News and Publications

Structure and Function of Cell Adhesion Molecules

B.A. Cunningham, K.L. Crossin, L.A. Krushel, A. Atkins, W. Zhou, E. Little, P. Vanderklish, G.M. Edelman

Cell adhesion molecules (CAMs) are classified structurally into 3 families: the N-CAM family, the calcium-dependent CAMs or cadherins, and the selectins. All members of the N-CAM family contain multiple immunoglobulin-like (Ig-like) domains, and many contain repeats resembling the type III repeats found in fibronectin. A variety of data indicate that different domains participate in different functions. Extracellular domains are involved in homophilic and heterophilic binding, and intracellular regions are involved in intracellular signaling and in interactions with the cytoskeleton. We are examining the functions of all of these domains in the neural CAM (N-CAM) and in a subfamily of neural CAMs represented by neuron-glia CAM, neuron-glia CAM--related molecule, and L1.

N-CAM mediates homophilic binding between cells, but the mechanism by which N-CAM on one cell interacts with N-CAM on another cell is unknown. Our previous findings suggested that N-CAM--N-CAM binding involves all 5 Ig-like domains that interact pairwise in an antiparallel orientation. To extend these results, we examined the biological activity of each of the recombinant N-CAM proteins in an astrocyte proliferation assay. We found that all N-CAM Ig-like domains inhibited astrocyte proliferation in parallel with their ability to influence N-CAM binding. The proliferation of other cells expressing N-CAM was also inhibited by the addition of N-CAM. In contrast, the proliferation of astrocytes from knockout mice lacking N-CAM was not inhibited by added N-CAM. These findings strongly support the hypothesis that the binding of soluble N-CAM to N-CAM on the surface of astrocytes leads to decreased proliferation.

Previous studies indicated that N-CAM is palmitoylated, but the sites of acylation were not localized. We have now shown that the cytoplasmic domain of full-length N-CAM becomes palmitoylated after transfection into COS-7 cells and that this acylation is on the closely spaced cysteines in the cytoplasmic domain of N-CAM. Moreover, when cDNAs encoding only the cytoplasmic domain were transfected into cells, the proteins were palmitoylated and even though lacking a membrane-spanning segment became associated with membranes. Site-directed mutagenesis of all 4 cysteines to serines eliminated both the palmitoylation and the association with the membrane.

Additional studies showed that not all of the cysteines are palmitoylated and that at least two are required for optimal association with the membrane. Mutation of the cysteines in full-length N-CAM did not affect the ability of the protein to promote aggregation when transfected into COS-7 cells. On the basis of these results, we suggest that the primary role of palmitoylation is to provide a second anchor in the plasma membrane. This attachment could organize the cytoplasmic region for interaction with other molecules to determine subcellular localization or affect signaling events caused by N-CAM--mediated adhesion. To ascertain the cytoplasmic molecules with which N-CAM interacts, we used the cytoplasmic domain of N-CAM in a yeast 2-hybrid screening system. Candidate clones have been isolated and are being characterized.

To extend these studies and to develop assays for analyses of the effects of the cytoplasmic regions of other CAMs on cell behavior, we have generated DNA constructs for expression in eukaryotic cells. Constructs expressing the N-CAM cytoplasmic region, with and without its transmembrane segment, and the cytoplasmic region of L1 with its transmembrane region plus various amounts of its extracellular domain are being tested in a variety of cells. Additional biological assays are being developed to measure the formation and stability of synapses. Organotypic cultures of hippocampus are being used to develop assays to correlate overexpression of mutant CAMs with variables such as synaptic density, dendritic spine morphology, postsynaptic density topography, and qualities of the excitatory postsynaptic potential. Preliminary experiments have established that transfected cell populations can be detected in slice cultures by means of a green fluorescent protein reporter and that immunogold labeling of myc-tagged constructs can be used to detect individual synapses at which the dominant-negative proteins have been expressed.

Chemical studies to delineate in detail the mechanism by which N-CAM mediates homophilic binding are continuing. Circular dichroism spectroscopy indicated that recombinant proteins corresponding to the Ig-like domains individually and in various combinations are folded, and these proteins are being isotopically labeled for structural analysis with nuclear magnetic resonance methods. The third Ig-like domain has the strongest influence on N-CAM binding, and we have shown that this domain can bind to itself. Constructs encoding variants of the domain have been prepared, and 1 variant appears to be properly folded and capable of forming dimers. This protein is being examined by nuclear magnetic resonance methods in collaboration with J. Dyson and P. Wright of the Department of Molecular Biology. Linear combinations of Ig-like domains I--II and I--V have been prepared. These proteins also appear to be properly folded, and we are attempting to crystallize them.