COLOCALIZATION OF THE HEMOPHILIC FINDING SITE AND THE NEURITOGENIC ACTIVITY OF THE CELL-ADHESION MOLECULE L1 TO ITS 2ND IG-LIKE DOMAIN

The cell adhesion molecule L1 has been implicated in mediating cell-ce ll adhesion and in promoting neurite outgrowth. The extracellular regi on of L1 contains six immunoglobulin (Ig)-like domains in the amino-te rminal region, followed by five fibronectin type III-like repeats. L1 is capable of undergoing hemophilic binding as well as heterophilic in teractions. To map the hemophilic binding domain in L1, three glutathi one S-transferase (GST) fusion proteins (GST-Ig1-2-3, GST-Ig4-5-6, and GST-Fn) were prepared and coupled to Covaspheres and their hemophilic binding activity was determined using the Covasphere-to-substratum bi nding assay. Only GST-Ig1-2-3 was capable of hemophilic binding. Next, His-tagged recombinant Ig-domain proteins (His-Ig1-2, His-Ig1, and Hi s-Ig2) were expressed and subjected to similar assays. Only His-Ig1-2 and His-Ig2 were capable of hemophilic interactions. Binding of His-Ig 2-conjugated Covaspheres to substrate-coated His-Ig2 was inhibited by anti-Ig1-2-3 Fab and soluble His-Ig2. These results indicate that the L1 hemophilic binding site resides within Ig2. To examine effects of t hese L1 recombinant proteins on neurite outgrowth, neural retinal cell s were cultured on different substrate-coated fusion proteins. Both GS T-Ig1-2-3 and His-Ig2 were potent inducers of neurite extension. These results thus indicate that the L1 Ig-like domain 2 alone is sufficien t to mediate L1-L1 interaction and promote neurite outgrowth from reti nal cells.