Abstract
The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains. Here, we have used a panel of recombinant proteins in a bead binding assay, as well as transfected and primary cells, to clarify the molecular mechanism of N-CAM homophilic binding. The entire extracellular region of N-CAM mediated bead aggregation in a concentration- and temperature-dependent manner. Interactions of the N-terminal Ig domains, Ig1 and Ig2, were essential for bead binding, based on deletion and mutation experiments and on antibody inhibition studies. These findings were largely in accord with aggregation experiments using transfected L cells or primary chick brain cells. Additionally, maximal binding was dependent on the integrity of the intramolecular domain-domain interactions throughout the extracellular region. We propose that these interactions maintain the relative orientation of each domain in an optimal configuration for binding. Our results suggest that the role of Ig3 in homophilic binding is largely structural. Several Ig3-specific reagents failed to affect N-CAM binding on beads or on cells, while an inhibitory effect of an Ig3-specific monoclonal antibody is probably due to perturbations at the Ig2-Ig3 boundary. Thus, it appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domain-domain interactions.
Highlights
The mechanism by which the neural cell adhesion molecule, N-CAM, mediates homophilic interactions between cells has been variously attributed to an isologous interaction of the third immunoglobulin (Ig) domain, to reciprocal binding of the two N-terminal Ig domains, or to reciprocal interactions of all five Ig domains
It appears that reciprocal interactions between Ig1 and Ig2 are necessary and sufficient for N-CAM homophilic binding, but that maximal binding requires the quaternary structure of the extracellular region defined by intramolecular domaindomain interactions
The Fc fusion facilitated high affinity binding of the recombinant proteins to protein A-coated beads through their C termini, thereby orienting the N-CAM domains in a manner similar to that observed on cells
Summary
N-CAM, neural cell adhesion molecule; BSA, bovine serum albumin; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody; Fn III, fibronectin type III domain. We have utilized a bead aggregation assay as a semiquantitative tool for studying the comparatively weak interactions of cell adhesion molecules. In this assay, extracellular regions of N-CAM, expressed as C-terminal fusion proteins with the Fc fragment of human IgG, were directionally coupled to Protein A microspheres to approximate both the orientation and the multivalent nature of adhesion molecules on the cell surface. The results of the bead studies indicate that the reciprocal interaction between Ig1 and Ig2 is necessary for robust N-CAM-mediated bead aggregation, and similar results were obtained with primary and transfected cells. The data suggest that intramolecular domain-domain interactions are required for optimal presentation of the molecule on the cell surface for adhesive interactions
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