DOMINANT-NEGATIVE EFFECT ON ADHESION BY MYELIN PO PROTEIN TRUNCATED IN ITS CYTOPLASMIC DOMAIN

The myelin Po protein is believed to hold myelin together via interact ions of both its extracellular and cytoplasmic domains. We have alread y shown that the extracellular domains of Po can interact in a hemophi lic manner(Filbin, M,T., F.S. Walsh, B,D. Trapp: J,A. Pizzey, and G.I. Tennekoon. 1990. Nature (Lond). 344:871-872). In addition, we have sh own that for this hemophilic adhesion to take place, the cytoplasmic d omain of Po must be intact and most likely interacting with the cytosk eleton; Po proteins truncated in their cytoplasmic domains are not adh esive (Wong, M.H., and M.T. Filbin, 1994. J. Cell Biol. 126:1089-1097) . To determine if the presence of these truncated forms of Po could ha ve an effect on the functioning of the full-length Po, we coexpressed both molecules in CHO cells. The adhesiveness of CHO cells expressing both full-length Po and truncated Po was then compared to cells expres sing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surf ace of the cell in approximately equal amounts as shown by an ELISA an d surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other c ell lines expressing only full-length Po that we had already shown to be adhesive Therefore, there should be sufficient levels of full-lengt h Po at the surface of these coexpressors to measure adhesion of Po. H owever, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were ind istinguishable from the control transfected cells not expressing Po. I n contrast, in the same time, the cells expressing only the full-lengt h Po had formed large aggregates. This indicates that the truncated fo rms of Po have a dominant-negative effect on the adhesiveness of the f ull-length Po, Furthermore, from cross-linking studies, full-length Po , when expressed alone but not when coexpressed with truncated Po, app ears to cluster in the membrane. We suggest that truncated Po exerts i ts dominant-negative effect by preventing clustering of full-length Po . We also show that colchicine, which disrupts microtubules. prevents adhesion of cells expressing only the full-length Po, This strengthens our suggestion that an interaction of Po with the cytoskeleton, eithe r directly or indirectly, is required for adhesion to take place.