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Differential effects of glial cell line-derived neurotrophic factor and neurturin on NG108-15 cells

by Lee, Hui-kwan

Abstract (Summary)
(Uncorrected OCR) Abstract of thesis entitled

Differential Effects of Glial Cell Line-Derived Neurotrophic Factor and Neurturin on NGI08-15 Cells

Submitted by

Lee Hui K wan, 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 affmity binding of RET to its cognate ligands is mediated by complexing with a member of glycosyl-phosphatidylinositollinked protein termed GDNF family receptor alpha: GFRal-4. Each GFLs forms a preferred ligand-coreceptor complex. GDNF preferably bind to GFRal while NTN, ARTN and PSP binds to GFRa2,3 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 GFRal but GFRa2 expression was below the level of detection. The ligand-coreceptor complex

being investigated was therefore GDNF-GFRal and NTN-GFRal. I found that RET9 and RET5l formed homodimers instead of heterodimers upon ligand stimulation by immunoprecipitation and western blotting. Intriguingly, RET5l 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 Erkl/2 activation was found in GDNF-treated cells. On the other hand, a rapid and sustained Erkl/2 activation was observed in NTN -treated cells. Phosphorylation of PLCy induced by NTN was more prolonged and pronounced whereas a weaker PLCy phosphorylation was induced by GDNF. For the biological responses, NTN but not GDNF stimulated differentiation through PLCy 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 Ml chain and stress-indue ed-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.

Bibliographical Information:

Advisor:

School:The University of Hong Kong

School Location:China - Hong Kong SAR

Source Type:Master's Thesis

Keywords:university of hong kong dissertations cell differentiation neurotrophic functions cellular signal transduction

ISBN:

Date of Publication:01/01/2004

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