Mutoh T, Hamano T, Tokuda A, Kuriyama M
The Second Department of Internal Medicine, Faculty of Medicine, Fukui Medical University, Japan.
Glycoconj J. 2000 Mar-Apr;17(3 -4):233-7. doi: 10.1023/a:1026597408790.
Our previous studies have shown that acidic glycosphingolipid, ganglioside GM1 (GM1), is an endogenous regulator of high affinity nerve growth factor receptor, Trk, which is an essential factor for the normal development and differentiation of neuronal cells by forming a complex with Trk. GM1 is also known to be a major constituent of caveola or glycosphingolipid-enriched microdomain (GEM) of the plasma membrane. In order to study the effect of the glycosylation of Trk on the formation of GM1-Trk complex and subcellular distribution of this protein, we generated PC12 cells stably overexpressing Trk (PCtrk). Pretreatment of this stable clones with tunicamycin, a potent inhibitor of N-glycosylation, caused the appearance of unglycosylated Trk core protein. These unglycosylated Trk can hardly respond to its ligand, NGF. Sucrose density gradient analysis revealed that unglycosylated Trk core protein was recovered in high density fractions, whereas most of GM1 is present in low density fractions corresponding to caveola or GEM fractions. Moreover, these unglycosylated Trk proteins lose their ability to form a complex with GM1, although GM1 is present in the same high density fractions. These data strongly suggest that spatial segregation of GM1 from the Trk protein by the inhibition of the glycosylation of Trk might be an important molecular mechanism for the unresponsiveness to NGF. Moreover, the binding site of GM1 in the Trk protein might act as an important determinant for the normal trafficking of the Trk protein within the cells.
我们之前的研究表明，酸性糖鞘脂神经节苷脂GM1是高亲和力神经生长因子受体Trk的内源性调节剂，Trk通过与GM1形成复合物，对神经元细胞的正常发育和分化至关重要。GM1也是细胞膜小窝或富含糖鞘脂微区（GEM）的主要成分。为了研究Trk糖基化对GM1-Trk复合物形成及该蛋白亚细胞分布的影响，我们构建了稳定过表达Trk的PC12细胞（PCtrk）。用衣霉素（一种有效的N-糖基化抑制剂）预处理这些稳定克隆，导致未糖基化的Trk核心蛋白出现。这些未糖基化的Trk几乎不能对其配体神经生长因子（NGF）作出反应。蔗糖密度梯度分析显示，未糖基化的Trk核心蛋白存在于高密度组分中，而大多数GM1存在于对应于小窝或GEM组分的低密度组分中。此外，尽管GM1存在于相同的高密度组分中，但这些未糖基化的Trk蛋白失去了与GM1形成复合物的能力。这些数据有力地表明，通过抑制Trk糖基化使GM1与Trk蛋白空间分离，可能是对NGF无反应的重要分子机制。此外，GM1在Trk蛋白中的结合位点可能是Trk蛋白在细胞内正常运输的重要决定因素。
