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This dissertation, Differential Effects of Glial Cell Line-derived Neurotrophic Factor and Neurturin on NG108-15 Cells by Hui-kwan, Rebecca, Lee, 李曉鈞, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Differential Effects of Glial Cell Line-Derived Neurotrophic Factor and Neurturin on NG108-15 Cells Submitted by Lee Hui Kwan, Rebecca for the degree of Master of Philosophy at The University of Hong Kong in December 2003 The RET proto-oncogene encodes a receptor tyrosine kinase which is important for the development of the enteric nervous system and the kidney. Alternate splicing of RET generates three isoforms of 1072, 1106 and 1114 amino acids. The long isoform of 1114 amino acids, referred as RET51, contains 51 unique amino acids at the carboxyl terminus that are replaced by 43 amino acids in RET43 and nine residues in RET9. The RET9 and RET51 transcripts are relatively more abundant. RET activation is triggered by members of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) consisting of GDNF, neurturin (NTN), artemin (ARTN) and persephin (PSP). High affinity binding of RET to its cognate ligands is mediated by complexing with a member of glycosyl-phosphatidylinositol linked protein termed GDNF family receptor alpha: GFRα1-4. Each GFLs forms a preferred ligand-coreceptor complex. GDNF preferably bind to GFRα1 while NTN, ARTN and PSP binds to GFRα2,3 and 4, respectively. To better understand the distinct signaling profiles generated by GDNF and NTN, a neuroblastoma/glioma hybrid cell line, NG108-15, was used in the present study. NG108-15 cells were found to express high levels of RET and GFRα1 but GFRα2 expression was below the level of detection. The ligand-coreceptor complex being investigated was therefore GDNF-GFRα1 and NTN-GFRα1. I found that RET9 and RET51 formed homodimers instead of heterodimers upon ligand stimulation by immunoprecipitation and western blotting. Intriguingly, RET51 proteins may be translocated into the nucleus after NTN stimulation from immunocytochemical studies. Tyrosine (Y) 905, 1015, 1062 and 1096 are the main tyrosine phosphorylation sites in RET. Both GDNF and NTN induced a rapid phosphorylation of these sites. Rapid and transient Erk1/2 activation was found in GDNF-treated cells. On the other hand, a rapid and sustained Erk1/2 activation was observed in NTN-treated cells. Phosphorylation of PLCγ induced by NTN was more prolonged and pronounced whereas a weaker PLCγ phosphorylation was induced by GDNF. For the biological responses, NTN but not GDNF stimulated differentiation through PLCγ activation. On the other hand, GDNF was found to act as a survival factor. There was no observable effect of GDNF and NTN on proliferation and cell migration. In order to identify different substrates of phosphorylation triggered by GDNF and NTN, mass spectrometry was used. Several phosphorylated proteins were identified in both untreated and NTN-treated cells. These included heat shock protein 90-beta, heat shock cognate 71kD protein and myosin heavy chain. Ribonucleoside-diphophate reductase M1 chain and stress-induced-phosphoprotein 1 were only found to be phosphorylated in untreated cells. In nuclear fraction of GDNF-treated cells, myc far upstream-binding protein was phosphorylated. The roles of these proteins in RET signaling remain to be determined. DOI: 10.5353/th_b2777136 Subjects: Cell differentiationNeurotrophic functionsCellular signal transduction

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