Abstract
The neural cell adhesion molecule NCAM mediates intercellular adhesion by homophilic binding and its homophilic binding site has been mapped to a decapeptide sequence 243-KYSFNYDGSE-252 located within the third immunoglobulin-like domain of chick NCAM. To investigate the relationship between homophilic binding and NCAM-dependent neurite outgrowth, mutations were created in the binding site of NCAM-140 cDNA. Mutant NCAMs were expressed in L cells, and their ability to promote neurite outgrowth from chick retinal ganglion cells was assayed in coculture systems. Mutations that resulted in the loss of NCAM homophilic binding failed to promote neurite outgrowth from retinal cells. Alternatively, synthetic peptides containing the decapeptide sequence of the homophilic binding site were used to block NCAM homophilic interaction. Peptides that inhibited NCAM-NCAM binding also blocked neurite elongation. However, the peptide P5 (243-KYSFNYDGSELIIKKVDKSDE-263), despite being an inhibitor of NCAM-NCAM binding, induced the sprouting of multiple neurites. Moreover, peptide P5 stimulated a 2-fold increase in neurite-bearing cells, suggesting that P5 is a potent inducer of neurite outgrowth. Only E4-E6 retinal cells could be induced by P5, corresponding closely to their NCAM-responsive embryonic stages. The P5 effects were inhibited by pertussis toxin, indicating the involvement of a G-protein-dependent pathway. Taken together, these results provide evidence for a direct role of the NCAM homophilic binding site in the regulation of neurite outgrowth.
Highlights
The Homophilic Binding Siteof the Neural Cell Adhesion Molecule NCAM Is Directly Involved in Promoting Neurite Outgrowthfrom Cultured Neural Retinal Cells*
Taketon- binding site plays in this importanbtiological event. The matgether, these results provide evidence for a direct rtoelreisoffurther complicated by observations that NCAM is cathe NCAM homophilic neurite outgrowth
The results indicate that the integrityof the sequence KYSFNYDGSE is necessary for supporting NCAM-induced neurite outgrowth and that the homophilic binding site participates directly by transducing morphogenetic signals that regulate neuriteoutgrowth
Summary
Materials--Trypsin, a-MEM,N2-supplement,andpertussistoxin were obtained from GIBCO (Toronto, ON). DiI was obtained from Molecular Probes (Eugene, OR). Active esters of amino acids for peptide synthesis wereobtained from Milligen (Millipore).All other reagents wereof the highest chemical grade. Solid-phase Peptide Synthesis-Oligopeptides were synthesized by classical solid-phase methods (40a) s described previously [33]. Synthesis was carried out on an Applied Biosystems model 430 automated peptide synthesizer. Peptides were solubilized and desalted on a P2 column equilibrated in 0.1 M NH,HCO,. The process of gel filtration and lyophilization was rou-. Peptide solutions were freshly prepared before each experiment and adjusted to neutral piHf n, ecessary. Peptide samples were sonicated to disperse aggregatesbefore use. Details of primers and strategies employed have been described elsewhere [34].
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