The cytoplasmic domain of the myelin Po protein influences: The adhesive interactions of its extracellular domain

Abstract

The extracellular domain of the myelin Po protein is believed to engage in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that Po 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 Po must be intact for the extracellular domains to adhere, we compared the adhesive capabilities of Po proteins truncated at the COOH-terminal to the full-length Po protein. Po 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 Po proteins at the surface as did a cell line expressing the full-length Po. 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 Po had formed large aggregates, while the cells expressing the truncated Po molecules were still mostly single cells indistinguishable from the control cells. Furthermore, 25-30% of the full-length Po was insoluble in NP40, indicative of an interaction with the cytoskeleton, whereas only 5-10% of Po lacking 52 amino acids and none of Po lacking 59 amino acids were insoluble. These results suggest that for the extracellular domain of Po to behave as a homophilic adhesion molecule, its cytoplasmic domain must be intact, and most probably, it is interacting with the cytoskeleton.