Gly-Gly)-Gly], was used as a membrane-anchored integrin ligand. It is shown by fluorescence microscopy and cryo electron microscopy that integrin reconstituted into phospholipid vesicles binds to vesicles decorated with the lipopeptide, forming regularly spaced bridges between the two kinds of vesicles. The novel integrin-specific ligand allows establishment of new model systems for systematic studies of the self-organization of integrin clusters and focal adhesion complexes.