Structural analysis of the sixth immunoglobulin-like domain of mouse neural cell adhesion molecule L1 and its interactions with alpha(v)beta3, alpha(IIb)beta3, and alpha5beta1 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)beta3, alpha(IIb)beta3, and alpha5beta1 integrins. The domain was found to have the expected beta-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)beta3, alpha(IIb)beta3, and alpha5beta1 integrins, suggesting specific interactions with these ligands. However, only the interaction to alpha(v)beta3 could be inhibited in a concentration-dependent manner by an RGD-containing peptide, and the IC50 was determined to be approximately 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)beta3. Our findings indicate an RGD-independent mechanism for the interactions to alpha(IIb)beta3 and alpha5beta1, as no involvement of any RGD motif could be demonstrated.
Blaess
S
S
Department of Biophysical Chemistry, Biozentrum, University of Basel, Switzerland.
Kammerer
R A
RA
Hall
H
H
D000595:Amino Acid Sequence; D000818:Animals; D004347:Drug Interactions; D007136:Immunoglobulins / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D019006:Neural Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D009842:Oligopeptides / Q000235:genetics / Q000502:physiology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism / Q000502:physiology*; D017469:Receptors, Fibronectin / Q000378:metabolism / Q000502:physiology*; D019030:Receptors, Vitronectin / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins
D007136:Immunoglobulins; D019006:Neural Cell Adhesion Molecules; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D017469:Receptors, Fibronectin; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid