Journal of Neurochemistry, 2001, 76, 1552±1564 PC12 cells utilize the homophilic binding site of L1 for cell2cell adhesion but L1± avb3 interaction for neurite outgrowth Paul M. Yip and Chi-Hung Siu Banting and Best Department of Medical Research and Department of Biochemistry, University of Toronto, Toronto, Canada Abstract Treatment of PC12 cells with nerve growth factor induces their differentiation into sympathetic neuron-like cells and the concomitant expression of the neural cell adhesion molecule L1, a member of the Ig superfamily. To investigate the mechanism of L1-stimulated neurite outgrowth in PC12 cells, substrate-immobilized fusion proteins containing different extracellular domains of L1 were assayed for their neurito- genic activity. Surprisingly, domain Ig2 of L1, which was previously found to contain both homophilic binding and neuritogenic activities, failed to promote neurite outgrowth. In contrast, L1-Ig6 stimulated neurite outgrowth from PC12 cells. Despite this, homotypic binding of PC12 cells was signi®cantly inhibited by antibodies against L1-Ig2, indicating that L1±L1 binding contributed to the intercellular adhesiveness of PC12 cells, but L1-stimulated neurite outgrowth depends on heterophilic interactions. Thus, PC12 cells provide a valuable model for the study of these two distinct functions of L1. Mutagenesis of L1-Ig6 highlighted the importance of the Arg-Gly-Asp motif in this domain for neuritogenesis. Inhibition studies using cyclic Arg-Gly-Asp-containing peptide and anti- integrin antibodies suggested the involvement of avb3 integrin. Furthermore, neurite outgrowth stimulated by L1-Ig6 was inhibited by lavendustin A and the MEK inhibitor PD98059, suggesting a signaling pathway that involves tyrosine kinase activation and the mitogen-activated protein kinase cascade. Keywords: cell adhesion molecule, GST-fusion proteins, kinase inhibitors, neuritogenesis, signaling pathway. J. Neurochem. (2001) 76, 1552±1564. The development of the nervous system requires the proper projection of elongating axons to their correct targets. Several types of molecules, including cell adhesion molecules (CAMs), diffusible factors and components of the extracellular matrix, in¯uence the direction and move- ment of the neuronal growth cone (Tessier-Lavigne and Goodman 1996). CAMs that function as cell surface receptors on neurons include members of the Ig superfamily, integrins and cadherins. The cell adhesion molecule L1 is a member of the Ig superfamily of adhesion receptors (Moos et al. 1988) and is expressed predominantly in the nervous system (Rathjen and Schachner 1984). L1 is a potent substrate for neurite outgrowth (Lagenaur and Lemmon 1987; Lemmon et al. 1989) and participates in several other developmental processes such as myelination (Martini and Schachner 1986), cell migration (Lindner et al. 1983), axonal fasciculation (Fischer et al. 1986), growth cone morphology (Payne et al. 1992) and memory (Lu Èthi et al. 1994). The importance of L1 in neuronal development has been borne out by studies on L1-null mice, which show malformations of the nervous system (Fransen et al. 1998; Demyanenko et al. 1999). The cDNAs of L1 have been cloned for several vertebrate and invertebrate species (for a review, see Hortsch 1996). The N-terminus of L1 contains six Ig-like domains, followed by ®ve ®bronectin (FN) type III repeats, a single transmembrane domain, and a short cytoplasmic tail. L1 is highly conserved among mammals, such that the human and mouse L1 amino acid sequences share 87% identity (Reid and Hemperly 1992), whereas the rat (NILE) and mouse L1 sequences share 96% identity (Miura et al. 1991). A large number of mutations in the human L1 gene has been implicated in X-linked hydrocephalus and related 1552 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1552±1564 Received August 31, 2000; revised manuscript received November 13, 2000; accepted November 14, 2000. Address correspondence and reprints requests to Dr C.-H. Siu, Charles H. Best Institute, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada. E-mail: chi.hung.siu@utoronto.ca Abbreviations used: a-MEM, a-minimal essential medium; BSA, bovine serum albumin; CAM, cell adhesion molecule; DiI, 1,1 0 - dioctadecyl-3,3,3 0 ,3 0 -tetramethylindocarbocyanine perchlorate; FGF, ®broblast growth factor; FN, ®bronectin; GST, glutathione S-transfer- ase; HBSS, Hanks' balanced salt solution; MAP kinase, mitogen- activated protein kinase; NCAM, neural cell adhesion molecule; NGF, nerve growth factor; RGD, Arg-Gly-Asp; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate.