Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry Li /HNK-1 Carbohydrate- and flu Integrin-Dependent Neural Cell Adhesion to Laminin- 1 Heike Hall, *Salvatore Carbonetto, and Melitta Schachner Department of Neurobiology, Swiss Federal Institute of Technology, Zurich, Switzerland; and *Centerfor Neuroscience, Montreal General Hospital, Montreal, Quebec, Canada Abstract: We have shown recently that mouse small cer- ebellar neurons adhere to a short amino acid sequence of the G2 domain of the laminin al chain via the cell surface-expressed HNK-i carbohydrate. Therefore, we were interested in identifying glycoproteins carrying the HNK-1 carbohydrate at the cell surface of these neurons. Adhesion of small cerebellar neurons to laminin is partially dependent on Ca 2~, Mn2-, and Mg2~, indicating the involvement of integrins, which were identified as ~3i, a3, and a6. They could be shown to bind to laminin by a /31 - dependent adhesion mechanism. None of these subunits was found to carry the HNK-1 carbohydrate. HNK-1-im- munoreactive glycoproteins were immunoprecipitated and shown to consist of predominantly one molecular species, which was identified as the neural cell recogni- tion molecule Li. Li was demonstrated to bind in a con- centration-dependent and saturating manner to laminin. The binding could be partially inhibited by Fab fragments of monoclonal antibodies against the HNK-1 carbohy- drate and against the Ig-like domains of Li. Furthermore, antibodies to the Ig-like domains of Li and ~31integrin inhibited partially cell adhesion to laminin. Determination of the association of Li, ~31 integrin, and the HNK-i car- bohydrate on the cell surface of live cerebellar neurons by antibody-induced patching and copatching revealed HNK-i to be linked to Li, but less so to /31 integrin. However, only negligible association was found between Li and /31 integrin. Furthermore, it could be shown that adhesion to laminin is mediated by Li/HNK-i- and /3i integrin-dependent mechanisms that act at least partially independent of each other. Key Words: HNK-i carbohy- drate— Laminin—lntegrins—L1 —Cell adhesion. J. Neurochem. 68, 544—553 (1997). laminin are mediated by integrins (for reviews, see Reichardt and Tomaselli, 1991; Hynes, 1992; Hynes and Lander, 1992). Laminin from mouse EHS sarcoma consists of the al, /31, and yl chains, which assemble into a cruci- form structure in which the three chains form an a- helical coiled-coil for most of the long arm, leaving free three short arms (Timpi et al., 1979; Beck et al., 1990, 1991). Distinct functional domains of laminin were found to interact with integrins (Kramer et al., 1989; Aumailley et al., 1990; Hall et al., 1990; Son- nenberg et al., 1991b; Gehlsen et al., 1992). For exam- ple, the region for al/31 binding has been localized to the N-terminus of the long arm (Colognato-Pyke et al., 1995). av/31 recognizes the cryptic RGD site within the al chain of the P1 fragment (Tashiro et al., 1991), and a6/3 1 interacts with the E8 fragment that comprises the C-terminal part of the long arm and the first two globular domains of the a 1 chain (Sonnen- berg et al., 1990, 1991b). A 21-amino-acid peptide from the G5 domain of the laminin a 1 chain has been proposed to be an a3/3l integrin binding sequence (Gehlsen et al., 1992; Pottarmalai et al., 1996). Previous studies have revealed that early postnatal small cerebellar neurons adhere mainly through the HNK-1 carbohydrate on the cell surface to the G2 domain of the laminin al chain (Hall et al., 1995; H. Hall et al., manuscript submitted for publication). This carbohydrate was defined by reaction with the mono- clonal antibody HNK- 1 as 3 ‘-sulfoglucuronylneolacto- Interactions of neural cells with extracellular matrix molecules have been proposed to be important for the ontogenetic formation of the nervous system (Sanes, 1989; Reichardt and Tomaselli, 1991; Letourneau, 1992; Letourneau et al., 1994). Among the prominent components of the extracellular matrix, laminin has been shown to be involved in cell adhesion and spread- ing and in promotion of neurite outgrowth (for review, see Timpl and Brown, 1994; Dziadek, 1995; Gum- biner, 1996). Most of the cellular interactions with Received July 3, 1996; revised manuscript received September 24, 1996; accepted September 25, 1996. Address correspondence and reprint requests to Dr. M. Schachner at Department of Neurobiology, Swiss Federal Institute of Technol- ogy, Honggerberg, 8093 Zurich, Switzerland. The present address of Dr. H. Hall is Department of Biophysical Chemistry, Biocenter of the University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland. Abbreviations used: BME, basal medium Eagle’s; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; HBSS, Hanks’ balanced salt solution; HRP, horseradish peroxidase; PAGE, poly- acrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TRITC, tetramethylrhodamine isothio- cyanate. 544