Isoforms of the granulocyte-macrophage colony-stimulating factor receptor

This thesis describes the identification and characterisation of a novel isoform of the GM-CSF receptor alpha subunit (GM-CSFRα). The novel isoform contains an insertion of 102bp encoding 34 amino acids between the transmembrane region and WSXWS domain. The insertion is unusual because it appears to have arisen through retroposition of an Alu repetitive element within the coding sequence of the gene. Subcloning and sequencing of a cosmid containing the GM-CSFRα gene revealed the presence of consensus splice donor acceptor sites flanking the insertion confirming that the isoform is a product of alternative RNA splicing. Analysis of primary human haemopoietic cells and cell lines by RT- PCR did not reveal any differences in expression of the novel isoform during differentiation or in myeloid leukaemias. Expression of GM-CSFRα4 in COS-7 cells showed that compared to the dominant GM-CSFRα1 species, the protein is expressed at a low level. When cotransfected with the βc subunit of the GM-CSFR, the novel isoform gives rise to a receptor of intermediate affinity in contrast to GM-CSFRα1/βc where dual high and low affinity GM-CSF binding results. Altering the ratio of GM-CSFRα/β cDNA in the transfection experiments showed that the switch in affinity results from altered stoicheiometry of the α and β subunits. When α subunits are limiting, intermediate affinity binding results whilst in conditions of α subunit excess, dual high and low affinity binding is seen. Analysis of GM-CSFRα mRNA and protein levels in cells transfected with GM-CSFRα1 and GM-CSFRα4 showed that the reduced expression of the later is mediated by a post transcriptional mechanism