Qualitative and quantitative changes in neural cell adhesion molecule (N-CAM) protein and mRNA forms were measured during myogenesis in G8-1 and C2 cell lines. Indirect immunofluorescence assay showed that N-CAM was constitutively expressed by myoblasts in culture and that myotubes appeared to be stained more strongly. These changes were quantified using a dot blot assay. N-CAM levels increased almost 4-fold in G8-1 cells and 15-fold in C2 cells during myogenesis. The kinetics of accumulation of N-CAM were not coordinate with other muscle markers such as creatine kinase or acetylcholine receptor levels, since N-CAM accumulated significantly ahead of these markers. Immunoblotting showed that myogenesis was not associated with changes in the extent of sialylation of N-CAM. However, distinct changes in desialo forms were observed after neuraminidase treatment. Myogenesis was accompanied by increases in 125- and 155-kD desialo forms with minor changes in 120- and 145-kD forms. Biosynthetic labeling studies showed that myoblasts specifically expressed a transmembrane isoform of 145 kD that was phosphorylated and was down-regulated in myotubes. Pulse-chase analysis of myotubes showed that the 120-kD isoform and an isoform of 145 kD that co-migrated with, but was distinct from, the 145 kD transmembrane isoform of myoblasts were precursors of the 125- and 155-kD isoforms, respectively, that accumulated in myotubes. The 125- and 155-kD isoforms in myotubes are linked to the cell membrane via phosphatidylinositol linkage and can be released by phospholipase C. Indirect immunofluorescence analysis showed that phosphatidylinositol specific phospholipase C specifically released N-CAM from the myotube membrane generating N-CAM-free myotubes, while myoblasts were unaffected by this treatment. Three N-CAM mRNA species were observed in mouse muscle cell lines. Myoblasts were characterized by their expression of 6.7- and 5.2-kb transcripts while myotubes express 5.2- and 2.9-kb transcripts. Thus, myogenesis is qualitatively associated with a down regulation of the 6.7-kb transcript and an up regulation of the 5.2- and 2.9-kb transcript.
Differential expression of mouse neural cell-adhesion molecule (N-CAM) mRNA species during brain development and in neural cell lines
