The carboxyl-terminal valine residues of ProTGF alpha are required for its efficient maturation and intracellular routing
The carboxyl-terminal valine residues of ProTGF alpha are required for its efficient maturation and intracellular routing
复制标题
DOI：
10.1091/mbc.8.8.1619
复制
发表时间：
1997-08-01
影响因子：
3.3
通讯作者：
Lee, DC
中科院分区：
文献类型：
--
作者：
Briley, GP;Hissong, MA;Lee, DC
转化生长因子- α (TGF - α)的可溶性形式是由一个完整的膜糖蛋白前体（proTGF - α）的蛋白水解加工而来的。先前的研究表明，phobol酯诱导的CHO细胞中proTGF α的裂解依赖于位于前体细胞质结构域羧基端的缬氨酸残基的存在。我们重新评估了这一需求，将表位标记的构建物引入到正常表达和处理TGF α的转化大鼠肝上皮细胞中。我们发现缺乏末端缬氨酸残基的proTGF α突变体成熟到完全糖基化的形式大大减少。此外，它们在细胞表面的含量大大降低，而是在内质网中积累。与这些结果一致，酶联免疫吸附试验（ELISA）和Western blot分析显示，在表达突变结构的细胞条件下，培养基中很少或没有可溶性TGF - α。最后，截断的proTGF α突变体缺乏大部分细胞质结构域，但保留羧基末端缬氨酸，以接近正常的方式进行加工和切割。这些结果，其中一些在CHO细胞中重现，表明羧基末端缬氨酸的主要作用是确保前体的正常成熟和路由。
Soluble forms of transforming growth factor-alpha (TGF alpha) are derived by proteolytic processing of an integral membrane glycoprotein precursor (proTGF alpha). Previous studies indicated that phorbol ester-induced cleavage of proTGF alpha in CHO cells is dependent on the presence of a valine residue located at the carboxyl terminus of the precursor's cytoplasmic domain. We reassessed this requirement with epitope-tagged constructs introduced into transformed rat liver epithelial cells that normally express and process TGF alpha. We found that proTGF alpha mutants lacking the terminal valine residues showed greatly reduced maturation to the fully glycosylated form. Additionally, they were present at substantially reduced levels on the cell surface and, instead, accumulated in the endoplasmic reticulum. Consistent with these results, enzyme-linked immunosorbant assay (ELISA) and Western blot analyses revealed little or no soluble TGF alpha in medium conditioned by cells expressing the mutant constructs. Finally, a truncated proTGF alpha mutant lacking most of the cytoplasmic domain but retaining a carboxyl-terminal valine was processed and cleaved in a near-normal manner. These results, some of which were reproduced in CHO cells, indicate that the predominant effect of the carboxyl-terminal valines is to ensure normal maturation and routing of the precursor.