Colocalization of the Homophilic Binding Site and the Neuritogenic Activity of the Cell Adhesion Molecule L1 to Its Second Ig-like Domain

doi:10.1074/jbc.270.49.29413

Colocalization of the Homophilic Binding Site and the Neuritogenic Activity of the Cell Adhesion Molecule L1 to Its Second Ig-like Domain (*)

From the Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada

¶ To whom correspondence should be addressed:
Charles H. Best Institute, University of Toronto, 112 College St., Toronto, Ontario M5G 1L6, Canada.
Tel.: 416-978-8766; Fax: 416-978-8528; chi.hung.siu{at}utoronto.ca.

Abstract

The cell adhesion molecule L1 has been implicated in mediating cell-cell adhesion and in promoting neurite outgrowth. The extracellular region of L1 contains six immunoglobulin (Ig)-like domains in the amino-terminal region, followed by five fibronectin type III-like repeats. L1 is capable of undergoing homophilic binding as well as heterophilic interactions. To map the homophilic binding domain in L1, three glutathione S-transferase (GST) fusion proteins (GST-Ig1-2-3, GST-Ig4-5-6, and GST-Fn) were prepared and coupled to Covaspheres and their homophilic binding activity was determined using the Covasphere-to-substratum binding assay. Only GST-Ig1-2-3 was capable of homophilic binding. Next, His-tagged recombinant Ig-domain proteins (His-Ig1-2, His-Ig1, and His-Ig2) were expressed and subjected to similar assays. Only His-Ig1-2 and His-Ig2 were capable of homophilic interactions. Binding of His-Ig2-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 homophilic binding site resides within Ig2. To examine effects of these L1 recombinant proteins on neurite outgrowth, neural retinal cells were cultured on different substrate-coated fusion proteins. Both GST-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 sufficient to mediate L1-L1 interaction and promote neurite outgrowth from retinal cells.

Footnotes

↵^§ Recipient of a Medical Research Council Studentship.
↵(*) This work was supported by Operating Grant MT-11443 from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵⁽¹⁾ The abbreviations used are:

Ig

immunoglobulin

NCAM

neural cell adhesion molecule

PCR

polymerase chain reaction

GST

glutathione S-transferase

PBS

phosphate-buffered saline

DTT

dithiothreitol

BSA

bovine serum albumin

HBSS

Hank's balanced salt solution

CEA

carcinoembryonic antigen

FGF

fibroblast growth factor

NgCAM

neuron-glia cell adhesion molecule.
- Received July 18, 1995.
- Revision received September 13, 1995.