MECHANISM OF HEMOPHILIC BINDING MEDIATED BY THE NEURAL CELL-ADHESION MOLECULE NCAM - EVIDENCE FOR ISOLOGOUS INTERACTION

We have previously shown that a decapeptide sequence between Lys-243 a nd Glu-252 (KYSFNYDGSE) in the third immunoglobulin (Ig) like domain o f chick neural cell adhesion molecule NCAM is directly involved in NCA M-to-NCAM binding. To identify the domain that interacts with this dec apeptide sequence, the Ig-like domain 3 of NCAM was expressed in bacte ria and the refolded protein was assayed for its NCAM binding activity . Covaspheres conjugated with domain 3 protein bound to a substratum c oated with either NCAM or the domain 3 protein, suggesting that NCAM-N CAM binding is mediated by interactions between domain 3 sequences on apposing molecules. Further studies were carried out using a cell-to-s ubstratum binding assay. Mouse L cells stably transformed with differe nt deletion constructs of NCAM were assayed for their ability to attac h to substratum coated with different peptide conjugates. The results indicated that a site in NCAM Ig-like domain 3 bound specifically to t he decapeptide sequence. To identify this site, cells expressing mutan t NCAMs with alterations in the amino acid sequence of the hemophilic binding site were subjected to the same cell-to substratum assay. Muta nt NCAMs that had lost their hemophilic binding activity also failed t o attach to the peptide substrate. Taken together, these results sugge st that the NCAM hemophilic binding site interacts isologously with th e same sequence on apposing molecules.