MacDonald J I, Verdi J M, Meakin S O
The John P. Robarts Research Institute, Neurodegeneration Group, London, Ontario, Canada.
J Mol Neurosci. 1999 Aug-Oct;13(1-2):141-58. doi: 10.1385/JMN:13:1-2:141.
Many responses to nerve growth factor (NGF) are regulated through the receptor tyrosine kinase trkA. To understand more fully the functions of trkA in NGF responsive cells, we have expressed the intracellular domain of rat trkA as a fusion protein with the yeast gal4 transcription factor, and used the fusion protein to probe rat and mouse cDNA libraries by the yeast two-hybrid system. We have identified a direct interaction between the intracellular domain of trkA and two members of the intermediate filament (IF) family of proteins, the guanine-nucleotide exchange protein Ras-GRF1, the p162 subunit of eIF3, and the beta-6 proteasome subunit. The interactions are dependent on an active trkA kinase, and RasGRF1, the beta-6 proteasomal subunit, and peripherin are directly phosphorylated by trkA. The interaction with trkA is not affected by mutations at either Tyr499 or Tyr794, the two major phosphotyrosine residues essential to the activation and receptor binding of Shc, FRS-2/SNT, and phospholipase Cgamma-1, and it is highly specific in vitro for trkA, with little or no binding observed with trkB and/or trkC. The results show that trkA may play a regulatory role in a variety of cellular functions in addition to neuritogenesis, including regulated protein degradation and transcriptional activation.
对神经生长因子（NGF）的许多反应是通过受体酪氨酸激酶trkA来调节的。为了更全面地了解trkA在NGF反应性细胞中的功能，我们将大鼠trkA的细胞内结构域作为与酵母gal4转录因子的融合蛋白进行表达，并利用该融合蛋白通过酵母双杂交系统探测大鼠和小鼠的cDNA文库。我们已经确定trkA的细胞内结构域与中间丝（IF）蛋白家族的两个成员、鸟嘌呤核苷酸交换蛋白Ras-GRF1、eIF3的p162亚基以及β-6蛋白酶体亚基之间存在直接相互作用。这些相互作用依赖于活性trkA激酶，并且RasGRF1、β-6蛋白酶体亚基和外周蛋白可被trkA直接磷酸化。与trkA的相互作用不受Tyr499或Tyr794突变的影响，这两个主要的磷酸酪氨酸残基对Shc、FRS-2/SNT和磷脂酶Cγ-1的激活和受体结合至关重要，并且在体外对trkA具有高度特异性，与trkB和/或trkC几乎没有或没有结合。结果表明，trkA除了在神经突形成中可能在多种细胞功能中发挥调节作用，包括调节蛋白质降解和转录激活。
