Structural requirements for neural cell adhesion molecule-heparin interaction.
Two biological domains have been identified in the amino terminal region of the neural cell adhesion molecule (NCAM): a homophilic-binding domain, responsible for NCAM-NCAM interactions, and a heparin-binding domain (HBD). It is not known whether these two domains exist as distinct structural entities in the NCAM molecule. To approach this question, we have further defined the relationship between NCAM-heparin binding and cell adhesion. A putative HBD consisting of two clusters of basic amino acid residues located close to each other in the linear amino acid sequence of NCAM has previously been identified. Synthetic peptides corresponding to this domain were shown to bind both heparin and retinal cells. Here we report the construction of NCAM cDNAs with targeted mutations in the HBD. Mouse fibroblast cells transfected with the mutant cDNAs express NCAM polypeptides with altered HBD (NCAM-102 and NCAM-104) or deleted HBD (HBD-) at levels similar to those of wild-type NCAM. Mutant NCAM polypeptides purified from transfected cell lines have substantially reduced binding to heparin and fail to promote chick retinal cell attachment. Furthermore, whereas a synthetic peptide that contains both basic amino acid clusters inhibits retinal-cell adhesion to NCAM-coated dishes, synthetic peptides in which either one of the two basic regions is altered to contain only neutral amino acids do not inhibit this adhesion. These results confirm that this region of the NCAM polypeptide does indeed mediate not only the large majority of NCAM's affinity for heparin but also a significant portion of the cell-adhesion-mediating capability of NCAM.
Reyes
A A
AA
Division of Basic Research, Children's Hospital Research Foundation, Cincinnati, Ohio 54229.
Akeson
R
R
Brezina
L
L
Cole
G J
GJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002642:Chick Embryo; D005455:Fluorescent Antibody Technique; D006493:Heparin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007118:Immunoassay; D015151:Immunoblotting; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D014162:Transfection
D015816:Cell Adhesion Molecules, Neuronal; D010455:Peptides; D006493:Heparin