Sawyer S T
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
J Biol Chem. 1989 Aug 5;264(22):13343-7.
The structure of the erythropoietin receptor has been identified in this laboratory as two proteins of 100 and 85 kDa by cross-linking 125I-erythropoietin (125I-EP) to the surface of erythroid cells purified from the spleens of mice infected with the anemia strain of Friend virus. This study investigates the relatedness of these two proteins and the possibility that these proteins are subunits of the functional receptor for EP. Other workers have claimed that the 100- and 85-kDa proteins are bridged by disulfide bonds. This most likely is an artifact due to the insolubility of the cross-linked membrane. Proteolytic digestion by the method of Cleveland (Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102-1106) resulted in identical fragments from the 100- and 85-kDa proteins, which strongly suggests that the primary amino acid sequence of these two proteins is similar if not identical. Increasing the number of protease inhibitors during the preparation of membranes and the binding and cross-linking steps increased the ratio of 100-kDa protein labeled compared to the 85-kDa protein. Together these results suggest that the 85-kDa protein is derived by proteolytic cleavage of the 100-kDa receptor for EP. It is not clear whether the 100-kDa protein can bind EP in the absence of the 85-kDa protein.
在本实验室中，通过将¹²⁵I-促红细胞生成素（¹²⁵I-EP）交联到从感染了贫血株Friend病毒的小鼠脾脏中纯化的红系细胞表面，已鉴定出促红细胞生成素受体的结构为两种分子量分别为100 kDa和85 kDa的蛋白质。本研究调查了这两种蛋白质的相关性以及它们是促红细胞生成素功能性受体亚基的可能性。其他研究人员声称，100 kDa和85 kDa的蛋白质通过二硫键相连。这很可能是由于交联膜不溶性导致的假象。采用克利夫兰法（Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli, U. K. (1977) J. Biol. Chem. 252, 1102 - 1106）进行蛋白酶消化，结果显示100 kDa和85 kDa的蛋白质产生了相同的片段，这强烈表明这两种蛋白质的一级氨基酸序列即便不完全相同也非常相似。在制备膜以及结合和交联步骤中增加蛋白酶抑制剂的数量，与85 kDa蛋白质相比，标记的100 kDa蛋白质的比例增加。这些结果共同表明，85 kDa的蛋白质是由促红细胞生成素100 kDa受体经蛋白酶切割产生的。尚不清楚在没有85 kDa蛋白质的情况下，100 kDa蛋白质是否能够结合促红细胞生成素。
