Potter, Sarah Jane
Molecular analysis of human CD23 (Fc[epsilon]RII) protein isoform function.
PhD thesis, University of Glasgow.
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In the research described in this thesis, the trafficking of each CD23 isoform was studied in detail in both wild-type and mutant CD23 proteins, using an identical cell system and the same method of CD23 ligation for both isoforms. Confocal microscopic analysis demonstrated intracellular sorting differences to exist between the two isoforms, with CD23a utilising the endocytic pathway, and CD23b following both the endocytic and phagocytic pathways in a B-cell line. Site-directed mutagenesis was used to investigate a number of potentially key residues present in the unique N-terminal tail of each isoform. The serine groups at positions 7 and 5 in the CD23a and b isoforms, respectively, and the NNP tri-peptide motif in the b isoform were found to be necessary for accurate trafficking of these proteins. The discovery that the CD23 isoforms utilise different trafficking pathways corroborates the hypothesis that CD23a and CD23b may have functionally different roles.
Current models of human CD23 signalling link the CD23a isoforms to a cAMP-generating pathway and the CD23b isoform to stimulation of inositol-1,4,5-trisphosphate production and calcium mobilisation. The ability of CD23 to transmit a signal within various cells and the fact it has a very short cytoplasmic tail, of only 23 amino acids and bereft of catalytic motifs, strongly suggests that CD23 may associate with other molecules involved in signal transduction. The divergence in the signalling pathways associated with each CD23 isoform has been attributed to the unique amino acids at the N-terminal cytoplasmic tails, as the remainder of the proteins are identical. However, there is no definitive evidence to link directly CD23 to any of these individual signalling pathways. The research presented in this thesis utilises the yeast two-hybrid assay to investigate binding partners for the N-terminal tail of CD23. Filamin A was identified as a potential interacting protein, and was found to interact with both the CD23a and CD23b isoforms. It is hypothesised that CD23 interacts with filamin A, which functions as an adaptor protein, to enable downstream responses to CD23 through connections to the appropriate signalling pathways in an isoform-specific manner.
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