Structural analysis of the sixth immunoglobulin-like domain of mouse neural cell adhesion molecule L1 and its interactions with αvβ3, αIIbβ3, and α5β1 integrins

Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin-like domain in an RGD-dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid-phase binding studies to alpha v beta 3, alpha IIb beta 3, and alpha 5 beta 1 integrins. The domain was found to have the expected P-sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single-step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin-like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration-dependent and saturable way to alpha v beta 3, alpha IIb beta 3, and alpha 5 beta 1 integrins, suggesting specific interactions with these ligands. However, only the interaction to alpha v beta 3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC50 was determined to be similar to 20 nM. Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562-564 is involved in the interaction to alpha v beta 3, Our findings indicate an RGD-independent mechanism for the interactions to alpha IIb beta 3 and alpha 5 beta 1, as no involvement of any RGD motif could be demonstrated.