Mechanism of homophilic binding mediated by the neural cell adhesion molecule NCAM. Evidence for isologous interaction.
We have previously shown that a decapeptide sequence between Lys-243 and Glu-252 (KYSFNYDGSE) in the third immunoglobulin (Ig)-like domain of chick neural cell adhesion molecule NCAM is directly involved in NCAM-to-NCAM binding. To identify the domain that interacts with this decapeptide sequence, the Ig-like domain 3 of NCAM was expressed in bacteria and the refolded protein was assayed for its NCAM binding activity. Covaspheres conjugated with domain 3 protein bound to a substratum coated with either NCAM or the domain 3 protein, suggesting that NCAM-NCAM binding is mediated by interactions between domain 3 sequences on apposing molecules. Further studies were carried out using a cell-to-substratum binding assay. Mouse L cells stably transformed with different deletion constructs of NCAM were assayed for their ability to attach to substratum coated with different peptide conjugates. The results indicated that a site in NCAM Ig-like domain 3 bound specifically to the decapeptide sequence. To identify this site, cells expressing mutant NCAMs with alterations in the amino acid sequence of the homophilic binding site were subjected to the same cell-to-substratum assay. Mutant NCAMs that had lost their homophilic binding activity also failed to attach to the peptide substrate. Taken together, these results suggest that the NCAM homophilic binding site interacts isologously with the same sequence on apposing molecules.
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002448:Cell Adhesion; D015816:Cell Adhesion Molecules, Neuronal / Q000378:metabolism*; D002449:Cell Aggregation; D002645:Chickens; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D015816:Cell Adhesion Molecules, Neuronal; D009842:Oligopeptides; D011994:Recombinant Proteins