Ferro F E, Kozak S L, Hoatlin M E, Kabat D
Department of Biochemistry and Molecular Biology, School of Medicine, Oregon Health Sciences University, Portland 97201-3098.
J Biol Chem. 1993 Mar 15;268(8):5741-7.
The membrane glycoprotein (gp55) encoded by the env gene of Friend spleen focus-forming virus (SFFV) causes mitogenesis of infected erythroblasts and is inefficiently (3-5%) processed from the rough endoplasmic reticulum (RER) to plasma membranes. Recent evidence suggested that gp55 binds to erythropoietin receptors (EpoR) in the RER, and it was proposed that this intracellular interaction causes mitogenesis (Li, J.-P., D'Andrea, A. D., Lodish, H. F., and Baltimore, D. (1990) Nature 343, 762-764). Other evidence has indicated that the plasma membrane component (gp55P) can also complex with EpoR. To identify the site of functional complexes and to study the factors that control their formation, we analyzed eight SFFV env mutants that contain in-frame deletions or linker insertions. The three nonpathogenic mutants encode gp55s that fail to leave the RER, whereas the five pathogenic mutants encode glycoproteins that occur on cell surfaces. Although BaF3 hematopoietic cells are interleukin 3 (IL-3)-dependent, a cellular derivative BaF3/EpoR that contains EpoR survives and grows in either IL-3 or erythropoietin (Epo). The five pathogenic SFFV env mutants converted BaF3/EpoR but not BaF3 cells to growth factor independence, whereas the nonpathogenic mutants did not eliminate growth factor requirements of any cells. Studies using 125I-Epo and the covalent cross-linking reagent disuccinimidyl suberate provided unambiguous evidence for ternary complexes of 125I-Epo.EpoR.gp55P on surfaces of all factor-independent cells. Size of the cell surface complex was correspondingly reduced in the case of a newly isolated SFFV mutant that encodes a severely truncated (M(r) approximately 41,000) but mitogenically active env glycoprotein. Our results support the hypothesis that activation of EpoR by the SFFV env glycoprotein occurs exclusively on cell surfaces.
弗瑞德脾集落形成病毒（SFFV）的env基因编码的膜糖蛋白（gp55）可引起受感染的成红细胞发生有丝分裂，并且从粗面内质网（RER）到质膜的加工效率较低（3 - 5%）。最近的证据表明，gp55在RER中与促红细胞生成素受体（EpoR）结合，有人提出这种细胞内相互作用会导致有丝分裂（Li, J.-P., D'Andrea, A. D., Lodish, H. F., and Baltimore, D. (1990) Nature 343, 762 - 764）。其他证据表明，质膜成分（gp55P）也能与EpoR形成复合物。为了确定功能复合物的形成位点并研究控制其形成的因素，我们分析了八个含有框内缺失或接头插入的SFFV env突变体。三个无致病性的突变体编码的gp55无法离开RER，而五个有致病性的突变体编码的糖蛋白出现在细胞表面。尽管BaF3造血细胞依赖白细胞介素3（IL - 3），但含有EpoR的细胞衍生物BaF3/EpoR在IL - 3或促红细胞生成素（Epo）中都能存活并生长。五个有致病性的SFFV env突变体使BaF3/EpoR细胞而非BaF3细胞不再依赖生长因子，而无致病性的突变体并未消除任何细胞对生长因子的需求。使用125I - Epo和共价交联剂辛二酸二琥珀酰亚胺酯进行的研究为所有不依赖因子的细胞表面上125I - Epo.EpoR.gp55P三元复合物提供了明确证据。对于一个新分离的SFFV突变体，其编码一种严重截短的（M(r)约为41,000）但具有有丝分裂活性的env糖蛋白，细胞表面复合物的大小相应减小。我们的结果支持这样的假设，即SFFV env糖蛋白对EpoR的激活仅发生在细胞表面。
