Barker P A, Miller F D, Large T H, Murphy R A
Department of Anatomy and Cell Biology, University of Alberta, Edmonton, Canada.
J Biol Chem. 1991 Oct 5;266(28):19113-9.
These studies were initiated to determine whether the soluble, truncated form of the nerve growth factor (NGF) receptor arises from post-translational processing of the intact, membrane-bound receptor or from an alternatively spliced mRNA. Pulse-chase analysis of cultured primary rat Schwann cells coupled with immunoprecipitations using antibodies to the intracellular and extracellular domains of the receptor were used to monitor receptor production. Three forms of the NGF receptor (80, 83, and 85 kDa) displaying a precursor product relationship were detected over the 2-h chase period; only the 85-kDa species was detected on the cell surface. Truncated receptors (50 and 52 kDa) were detected in conditioned media 5 h after cell labeling but were never observed intracellularly. Polymerase chain reaction and RNase protection analyses of NGF receptor mRNA targeted toward the coding region for the transmembrane domain detected no splice variants that could generate truncated receptor, and media conditioned by fibroblasts transfected with rat receptor cDNA, in which splicing cannot occur, nonetheless contained the truncated receptor protein. Taken together, these results suggest that the truncated NGF receptor does not arise as a distinct translation product but rather from a post-translational modification of the intact, surface-bound form of the protein.
开展这些研究是为了确定神经生长因子（NGF）受体的可溶性截短形式是源自完整的膜结合受体的翻译后加工，还是源自选择性剪接的mRNA。使用针对受体细胞内和细胞外结构域的抗体进行免疫沉淀，并结合对培养的原代大鼠雪旺细胞的脉冲追踪分析，来监测受体的产生。在2小时的追踪期内检测到三种呈现前体-产物关系的NGF受体形式（80、83和85 kDa）；仅在细胞表面检测到85 kDa的形式。在细胞标记5小时后的条件培养基中检测到截短的受体（50和52 kDa），但在细胞内从未观察到。针对跨膜结构域编码区的NGF受体mRNA进行聚合酶链反应和核糖核酸酶保护分析，未检测到可产生截短受体的剪接变体，并且用大鼠受体cDNA转染的成纤维细胞（其中不会发生剪接）所产生的条件培养基中仍然含有截短的受体蛋白。综上所述，这些结果表明截短的NGF受体并非作为一种独特的翻译产物产生，而是源自该蛋白质完整的表面结合形式的翻译后修饰。
