Masuda S, Nagao M, Takahata K, Konishi Y, Gallyas F, Tabira T, Sasaki R
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
J Biol Chem. 1993 May 25;268(15):11208-16.
Radioiodinated erythropoietin (Epo) was bound specifically to the cells of two non-erythroid clonal lines, PC12 and SN6, which expressed neuronal characteristics. The binding was time-, cell number-, and dose-dependent and was reversible. Although the cloned Epo receptor from PC12 cells (derived from rat adrenal medulla) was identical to that from rat erythroid cells, significant differences in the ligand binding properties between two cell lineages were found; 1) PC12 cells had a single class of binding sites with very low affinity (Kd = 16 nM), whereas erythroid cells had two classes of binding sites with different affinities (Kd = 95 pM for high affinity sites and 1.9 nM for low affinity sites), and 2) cross-linking experiments revealed one cross-linked product of 105 kDa for PC12 cells and two products of 140 and 120 kDa for erythroid cells. Taken together with additional results, the presence of a putative accessory protein(s) that may alter the ligand binding affinity through interaction with Epo receptor is discussed. The binding of Epo to PC12 cells caused a rapid increase in the cytosolic concentration of free calcium. The presence of EGTA had no effect on the Epo binding but completely inhibited the calcium increase, indicating that Epo stimulated the calcium influx from outside of the cells. The addition of Epo to the culture media of PC12 cells elevated the intracellular concentrations of monoamines.
放射性碘化促红细胞生成素（Epo）特异性结合于两种非红系克隆细胞系PC12和SN6的细胞，这两种细胞系具有神经元特征。这种结合具有时间、细胞数量和剂量依赖性，并且是可逆的。尽管来自PC12细胞（源自大鼠肾上腺髓质）的克隆促红细胞生成素受体与大鼠红系细胞的相同，但发现两个细胞谱系之间在配体结合特性上存在显著差异；1）PC12细胞具有一类亲和力非常低的结合位点（Kd = 16 nM），而红系细胞具有两类亲和力不同的结合位点（高亲和力位点的Kd = 95 pM，低亲和力位点的Kd = 1.9 nM），2）交联实验显示PC12细胞有一个105 kDa的交联产物，而红系细胞有140 kDa和120 kDa的两个产物。结合其他结果，讨论了可能存在通过与促红细胞生成素受体相互作用改变配体结合亲和力的假定辅助蛋白。促红细胞生成素与PC12细胞的结合导致游离钙的胞质浓度迅速增加。乙二醇双四乙酸（EGTA）的存在对促红细胞生成素的结合没有影响，但完全抑制了钙的增加，表明促红细胞生成素刺激了钙从细胞外流入。向PC12细胞的培养基中添加促红细胞生成素可提高单胺的细胞内浓度。
