Wong M H, Filbin M T
Department of Biological Sciences, Hunter College of the City University of New York, New York 10021, USA.
J Cell Biol. 1996 Sep;134(6):1531-41. doi: 10.1083/jcb.134.6.1531.
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.
髓磷脂Po蛋白被认为通过其细胞外和细胞质结构域的相互作用将髓磷脂结合在一起。我们已经表明，Po的细胞外结构域可以以嗜同种方式相互作用（菲尔宾，M.T.，F.S.沃尔什，B.D.特拉普，J.A.皮兹，和G.I.坦内科恩。1990年。《自然》（伦敦）。344:871 - 872）。此外，我们已经表明，为了发生这种嗜同种粘附，Po的细胞质结构域必须完整，并且很可能与细胞骨架相互作用；在其细胞质结构域中被截短的Po蛋白没有粘附性（王，M.H.，和M.T.菲尔宾，1994年。《细胞生物学杂志》。126:1089 - 1097）。为了确定这些截短形式的Po的存在是否会对全长Po的功能产生影响，我们在CHO细胞中共表达了这两种分子。然后将表达全长Po和截短Po的CHO细胞的粘附性与仅表达全长Po的细胞进行比较。在这些共表达细胞中，全长和截短的Po蛋白都进行了糖基化。如通过酶联免疫吸附测定（ELISA）和表面标记，随后进行免疫沉淀所显示的，它们以大致相等的量到达细胞表面。此外，细胞表面全长Po的量与我们已经表明具有粘附性的仅表达全长Po的其他细胞系相当。因此，在这些共表达细胞的表面应该有足够水平的全长Po来测量Po的粘附性。然而，通过聚集测定评估，共表达细胞没有粘附性。到60分钟时，它们没有形成大的聚集体，并且与未表达Po的对照转染细胞没有区别。相比之下，在相同时间内，仅表达全长Po的细胞已经形成了大的聚集体。这表明截短形式的Po对全长Po的粘附性具有显性负效应。此外，从交联研究来看，全长Po单独表达时会在膜中聚集，但与截短Po共表达时则不会。我们认为截短的Po通过阻止全长Po的聚集发挥其显性负效应。我们还表明，破坏微管的秋水仙碱会阻止仅表达全长Po的细胞的粘附。这强化了我们的观点，即Po与细胞骨架直接或间接的相互作用是发生粘附所必需的。
