THE CYTOPLASMIC DOMAIN OF THE MYELIN PO PROTEIN INFLUENCES THE ADHESIVE INTERACTIONS OF ITS EXTRACELLULAR DOMAIN

The extracellular domain of the myelin Po protein is believed to engag e in adhesive interactions and thus hold the myelin membrane compact. We have previously shown that Po can behave as a homophilic adhesion m olecule 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 o f Po must be intact for the extracellular domains to adhere, we compar ed the adhesive capabilities of Po proteins truncated at the COOH-term inal to the full-length Po protein. Po cDNAs lacking nucleotides codin g for the last 52 or 59 amino acids were transfected into CHO cells, a nd surface expression of the truncated proteins was assessed by immuno fluorescence, surface labeling followed by immunoprecipitation, and an ELISA. Cell lines were chosen that expressed at least equivalent amou nts of the truncated Po proteins at the surface as did a cell line exp ressing the full-length Po. The adhesive properties of these three cel l lines were compared. It was found that when a suspension of single c ells was allowed to aggregate for a period of 60 min, only the cells e xpressing the full-length Po had formed large aggregates, while the ce lls expressing the truncated Po molecules were still mostly single cel ls indistinguishable from the control cells. Furthermore, 25-30% of th e full-length Po was insoluble in NP40, indicative of an interaction w ith the cytoskeleton, whereas only 5-10% of Po lacking 52 amino acids and none of Po lacking 59 amino acids were insoluble. These results su ggest that for the extracellular domain of Po to behave as a hemophili c adhesion molecule, its cytoplasmic domain must be intact, and most p robably, it is interacting with the cytoskeleton.