Protein-protein interactions in GnRH receptor signalling

Abstract

Many signal transduction pathways in eukaryotic cells are controlled by the reversible assembly of proteins into signalling complexes that integrate and transmit signals from cell surface receptors to the cytoplasm and nucleus. Protein phosphorylation on serine, threonine and tyrosine residues plays a key role in these events by creating binding sites for modular protein-interaction domains to allow proteins to associate with one another, and regulating the activity of many enzymes and transcription factors.The extracellular signal regulated kinase (ERK) cascade is one of the most intensively studied signalling pathways in mammalian cells. ERK 1 and 2 have been shown to regulate gene expression programmes in response to extracellular signals, including gonadotrophin releasing hormone (GnRH), which binds to a G-protein coupled receptor (GPCR). However, many questions remain to be answered regarding the proximal events that lead to the activation of the ERK cascade by GnRH.A HEK 293 cell line, stably expressing the rat GnRH receptor, was 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 by expression of dominant negative cDNA constructs, ERK activation was found to be mediated by the Rho family GTPase Racl, and the non-receptor tyrosine kinases focal adhesion kinase (FAK) and Src. FAK was found to function as a tyrosine phosphorylated scaffold upon which key 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, twodimensional gel electrophoresis and matrix assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry, Src was found to associate with the lipid kinase diacylglycerol kinase ζ, (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.