Zhang Yong, Zhu Wei, Wang Yong-Gang, Liu Xiu-Jie, Jiao Li, Liu Xuan, Zhang Zhao-Huan, Lu Chang-Lin, He Cheng
Department of Neurobiology, Second Military Medical University, Shanghai, 200433, PR of China.
J Cell Sci. 2006 Apr 15;119(Pt 8):1666-76. doi: 10.1242/jcs.02845. Epub 2006 Mar 28.
RET receptor signalling is essential for glial-cell-line-derived neurotrophic factor (GDNF)-induced survival and differentiation of various neurons such as mesencephalic neurons. To identify proteins that mediate RET-dependent signaling, yeast two-hybrid screening was performed with the intracellular domain of RET as bait. We identified a new interaction between RET and the adapter protein SH2-Bbeta. Upon GDNF stimulation of PC12-GFRalpha1-RET cells (that stably overexpress GDNF receptor alpha1 and RET), wild-type SH2-Bbeta co-immunoprecipitated with RET, whereas the dominant-negative SH2-Bbeta mutant R555E did not. RET interacted with endogenous SH2-Bbeta both in PC12-GFRalpha1-RET cells and in rat tissues. Mutagenesis analysis revealed that Tyr981 within the intracellular domain of RET was crucial for the interaction with SH2-Bbeta. Morphological evidence showed that SH2-Bbeta and RET colocalized in mesencephalic neurons. Furthermore, functional analysis indicated that overexpression of SH2-Bbeta facilitated GDNF-induced neurite outgrowth in both PC12-GFRalpha1-RET cells and cultured mesencephalic neurons, whereas the mutant R555E inhibited the effect. Moreover, inhibition of SH2-Bbeta expression by RNA interference caused a significant decrease of GDNF-induced neuronal differentiation in PC12-GFRalpha1-RET cells. Taken together, our results suggest that SH2-Bbeta is a new signaling molecule involved in GDNF-induced neurite outgrowth.
RET受体信号传导对于胶质细胞源性神经营养因子（GDNF）诱导的多种神经元（如中脑神经元）的存活和分化至关重要。为了鉴定介导RET依赖性信号传导的蛋白质，以RET的细胞内结构域为诱饵进行了酵母双杂交筛选。我们鉴定出RET与衔接蛋白SH2-Bβ之间存在新的相互作用。在用GDNF刺激PC12-GFRα1-RET细胞（稳定过表达GDNF受体α1和RET）后，野生型SH2-Bβ与RET共免疫沉淀，而显性负性SH2-Bβ突变体R555E则没有。在PC12-GFRα1-RET细胞和大鼠组织中，RET均与内源性SH2-Bβ相互作用。诱变分析表明，RET细胞内结构域中的Tyr981对于与SH2-Bβ的相互作用至关重要。形态学证据显示，SH2-Bβ和RET在中脑神经元中共定位。此外，功能分析表明，SH2-Bβ的过表达促进了PC12-GFRα1-RET细胞和培养的中脑神经元中GDNF诱导的神经突生长，而突变体R555E则抑制了该效应。此外，RNA干扰抑制SH2-Bβ表达导致PC12-GFRα1-RET细胞中GDNF诱导的神经元分化显著降低。综上所述，我们的结果表明，SH2-Bβ是参与GDNF诱导的神经突生长的一种新的信号分子。
