G protein-coupled receptor (GPCR) signalling cascades are a highly conserved and important method of regulating a wide range of physiological processes and responses. The conserved nature of GPCR signalling throughout eukaryotic cells allows the use of model systems such as the pheromone-response pathway in the fission yeast Schizosaccharomyces pombe, to investigate the intricacies of the signalling interactions without the complexities associated with higher eukaryotic cells. This thesis describes the use of Sz. pombe to investigate the signalling characteristics and regulation of G protein α-subunits and the further characterisation of events within the pheromone-response pathway of Sz. pombe. Human Gα subunits were found not to couple to the Sz. pombe pheromone-response pathway as they failed to reach the plasma membrane. Such localisation is essential for the functions of the endogenous Gα subunit, Gpa1, which is targeted to the plasma membrane via myristoylation and palmitoylation. The Gpa1 N-terminal region was also found to influence volume of cells by influencing the length of the cell cycle. The regulation of Gpa1 activity was investigated by mutational analysis of residues with predicted involvement in Gα activation and GTP hydrolysis. Mutations within the conserved G5 loop of the nucleotide-binding pocket resulted in enhanced levels of spontaneous Gpa1 activation, due to destabilisation of GDP binding. Attenuating GTP hydrolysis was found to have both positive and negative effects upon Gpa1 signalling, leading to the hypothesis that each Gpa1 protein can only activate one effector protein per bound GTP molecule, before entering a GTP-bound inactive state. Subsequently, the acceleration of GTP hydrolysis by the regulator of G protein signalling (RGS) protein Rgs1, acts to enhance signalling at high levels of pheromone stimulation by freeing Gpa1 from this GTP-bound inactive state, to propagate further signalling reactions. High-affinity binding of the effector protein to GTP-bound Gpa1 may constituent the GTP-bound inactive state of Gpa1, necessitating the hydrolysis of GTP to dissociate the complex. Investigating the interactions of pheromone-signalling components revealed the importance of Ral2 in coordinating the activities of Gpa1 and Ras1 within the pheromone response pathway and suggested that Rgs1 may be the basis of a signalling complex at the plasma membrane. Additionally, the dominant negative activity of inactive Gpa1 mutants suggested the existence of a Gβγ-like protein within the pheromone-response pathway. The results presented in this thesis should allow further development of the Sz. pombe model system for investigating GPCR signalling reactions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:549501 |
| Date | January 2009 |
| Creators | Godfrey, Emma Louise |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/3150/ |
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