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Investigating the signalling characteristics of Gα subunits in the fission yeast Schizosaccharomyces pombe
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Godfrey, Emma L. (2009) Investigating the signalling characteristics of Gα subunits in the fission yeast Schizosaccharomyces pombe. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b2334402~S15
Abstract
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.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Schizosaccharomyces pombe, G proteins -- Research, Eukaryotic cells -- Research | ||||
Official Date: | August 2009 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Warwick Medical School | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Ladds, Graham | ||||
Sponsors: | Warwick Medical School (WMS) | ||||
Extent: | 430 leaves : ill., charts | ||||
Language: | eng |
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