Investigating the role of the Mam2 C-terminal tail in the pheromone-signalling pathway of Schizosaccharomyces pombe
McCann, Eilish Clare (2010) Investigating the role of the Mam2 C-terminal tail in the pheromone-signalling pathway of Schizosaccharomyces pombe. PhD thesis, University of Warwick.Full text not available from this repository.
Official URL: http://webcat.warwick.ac.uk/record=b2339747~S15
G protein-coupled receptors (GPCRs) allow cells to respond to extracellular stimuli
and are involved in virtually all major physiological processes in eukaryotes.
Consequently, aberrant GPCR signalling can lead to disease, making them attractive
candidates for research and the development of pharmacological interventions.
However, GPCR signalling responses in higher eukaryotes are complex, with the
presence of multiple signalling pathways hindering the isolation and study of specific
signalling components. The model organism, Schizosaccharomyces pombe, provides
a simplified system with which to investigate GPCR signalling in the pheromoneresponse
Pheromone stimulation in Sz. pombe causes the production of proteins necessary for
the mating process, including the carboxypeptidase Sxa2. This study utilises Sz.
pombe reporter strains in which sxa2 has been replaced with the bacterial reporter
lacZ, thus enabling quantification of the level of signalling through the pheromoneresponse
pathway. Using this system, the pheromone-responsive Mam2 receptor was
investigated, with specific focus on the large intracellular C-terminal tail.
Truncating Mam2 to remove the C-terminal tail altered signalling by increasing the
level of ligand-independent signalling and decreasing the level of maximal
signalling. Similar effects were observed when a regulator of G protein signalling,
Rgs1, was removed from strains containing the full-length Mam2 receptor. This
suggested a relationship between these two signalling components, which was
confirmed through yeast 2-hybrid analysis. Using this approach, an 8-residue section
of the Mam2 C-terminal tail was found to be necessary for a direct interaction with
Rgs1. Further characterisation of the Mam2 tail revealed an additional role for this
region in receptor sensitivity to pheromone stimulation. Mutational analysis
implicated three serine residues in receptor sensitivity, suggesting that the C-terminal
tail of Mam2 may contribute to receptor internalisation from the plasma membrane
therefore enabling desensitisation to prolonged pheromone stimulation.
Similar techniques applied to the study of Rgs1 revealed that two conserved DEP
domains in the N-terminus of Rgs1 are important for the interaction with the Mam2
C-terminal tail. Fluorescent tagging of Rgs1 and subsequent microscopic analysis
indicated that Rgs1 localises to endomembranous structures surrounding the nucleus,
contrary to predictions made by mathematical models developed by this group,
which suggest that Rgs1 requires plasma membrane-localisation in order to function.
A better understanding of the action of Mam2 and Rgs1 in the Sz. pombe pheromone
response can inform mathematical models and future studies involving the more
complex mammalian signalling cascades.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QP Physiology|
|Library of Congress Subject Headings (LCSH):||Schizosaccharomyces pombe, Pheromones, Cellular signal transduction|
|Official Date:||April 2010|
|Institution:||University of Warwick|
|Theses Department:||Department of Biological Sciences|
|Extent:||xviii, 262 leaves : ill., charts|
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