Regulation of C-type lectin-like receptors dectin-1 and CLEC-2 by tetraspanins
Tomlinson, Neil David (2010)
Ph.D. thesis, University of Birmingham.
Tetraspanins are a superfamily of glycoproteins that function as ‘organisers’ of membranes by clustering with each other to form tetraspanin-enriched microdomains, into which certain other receptors and signalling proteins are recruited and regulated. Tetraspanin microdomains have been implicated in a range of biological processes including cell signalling, adhesion, intracellular trafficking, cell-cell fusion and viral entry. The tetraspanin CD37 was recently shown to negatively regulate the C-type lectin-like receptor dectin-1, which is essential for innate immune responses to fungal pathogens. The aim of this thesis was to firstly develop a cell line model system to investigate the mechanism by which tetraspanins inhibit dectin-1, and to secondly extend this work to the dectin-1-related CLEC-2, which is essential for platelet thrombus formation and stability. Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay in the Jurkat T-cell line, transient over-expression of CD37 was found to powerfully inhibit dectin-1 signalling following stimulation with its ligand, β-glucan. Over-expression of other tetraspanins also inhibited dectin-1 signalling, but did not globally inhibit receptor signalling because the platelet collagen receptor, GPVI, was unaffected. Similar to dectin-1, CLEC-2 signalling in response to its ligand, the snake venom toxin rhodocytin, was also abrogated following tetraspanin over-expression. However, stable tetraspanin over-expression only partially reduced signalling. Moreover, knockdown of the major Jurkat cell tetraspanin, CD81, and deletion of the major platelet tetraspanin, CD9, did not affect dectin-1 and CLEC-2 signalling, respectively. In summary, the importance of transient tetraspanin over-expression for dectin-1 and CLEC-2 inhibition, and the fact that any tetraspanin can inhibit, suggests that tetraspanin microdomains are disrupted by the presence of one over-expressed tetraspanin. This leads to a failure of dectin-1 and CLEC-2 signalling by a mechanism that is not clear, but suggests that tetraspanin microdomains are important for signalling by these C-type lectin-like receptors.
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