Takahashi M, Asai N, Iwashita T, Isomura T, Miyazaki K, Matsuyama M
Department of Pathology, Nagoya University School of Medicine, Japan.
Oncogene. 1993 Nov;8(11):2925-9.
The ret proto-oncogene (proto-ret) encodes a receptor type tyrosine kinase with a cadherin-related sequence in the extracellular domain. To investigate whether the proto-Ret protein functions as a cell adhesion molecule like cadherins, we transfected the human proto-ret gene fused to the SV40 promoter or cytomegalovirus (CMV) promoter into mouse L cells in which cadherins are not expressed. Three transfectants with high levels of expression of the proto-Ret proteins were obtained. The proto-Ret proteins were expressed as 150 kDa and 170 kDa glycoproteins in transfectants as observed in human neuroblastoma cells. Cell fractionation experiments revealed that the 170 kDa protein but not the 150 kDa protein was detected predominantly in the plasma membrane fraction, indicating that the 170 kDa protein represents the mature glycosylated form of the proto-Ret protein present on the cell surface. Both 150 kDa and 170 kDa proto-Ret proteins showed tyrosine kinase activity in immunocomplex kinase assay. It is known that cadherins have Ca(2+)-dependent homophilic binding activity and are resistant to trypsinization in the presence of Ca2+. When L cells expressing the proto-Ret proteins were treated with trypsin in the presence of Ca2+, the 170 kDa protein was resistant to its digestion. On the other hand, it was completely digested in the presence of EGTA, suggesting the possibility that the proto-Ret protein interacts with Ca2+ like cadherins. However, the transfectants did not show clear adhesive properties in cell aggregation assays.
原癌基因ret（proto-ret）编码一种受体型酪氨酸激酶，其胞外结构域含有一个与钙黏蛋白相关的序列。为了研究原Ret蛋白是否像钙黏蛋白一样作为细胞黏附分子发挥作用，我们将与SV40启动子或巨细胞病毒（CMV）启动子融合的人原ret基因转染到不表达钙黏蛋白的小鼠L细胞中。获得了三个原Ret蛋白高表达的转染细胞株。在转染细胞中，原Ret蛋白表达为150 kDa和170 kDa的糖蛋白，这与在人神经母细胞瘤细胞中观察到的情况一致。细胞分级分离实验表明，170 kDa的蛋白而非150 kDa的蛋白主要在质膜组分中被检测到，这表明170 kDa的蛋白代表细胞表面存在的原Ret蛋白的成熟糖基化形式。在免疫复合物激酶测定中，150 kDa和170 kDa的原Ret蛋白均显示出酪氨酸激酶活性。已知钙黏蛋白具有Ca(2+)依赖的同源性结合活性，并且在有Ca2+存在时对胰蛋白酶消化具有抗性。当在有Ca2+存在的情况下用胰蛋白酶处理表达原Ret蛋白的L细胞时，170 kDa的蛋白对其消化具有抗性。另一方面，在有乙二醇双四乙酸（EGTA）存在时它被完全消化，这提示原Ret蛋白可能像钙黏蛋白一样与Ca2+相互作用。然而，在细胞聚集试验中转染细胞并未表现出明显的黏附特性。
