Abstract
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.
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Abbreviations
- BSA:
-
(bovine serum albumin)
- DMEM:
-
(Dulbecco's modified Eagle's medium)
- GDNF:
-
(glial cell line-derived neurotrophic factor)
- EGFP:
-
(enhanced green fluorescent protein)
- FBS:
-
(fetal bovine serum)
- PBS:
-
(phosphate-buffered saline)
- RTK:
-
(receptor tyrosine kinase)
- SDS-PAGE:
-
(sodium dodecyl sulfate polyacrylamide gel electrophoresis)
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Acknowledgements
We are grateful to Dr Carlos F Ibanez (Karolinska Institute, Sweden) for generously providing the RET cDNA. The Rap1GAP cDNA was a generous gift from Dr Patrick J Casey (Duke University Medical Center, USA). The GST-RalGDS-RBD plasmid (for detecting Rap1 activity) was a generous gift from Dr Chengbiao Wu (Stanford University School of Medicine, USA). This work was supported by the National Natural Science Foundation of China (30400123, 30570939, 30770657 and 30530240); National Key Basic Research Program (2006CB500702, 2007CB947100, 2011CB504401 and 2009ZX09311-001); Shanghai Rising-Star Program (05QMX1469) and Shanghai Metropolitan Fund for Research and Development (07DJ14005).
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Jiao, L., Zhang, Y., Hu, C. et al. Rap1GAP interacts with RET and suppresses GDNF-induced neurite outgrowth. Cell Res 21, 327–337 (2011). https://doi.org/10.1038/cr.2010.139
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DOI: https://doi.org/10.1038/cr.2010.139
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