A systems and molecular analysis of G protein-mediated signalling
Croft, Wayne D. (2012) A systems and molecular analysis of G protein-mediated signalling. PhD thesis, University of Warwick.
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The ability of cells to respond correctly to signals from their microenvironment is an
essential prerequisite of life. Many external signals are detected through G protein-coupled
receptor (GPCR) signalling pathways, which control all aspects of eukaryotic physiology.
Ligand-bound GPCRs initiate signalling by promoting exchange of GDP for GTP on the
Gα subunit of heterotrimeric G proteins, thereby facilitating activation of downstream
effectors. Signalling is terminated by the hydrolysis of GTP to GDP through intrinsic
GTPase activity of the Gα subunit, in a reaction catalysed by the regulator of G protein
signalling (RGS) proteins.
Due to the problem of complexity in higher eukaryotic GPCR signalling, the matingresponse
in Schizosaccharomyces pombe has been used to study GPCR signalling in isolation.
In vivo data from quantitative assays of reporter strains and live-cell
microscopy informs the development of an ordinary differential equation model of the signalling
pathway, first described by Smith et al., 2009.
The rate of nucleotide exchange on the Gα (Gpa1) is a key molecular mechanism controlling
duration and amplitude of signalling response. The in
uence of this is investigated
through characterisation of Gpa1 nucleotide exchange mutants and perturbation of reaction
rate parameters in the computational model. Further, this thesis also presents data
relating to the temporal and spatial regulation of Rgs1 (the sole RGS protein for Gpa1).
Using an inter-disciplinary approach, evidence is provided to suggest that an interaction
between Rgs1 and the C-terminal tail of the GPCR (Mam2) tethers Rgs1 to the plasma
membrane to facilitate its function.
Finally, quantification of signalling at the single cell level is described. Time-lapse livecell
uorescent reporter cells is optimised and single cell signalling response
quantified using image analysis software. Single cell quantification provides greater insight
into temporal dynamics, cell-to-cell variability, and highlights the existence of mechanisms
for cellular decision-making.
|Item Type:||Thesis or Dissertation (PhD)|
|Subjects:||Q Science > QP Physiology|
|Library of Congress Subject Headings (LCSH):||G proteins, Cellular signal transduction, Schizosaccharomyces pombe -- Physiology|
|Official Date:||May 2012|
|Institution:||University of Warwick|
|Theses Department:||Warwick Systems Biology Centre|
|Supervisor(s)/Advisor:||Ladds, Graham Robert ; Rand, David A.|
|Extent:||xviii, 283 leaves : ill., charts|
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