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Title: Expression and Characterisation of GPCRs in Schizosaccharomyces pombe
Author: Forfar, Rachel Alice Emma
ISNI:       0000 0001 3474 7071
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2007
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G protein-coupled receptors COPCRs) comprise the largest class of cell surface receptors and regulate a wide range of physiological processes in response to diverse stimuli. Accordingly, they are of great interest to the pharmaceutical industry, with -30% of marketed drugs targeting these receptors. However, the complex interplay between components of GPCR signalling pathways in mammalian cell systems inhibits the study of many aspects of these cascades. This thesis describes the use of Schizosaccharomyces p011lbe to simplify investigations into the expression and characterisation of both human and yeast GPCRs, and describes attempts to create a more sensitive strain for enhanced detection of signalling. Initially, the sxa2>lacZ reporter system was used to conduct studies of heterologously expressed AI and Aza adenosine receptors, but unfortunately these did not produce a functional response. Endeavours were made to increase receptor localisation at the plasma membrane by addition of a cleavable leader sequence, and to improve the interaction with the Oa subunit by exchanging IC3 loop from Mam2 into the adenosine receptors. However, no signalling was detected with these receptor alterations. . Additionally, a number of genetic modifications were made to the system in order to improve the detection of ·signalling. Modulation of the reporter strain pheromone response pathway was performed by creating a hyperactivated ras]Val17 strain, and this produced a two-fold increase in signalling through the endogenous Mam2 receptor. However, the ligand-independent signalling was also elevated, resulting in a similar signal:background ratio to the standard sxa2>lacZ reporter. Instead, simultaneous use of the lacZ and ura4 reporter genes in a 'double reporter' strain reduced ligand-independent levels of signalling, thereby substantially increasing the signal:background ratio. This was further enhanced through the addition of 6Azauracil, a potent inhibitor of Ura4 activity. With further alterations this strain could help detect signalling through the adenosine receptors. The dimerisation of two yeast GPCRs was investigated as a model for dimerisation of human receptors, since the adenosine receptors were not functional in Sz. p011lbe. Heterodimerisation of the related yeast receptors Mam2 and STE2 demonstrated an altered Oa-specificity compared to the respective homodimers, which may have implications in the signal transduction of human OPCR heterodimers. The results presented in this thesis should allow investigations to be extended into the dimerisation of human GPCRs in Sz. p011lbe and to determine the effects on their Gaspecificities.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available