THE FUNCTIONAL FORM OF THE ERYTHROPOIETIN RECEPTOR IS A 78-KDA PROTEIN - CORRELATION WITH CELL-SURFACE EXPRESSION, ENDOCYTOSIS, AND PHOSPHORYLATION
THE FUNCTIONAL FORM OF THE ERYTHROPOIETIN RECEPTOR IS A 78-KDA PROTEIN - CORRELATION WITH CELL-SURFACE EXPRESSION, ENDOCYTOSIS, AND PHOSPHORYLATION
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DOI：
10.1073/pnas.90.14.6849
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发表时间：
1993-07-15
影响因子：
11.1
通讯作者：
HANKINS, WD
中科院分区：
文献类型：
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作者：
SAWYER, ST;HANKINS, WD
在缺乏促红细胞生成素（EPO）的HC-D57小鼠红细胞白血病细胞中观察到丰富的70- 78 kda形式的促红细胞生成素受体（EPOR）。与64- kda和66-kDa的EPOR蛋白相比，这些高分子量形式的EPOR （hmm-EPOR）与EPO结合位点的数量和内吞作用密切相关。假设hmm-EPOR是EPOR的高度糖基化形式，出现在细胞表面，并代表至少一种生物活性EPOR的成分。一致的发现如下。(i)从HC-D57细胞中退出EPO后，只有hm - epor增加，与i -125标记的EPO结合增加10倍相关。此外，epo依赖性的I-125-EPO结合下调和hmm-EPOR消失同时发生，而66-kDa EPOR的量没有变化。（ii）在表达EPOR cDNA的COS细胞中检测到78 kda的EPOR。（iii）用抗nh2末端抗体探测这些细胞的完整表面，只恢复了78-kDa的EPOR。（iv）酶促去糖基化和去磷酸化表明，hmm-EPOR明显是由62 kda EPOR的额外n -链糖基化引起的。(v)在EPO存在的情况下，HC-D57细胞中hm - epor的转换速度加快了12倍（半衰期从3小时变为15分钟）。（vi）抗磷酸酪氨酸抗血清检测到epo依赖的78-kDa EPOR磷酸化。97 kda蛋白酪氨酸磷酸化的动力学与hmm-EPOR的占用和内化相关。综上所述，我们认为78 kda的EPOR直接参与了EPO的初始生物学作用。
An abundant 70- to 78-kDa form of the erythropoietin receptor (EPOR) was observed in HC-D57 murine erythroleukemia cells deprived of erythropoietin (EPO). In contrast to the 64- and 66-kDa EPOR proteins, these high molecular mass forms of EPOR (hmm-EPOR) correlated well with the number of binding sites and endocytosis of EPO. The hypothesis that hmm-EPOR are wore highly glycosylated forms of the EPOR, appear on the cell surface, and represent at least one component of the biologically active EPOR was tested. Consistent findings were as follows. (i) Only hmm-EPOR increased following withdrawal of EPO from HC-D57 cells, correlating with a 10-fold increase in binding of I-125-labeled EPO. In addition, the EPO-dependent downregulation of I-125-EPO binding and disappearance of hmm-EPOR occurred in parallel while the amount of 66-kDa EPOR did not change. (ii) The 78-kDa EPOR was detected in COS cells expressing EPOR cDNA. (iii) Probing of the intact surface of these cells with anti-NH2-terminal antibody recovered only the 78-kDa EPOR. (iv) Enzymatic deglycosylation and dephosphorylation showed that hmm-EPOR apparently resulted from additional N-linked glycosylation of a 62-kDa EPOR. (v) The hmm-EPOR turnover in HC-D57 cells was accelerated 12-fold in the presence of EPO (half-life changed from 3 hr to 15 min). (vi) Anti-phosphotyrosine antiserum detected an EPO-dependent phosphorylation of the 78-kDa EPOR. The kinetics of tyrosine phosphorylation of a 97-kDa protein correlated with the occupancy and internalization of hmm-EPOR. In summary, we suggest that the 78-kDa EPOR is directly involved in the initial biological actions of EPO.