Thesis (PhD)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Health and Human Performance. Title from resource description page. Includes bibliographical references.

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 109-144).

Thesis (M. Phil.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 89-106).

Thesis (M.Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 109-144) Also available in print.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 87-114).

Thesis (Ph. D.)--University of Hong Kong, 2006.

[Truncated abstract] G-protein coupled receptors (GPCRs) form one of the largest superfamilies of cell-surface receptors and respond to a vast range of stimuli including light, hormones and neurotransmitters. Although structurally similar, GPCRs are regulated by many diverse proteins, which allow the specific functions of each receptor to be carried out. This thesis focussed on two well-documented GPCRs, the thyrotropin releasing hormone receptor (TRHR) and gonadotrophin-releasing hormone receptor (GnRHR), which control the thyroid and reproductive endocrine pathways respectively. Although each of these anterior pituitary receptors is responsible for distinct physiological responses, both are integral to normal development and homeostasis. This thesis focused on three areas of GPCR regulation: ?-arrestin recruitment, transcription factor regulation and receptor up-regulation. The role of the cytoplasmic protein, ?-arrestin, has perhaps been previously underestimated in GPCR regulation, but it is now increasingly apparent that ?-arrestins not only inhibit further G-protein activation and assist in GPCR internalisation but also act as complex scaffolding platforms to mediate and amplify downstream signalling networks for hours after initial GPCR activation. It is therefore becoming increasingly important to be able to monitor such complexes in live cells over longer time-frames. ... Members of the E2F transcription family have been previously identified by this laboratory as potential GnRHR interacting proteins, via a yeast-2-hybrid screen and BRET. This thesis further investigated the role of E2F family members and demonstrates that a range of GPCRs are able to activate E2F transcriptional activity when stimulated by agonist. However, despite GnRHR displaying robust E2F transcriptional activation upon agonist stimulation, this did not result in any conclusive evidence for functional regulation, although it is possible E2F may modulate and assist in GnRHR trafficking. Furthermore it is apparent that E2F family members are highly redundant, as small effects in GnRHR binding and cell growth were only observed when protein levels of both E2F4 and E2F5 were altered. During the course of the investigation into the effect of E2F transcription on GPCR function, it was evident that long-term agonist stimulation of GnRHR had a profound effect on its expression. As this was explored further, it became clear that this agonist-induced up-regulation was both dose- and time-dependent. Furthermore, altering levels of intracellular calcium and receptor recycling/synthesis could modulate GnRHR up-regulation. In addition, an extremely sensitive CCD camera has been used for the first time to visualise the luciferase activity attributed to GnRHR up-regulation. Overall, this thesis demonstrates the complex nature of GPCR regulation. For the first time, long-term BRET analysis on ?-arrestin interactions with both classes of GPCRs has been examined in a variety of cellular formats. This has given valuable insights into the roles of phosphorylation and internalisation on ?-arrestin interaction. Additionally, this thesis has revealed that prolonged agonist exposure increases receptor expression levels, which has major implications for drug therapy regimes in the treatment of endocrine-related disorders and tumours.

Thesis (MSc (Biochemistry))--Stellenbosch University, 2008. / Gonadotropin-releasing hormone (GnRH) acting through the cognate GnRH receptor (GnRH-R) plays an important role in the regulation of mammalian reproductive function by regulating the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH). The sensitivity of pituitary gonadotropes to GnRH depends on the number of GnRH receptors present on the gonadotrope cell surface. GnRH-R is regulated at a transcriptional, post-transcriptional and post-translational level. Hormones such as GnRH and glucocorticoids (GCs) regulate GnRH-Rs in a time- and dose-dependent manner. Previous studies have shown that the GnRH-R promoter confers glucocorticoid-dependent activation via the activating protein 1 (AP-1) site in the nongonadotrope GGH3 cell line. The mechanism by which GCs regulate the GnRH-R promoter is not precisely known as the literature is contradictory. Therefore this study investigates the mechanism of transcriptional regulation of the mouse GnRH-R promoter in the mouse gonadotrope cell line LβT2, treated with the synthetic GC dexamethasone (dex). Assays used include promoter-reporter studies, Western blotting, endogenous mRNA expression studies, electrophoretic mobility shift assay (EMSA) as well as the in vivo chromatin immunoprecipitation (ChIP) assay. A transfected promoter-reporter plasmid containing 600 bp of the mouse GnRH-R promoter was used to investigate the effect of dex on transcriptional regulation. Previously it was determined in our laboratory that the GnRH-R promoter is activated via an AP-1 binding site in the LβT2 cell line, and is regulated in a time- and dose-dependent manner by dex. In the present study in the LβT2 cell line a small induction was indeed seen upon dex treatment. Cotransfecting a expression vector for rat GR succeeded in inducing a 2 fold positive dex response. Western blot analysis revealed that GR levels remain consistent even after 8 hours dex induction. The effect of dex on the endogenous GnRH-R gene was investigated by means of real-time RT-PCR. Dex did indeed upregulate the gene in a time-dependant manner. Maximal induction (7.4 fold) was obtained after at least 12 hours of dex treatment. Untreated LβT2 nuclear extracts were investigated using EMSA, for protein binding to the mouse GnRH-R promoter AP-1 binding site, and these proteins were identified as c- Fos and GR. This suggests that the GR interacts with the AP-1 transcription factor via a tethering mechanism to mediate the positive dex response. The results of an in vivo ChIP assay were consistent with this hypothesis, showing that the GR interacted with a genomic fragment containingthe AP-1 site, in response to dex. The transactivation of the GnRH-R promoter by means of the GR tethering to AP-1 has not been shown before in the LβT2 cell line.

Thesis (PhD (Biochemistry))--University of Stellenbosch, 2006. / Gonadotropin-releasing hormone (GnRH), acting via its cognate receptor (GnRHR) is the primary regulator of mammalian reproductive function. Pituitary sensitivity to GnRH can be directly correlated with GnRHR levels on the surface of the pituitary gonadotrope cells, which can be regulated at transcriptional, post-transcriptional and post-translational levels. This study investigated mechanisms of transcriptional regulation of mouse GnRHR expression in two mouse gonadotrope cell lines, αT3-1 and LβT2, using a combination of endogenous mRNA expression studies, promoter-reporter studies, a two-hybrid protein-protein interaction assay, Western blotting, and in vitro protein-DNA binding studies. In the first part of the study, the role of two GnRHR promoter nuclear receptor binding sites (NRSs) and their cognate transcription factors in basal and Protein Kinase A (PKA)-stimulated regulation of GnRHR promoter activity was investigated in αT3-1 cells. The distal NRS was found to be crucial for basal promoter activity in these cells. While the NRSs were not required for the PKA response in these cells, results indicate a modulatory role for the transcription factors Steroidogenic Factor-1 (SF-1) and Nur77 via these promoter elements. The second part of the study focused on elucidating the mechanism of homologous regulation of GnRHR transcription in LβT2 cells, with a view to defining the respective roles of PKA and Protein Kinase C (PKC) in the transcriptional response to GnRH. In addition, the respective roles of the NRSs, the cyclic AMP response element (CRE) and the Activator Protein-1 (AP-1) promoter cis elements, together with their cognate transcription factors, in basal and GnRH-stimulated GnRHR promoter activity, were investigated. Homologous upregulation of transcription of the endogenous gene was confirmed, and was quantified by means of real-time RTPCR. The GnRH response of the endogenous gene and of the transfected promoter-reporter construct required PKA and PKC activity, and the GnRH response of the promoter-reporter construct was found to be dependent on a functional AP-1 site. Furthermore, GnRH treatment resulted in increased binding of phosphorylated cAMP-response element binding protein (phospho-CREB) and decreased expression and binding of SF-1 to their cognate cis elements in vitro, and stimulated a direct interaction between SF-1 and CREB, suggesting that these events are also required for the full transcriptional response to GnRH. This study is the first providing detail regarding the mechanism of transcriptional regulation of GnRHR expression in LβT2 cells by GnRH. Based on results from this study, a model has been proposed which outlines for the first time the kinase pathways, the promoter cis elements and the cognate transcription factors involved in homologous regulation of GnRHR transcription in the LβT2 cell line. As certain aspects of this model have been confirmed for the endogenous GnRHR gene, the model is likely to be physiologically relevant, and provides new ideas and hypotheses to be tested in future studies.