Rap1GAP interacts with RET and suppresses GDNF-induced neurite outgrowth.
Glial cell line-derived neurotrophic factor (GDNF) was originally recognized for its ability to promote survival of midbrain dopaminergic neurons, but it has since been demonstrated to be crucial for the survival and differentiation of many neuronal subpopulations, including motor neurons, sympathetic neurons, sensory neurons and enteric neurons. To identify possible effectors or regulators of GDNF signaling, we performed a yeast two-hybrid screen using the intracellular domain of RET, the common signaling receptor of the GDNF family, as bait. Using this approach, we identified Rap1GAP, a GTPase-activating protein (GAP) for Rap1, as a novel RET-binding protein. Endogenous Rap1GAP co-immunoprecipitated with RET in neural tissues, and RET and Rap1GAP were co-expressed in dopaminergic neurons of the mesencephalon. In addition, overexpression of Rap1GAP attenuated GDNF-induced neurite outgrowth, whereas suppressing the expression of endogenous Rap1GAP by RNAi enhanced neurite outgrowth. Furthermore, using co-immunoprecipitation analyses, we found that the interaction between RET and Rap1GAP was enhanced following GDNF treatment. Mutagenesis analysis revealed that Tyr981 in the intracellular domain of RET was crucial for the interaction with Rap1GAP. Moreover, we found that Rap1GAP negatively regulated GNDF-induced ERK activation and neurite outgrowth. Taken together, our results suggest the involvement of a novel interaction of RET with Rap1GAP in the regulation of GDNF-mediated neurite outgrowth.
Jiao
Li
L
Institute of Neuroscience and Key Laboratory of Molecular Neurobiology of Ministry of Education, Neuroscience Research Center of Changzheng Hospital, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, China.
Zhang
Yong
Y
Hu
Chun
C
Wang
Yong-Gang
YG
Huang
Aijun
A
He
Cheng
C
D000818:Animals; D045744:Cell Line, Tumor; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology*; D047468:Immunoprecipitation; D051379:Mice; D009154:Mutation; D016501:Neurites / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D020798:Two-Hybrid System Techniques; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D051100:Glial Cell Line-Derived Neurotrophic Factor; D034741:RNA, Small Interfering; D051096:Proto-Oncogene Proteins c-ret; C495001:Ret protein, rat; D048049:Extracellular Signal-Regulated MAP Kinases; D020827:rap1 GTP-Binding Proteins