Different Extracellular Domains of the Neural Cell Adhesion Molecule (N-CAM) Are Involved in Different Functions

The neural cell adhesion molecule (N-CAM) engages in diverse functional roles in neural cell interactions. Its extracellular part consists of five Ig-like domains and two fibronectin type III homologous (type III) repeats. To investigate the functional properties of the different structural domains of the molecule in cell interactions and signal transduction to the cell interior, we have synthesized, in a bacterial expression system, the individual domains and tandem sets of individual domains as protein fragments. These protein fragments were tested for their capacity to influence adhesion and spreading of neuronal cell bodies, promote neurite outgrowth, and influence cellular migration patterns from cerebellar microexplants in vitro. Ig-like domains I and II and the combined type III repeats I-II were most efficient for adhesion of neuronal cell bodies, when coated as substrates. Neurite outgrowth was best on the substrate-coated combined type III repeats I-II, followed by the combined Ig-like domains I-V and Ig-like domain I. Spreading of neuronal cell bodies was best on substrate-coated combined type III repeats I-II, followed by Ig-like domain I and the combined Ig-like domains I-V. The cellular migration pattern from cerebellar microexplant cultures plated on a mixture of laminin and poly-L-lysine was modified by Ig-like domains I, III, and IV, while Ig-like domains II and V and the combined type III repeats I-II did not show significant modifications, when added as soluble fragments. Outgrowth of astrocytic processes from the explant core was influenced only by Ig-like domain I. Metabolism of inositol phosphates was strongly increased by Ig-like domain I and less by the Ig-like domains II, III, IV, and V, and not influenced by the combined type HI repeats I-II. Intracellular concentrations of Ca 2§ and pH values were increased only by the Ig-like domains I and II. Intracellular levels of cAMP and GMP were not influenced by any protein fragment. These experiments indicate that different domains of N-CAM subserve different functional roles in cell recognition and signal transduction, and are functionally competent without nervous system-derived carbohydrate structures. C ELL recognition plays an important role during the formation, maintenance, and regeneration of the nervous system. Several recognition molecules have been discovered to mediate cell-to-cell and cell-to-substratum interactions (for reviews see Edelman et al., 1990; Edelman and Crossin, 1991; Schachner, 1991). Among these, the neural cell adhesion molecule (N-CAM) ~ has been implicated in several types of neural cell interactions (Cunningham et al., 1987). N-CAM is involved in neuron-to-neuron and neuron-to-glia interactions in short-term adhesion assays (Keilhauer et al., 1985). It allows neurites to fasciculate (Rutishauser and Edelman, 1980; Fischer et al., 1986) and to interact with their muscle targets in the periphery (Grumet et al., 1982; Covault and Sanes, 1986; Covault et al., 1986). Formation of the retinal and tectal cytoarchitecture has also been attributed, at least in part, to N-CAM (Buskirk et al., 1980; Fraser et al., 1984). N-CAM is expressed early during 1. Abbreviation used in this paper: N-CAM, neural cell adhesion molecule. neural development at the time of neurulation (Crossin et al., 1985). Its functional role in this process, in particular neural induction, has, however, remained obscure (Jacobson and Rutishauser, 1986). It remains expressed by the majority of cell types of neuroectodermal origin during later stages of nervous system development and appears to subserve yet unknown functions in the adult nervous system, where it continues to be expressed, although at lower levels than at early developmental stages. N-CAM is expressed on neural crest cells before migration, ceases to be detectable during migration, and reappears once these cells have arrived at their final position (Thiery et al., 1982). After peripheral nerve transection, the temporal and spatial sequence of N-CAM expression is the same as during development, suggesting that N-CAM also plays a role in regeneration (Nieke and Schachner, 1985; Martini and Schachner, 1988). N-CAM is a recognition molecule that belongs to the immunoglobulin superfamily, carries one more and one less conserved fibronectin type Ill homologous repeat (Barthels 9 The Rockefeller University Press, 0021-9525/92/07/177/18 $2.00 The Journal of Cell Biology, Volume 118, Number 1, July 1992 177-194 177 on May 2, 2019 jcb.rupress. org Downloaded from http://doi.org/10.1083/jcb.118. 1.177 Published Online: 1 July, 1992 | Supp Info: