The myelin Po protein is believed to hold myelin together via interactions of both its extracellular and cytoplasmic domains. We have already shown that the extracellular domains of Po can interact in a homophilic 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 shown that for this homophilic adhesion to take place, the cytoplasmic domain of Po must be intact and most likely interacting with the cytoskeleton; Po proteins truncated in their cytoplasmic domains are not adhesive (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 have 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 expressing only full-length Po. In these coexpressors, both the full-length and the truncated Po proteins were glycosylated. They reached the surface of the cell in approximately equal amounts as shown by an ELISA and surface labeling, followed by immunoprecipitation. Furthermore, the amount of full-length Po at the cell surface was equivalent to other cell lines expressing only full-length Po that we had already shown to be adhesive. Therefore, there should be sufficient levels of full-length Po at the surface of these coexpressors to measure adhesion of Po. However, as assessed by an aggregation assay, the coexpressors were not adhesive. By 60 min they had not formed large aggregates and were indistinguishable from the control transfected cells not expressing Po. In contrast, in the same time, the cells expressing only the full-length Po had formed large aggregates. This indicates that the truncated forms of Po have a dominant-negative effect on the adhesiveness of the full-length Po. Furthermore, from cross-linking studies, full-length Po, when expressed alone but not when coexpressed with truncated Po, appears to cluster in the membrane. We suggest that truncated Po exerts its 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, either directly or indirectly, is required for adhesion to take place.

The extracellular domain of the myelin P0 protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that P0 can behave as a homophilic adhesion molecule through interactions of its extracellular domains (Filbin, M. T., F. S. Walsh, B. D. Trapp, J. A. Pizzey, and G. I. Tennekoon. 1990. Nature (Lond.) 344:871-872). To determine if the cytoplasmic domain of P0 must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of P0 proteins truncated at the COOH-terminal to the full-length P0 protein. P0 cDNAs lacking nucleotides coding for the last 52 or 59 amino acids were transfected into CHO cells, and surface expression of the truncated proteins was assessed by immunofluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amounts of the truncated P0 proteins at the surface as did a cell line expressing the full-length P0. The adhesive properties of these three cell lines were compared. It was found that when a suspension of single cells was allowed to aggregate for a period of 60 min, only the cells expressing the full-length P0 had formed large aggregates, while the cells expressing the truncated P0 molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length P0 was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of P0 lacking 52 amino acids and none of P0 lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of P0 to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.