Interaction of SH2-Bbeta with RET is involved in signaling of GDNF-induced neurite outgrowth.
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.
Zhang
Yong
Y
Department of Neurobiology, Second Military Medical University, Shanghai, 200433, PR of China.
Zhu
Wei
W
Wang
Yong-Gang
YG
Liu
Xiu-Jie
XJ
Jiao
Li
L
Liu
Xuan
X
Zhang
Zhao-Huan
ZH
Lu
Chang-Lin
CL
He
Cheng
C
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D049108:Cell Enlargement; D002460:Cell Line; D002478:Cells, Cultured; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000502:physiology*; D008636:Mesencephalon / Q000166:cytology / Q000378:metabolism; D008969:Molecular Sequence Data; D016501:Neurites / Q000502:physiology*; D016716:PC12 Cells; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D014162:Transfection; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D051100:Glial Cell Line-Derived Neurotrophic Factor; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human