Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.362585
Title: The expression and regulation of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR)
Author: Chopra, Rajesh
ISNI:       0000 0001 3548 0057
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1996
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Abstract:
The granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) consists of a specific α chain which binds ligand with low affinity and a β chain which confers high affinity binding. The aim of this thesis was to investigate the expression and regulation of the GM-CSFR. RNase protection assays were developed in order to detect the low abundance mRNA species. The GM-CSFRa mRNA expression was determined to be in the pg range and the β mRNA expression at least one log lower. GM- CSFRa mRNA expression was demonstrated in a number of haemopoietic cell lines and the expression of GM-CSFRa mRNA was consistently higher than that of GM-CSFRα mRNA, which is in keeping with the receptor protein expression. RNase protection assays of GM-CSFRα yielded extra bands in addition to the expected mRNA transcript. Characterisation studies, using RT-PCR, established the existence of at least two additional mRNA transcripts involving alternative splicing of the 5' untranslated region of the GM-CSFRa chain. Sequencing demonstrated that these isoforms were the result of a deletion of 24 nucleotides at the 3' end of exon 2 (exon 2b deleted isoform) and complete deletion of exon 2 (exon 2 deleted isoform). These isoforms were detected in primary haemopoietic cells, blasts from patients with acute myeloid leukaemia and malignant cell lines. Together the isoforms were more highly expressed than the full length sequence. Translation of constructs corresponding to the exon 2 deleted isoforms was assessed using an in-vitro reticulocyte lysate system. Deletion of exon 2 resulted in a significantly lower in vitro translation of the receptor protein compared to the full length sequence, whilst deletion of exon 2b results in higher translation of the a chain protein. These in vitro studies therefore identified putative regulatory sequences that may modulate translation of the GM-CSFRα chain. In order to investigate the underlying mechanisms by which the receptor expression is regulated, GM-CSFR binding and mRNA expression were investigated using two model systems: TF-1 cells undergoing down and up- regulation of surface receptors; and HL-60 cells undergoing DMSO-induced differentiation. The half-life of the GM-CSFR protein in TF-1 cells growing in steady state conditions in the presence of Epo and the translation inhibitor cycloheximide, was approximately 4 hours, which would indicate a constant nonligand mediated turnover of cell surface receptor protein. Incubation of the TF-1 cells in the presence of the transcription inhibitor actinomycin-D indicated that the half-life of the GM-CSFRα mRNA was between 1-3 hours and that of the GM-CSFR α mRNA greater than 8 hours. The cell surface expression of the specific GM-CSFRα and β chains and mRNA levels however, did not change in the presence of actinomycin D, implying post-transcriptional regulation of the GM- CSFR expression. In order to further clarify the nature of this post-transcriptional regulation, cell surface receptor expression in cells undergoing up-regulation of their receptor was measured. Up-regulation was inhibited in the presence of cycloheximide, but not by the lysosomotropic agent ammonium chloride, suggesting that new protein synthesis, rather than receptor recycling, is important when GM-CSFR expression is dynamically modulated. Uninduced HL-60 cells express low numbers of both low and high affinity receptors and on induction with DMSO for 5 days, the receptor numbers increased and both the GM-CSFRα and β mRNA levels showed a co-ordinate increase. This implies transcriptional regulation of the GM-CSFRα and β chains during differentiation. The different mechanisms described for GM-CSFR regulation may explain at least in part, how GM-CSF can act through its receptor to modulate long term differentiation events and at the same time augment mature cell function where the control mechanisms take place over a time course of minutes and hours.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.362585  DOI: Not available
Keywords: Genetics
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