| Glia cell line-derived neurotrophic factor (GDNF) family contains four members: GDNF, NRTN ( neurturin, ARTN ( artemin, PSPN (persephin), which could not only support the survival and development of mescencephalic dopaminergic neuron and motoneuron in the central nervous system, but also enhance the survival and differentiation of sympathetic and parasympathetic neuron, sensory neuron and enteric neuron in the peripheral nervous system. GDNF is also an important morphologic fator involved in the development of the kidney and plays a key role in the differentiation of spermatogonial cells. GDNF family ligands mediate signaling pathway through their corresponding receptors GFRαl-4 and their corporate receptor RET. After binding with their ligands, GFRal-4 would trigger the autophosphorylation of RET and then active the downstream signaling pathway such as Ras/MAPK, PI3-K and PLCy etc. RET, belonging to the receptor kinase family, is a transmembrane glycoprotein and contains extracellular, transmembrane and intracellular domains. The intracellular domain of RET, with the tyrosine kinase activity, provides the docking sites for many adaptor proteins. For example, the pY1062 in RET could at least bind to five dock proteins, such as She, FRS2, DOK4/5, IRS 1/2 and enigma, to induce different downstream signaling pathway either leading to neuronal growth or branching.According to the length of carboxyl terminal, SH2-B is classified as α,β,γand 6 isoforms. SH2-B contains multiple motifs participating in protein interactions, such as SH2 domain, PH domain, proline-riched domain and many potential phosphorylation sites. SH2-B was identified as a binding protein of the receptors for nerver growth factor, insulin, platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-1, hepatocyte growth factor, the cytokinereceptor-associated tyrosine kinase JAK2, and the Rl Fc, Rl rceptor. We have previously demonstrated that the intracellular domain of RET could bind to SH2-B 0 in vitro by using yeast two-hybrid system to screen a human brain cDNA library. In the present study, we sought to identify the region in SH2-B ?critical for the interaction with RET, to determine the capability of the interaction in vivo, and to investigate the function of the interaction.The main results of our research are as follows:1. The cDNAs for full length of wild type SH2-B 0 and its PH domain, SH2 domain were amplified by PCR. The dominant negative mutant of SH2-B P (R555E) was obtained by PCR based Quick-change mutagensis. All the cDNAs above were respectively cloned into the AD-vector to test their corresponding activity to bind to the intracelluar region of RET in the yeast two-hybrid system. Our result of p-galactosidase activity assay showed that the SH2 domain was both sufficient and necessary for SH2-B f3 to bind to RET, while the PH domain was incapable of association with RET.2. The results of co-immunoprecipitation assay revealed that only after the stimulation of GDNF, the endogenous SH2-B 0 could bind to over-expressed RET in PC12-rGFRal-RET cell, which constructed by our lab previously. In the wild type PC 12 cell, either with or without the stimulation of GDNF, the binding was undetectable. These results demonstrated that SH2-B P was capable of interacting with the activated RET in vivo.3. By using co-immunoprecipitation assay, we also found that there were not only co-existence but also association between the endogenous SH2-B 0 and RET in spinal and mesencephalic tissue lysates.4. To investigate the functional relevance of the interaction between RET and SH2-B P in PC 12 cells, the SH2-B 0 or its mutant (R555E) was transiently co-transfected with EGFP-N2 plasmid, which harbouring the cDNA for Green fluorescence protein (GFP), into the PC 12 - rGFRal- RET cell. Neurite ourgrowth assay based on the GFP positive cells revealed that the wild type SH2-B 0 group of PC 12 cells had much higher ratio of neurites bearing cells than the control group inresponse to GDNF stimulat...
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