Human embryonic kidney (HEK) 293 cells, stably expressing the rat GnRH receptor were used to investigate the mechanism of ERK activation by GnRH. ERK activation was found to be dependent on cell adhesion to the extracellular matrix, and required an intact actin cytoskeleton. Through the use of specific pharmacological inhibitors and expression of dominant negative cDNA constructs, ERK activation was found to be mediated by the Rho family GTPase Rac1, and the non-receptor tyrosine kinases Src and focal adhesion kinase (FAK). FAK was found to function as a tyrosine phosphorylated scaffold upon which components of the ERK cascade assembled. Having established a role for Src in the activation of ERK, a proteomics study was undertaken to identify novel Src binding proteins that may be involved in the regulation of GnRH receptor signalling. Through a combination of immune-precipitation, two-dimensional gel electrophoresis, and matrix assisted laser desorption ionisation – time of flight mass spectrometry, Src was found to associate with the lipid kinase diacylglycerol kinase zeta (DGKζ). This interaction was found to be required for GnRH to stimulate DGKζ enzyme activity. By phosphorylating the second messenger molecule diacylglycerol to produce phosphatidic acid, DGKζ may play an important role in regulating GnRH receptor signalling. In this thesis, a potential mechanism of ERK activation is described for the GnRH receptor, with Src playing a key role in this pathway. In addition, Src was found to be involved in the activation of DGKζ, and is therefore implicated in the regulation of diacylglycerol signalling. This is the first report of an interaction between Src and DGKζ.
|Publisher||University of Edinburgh|
|Source Sets||Ethos UK|
|Type||Electronic Thesis or Dissertation|
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