Molecular Brain Research Group, Laboratory of Neural Signaling, Robarts Research Institute, University of Western Ontario, 100 Perth Drive, London, ON, Canada N6A 5K8.
J Mol Neurosci. 2013 Jan;49(1):38-51. doi: 10.1007/s12031-012-9847-9. Epub 2012 Jun 29.
We previously demonstrated that the guanine nucleotide exchange factor, RasGrf1, binds nerve growth factor (NGF)-activated TrkA and facilitates neurotrophin-induced neurite outgrowth in PC12 cells. RasGrf1 can activate both Ras and Rac, via intrinsic Cdc25 and DH domains, respectively, suggesting that the activation of both could contribute to this process. Previous studies have assayed constitutive neurite outgrowth following RasGrf1 over-expression in PC12 cells, in either the absence or presence of ectopic H-Ras, and have suggested an essential role for either Ras or Rac depending on the presence of H-Ras over-expression. In contrast, in this study, we have addressed the mechanism of how RasGrf1 facilitates neurite outgrowth in response to the neurotrophins, NGF and BDNF. Using Ras/Rac activation assays and site-directed RasGrf1 mutants, we find that both Ras and Rac are essential to neurotrophin-induced neurite outgrowth. Moreover, we find that H-Ras over-expression rescues the loss of neurite outgrowth observed with a Rac minus mutant and that RasGrf1 differentially stimulates NGF-dependent activation of Rac or Ras, depending on cell type. Collectively, these studies clarify the mechanism of how RasGrf1 expression facilitates neurotrophin-induced neurite outgrowth. Moreover, they suggest that H-Ras over-expression should be used with caution to measure phenotypic responses.
我们之前已经证明，鸟嘌呤核苷酸交换因子 RasGrf1 与神经营养因子（NGF）激活的 TrkA 结合，并促进 PC12 细胞中神经营养因子诱导的轴突生长。RasGrf1 可以通过内在的 Cdc25 和 DH 结构域分别激活 Ras 和 Rac，这表明这两种蛋白的激活都可能有助于这一过程。以前的研究已经在没有或存在异位 H-Ras 的情况下，在 PC12 细胞中检测了 RasGrf1 过表达后诱导的轴突生长，并且根据 H-Ras 过表达的存在，提出了 Ras 或 Rac 的组成性激活的重要作用。相比之下，在这项研究中，我们研究了 RasGrf1 如何促进神经营养因子诱导的轴突生长的机制。通过 Ras/Rac 激活测定和 RasGrf1 突变体，我们发现 Ras 和 Rac 对于神经营养因子诱导的轴突生长都是必需的。此外，我们发现 H-Ras 过表达可以挽救 Rac 缺失突变体中观察到的轴突生长丧失，并且 RasGrf1 根据细胞类型差异地刺激 NGF 依赖性 Rac 或 Ras 的激活。总的来说，这些研究阐明了 RasGrf1 表达如何促进神经营养因子诱导的轴突生长的机制。此外，它们表明应该谨慎使用 H-Ras 过表达来测量表型反应。
