Pseudoexons are intronic sequences that meet the bioinformatic criteria that define true exons. They outnumber their true counterparts several fold and the reasons for their lack of recognition by the splicing machinery remain unclear. A naturally occurring mutation that activates one such pseudoexon in the growth hormone receptor (GHR) gene has been described. This thesis utilises the mutation as a model of pseudoexon suppression and activation. The aims of the work were to: 1. Establish an in-vitro model of GHR pseudoexon splicing. 2. Identify sequence elements important to the suppression of the pseudoexon. 3. Identify nuclear factors important to the suppression of the pseudoexon. 4. Establish a cell culture system to parallel the in-vitro system. The results show successful establishment of an in vitro and in vivo model of GHR pseudoexon splicing. Deletional experiments using the in vitro splicing system identified silencing elements within the pseudoexon sequence itself. Immunoprecipitation experiments in vitro and RNA interference experiments in vivo implicate heterogenous nuclear ribonucleoprotein El (hnRNP El) as a potential trans-acting silencing factor. Further immunoprecipitation experiments indicate that the spliceosomal factor, Ul small nuclear ribonucleoprotein (Ul snRNP), may also have a role in pseudoexon suppression. Ul snRNP was found to have similar interactions with a panel of other pseudoexons and rarely used alternative exons. We propose a model in which Ul snRNP interacts with silencing factors to block pseudoexon 5' splice-site usage. We suggest that this interaction may occur temporally earlier than true splice site interactions and in a different conformation. Such a temporal and conformationally different interaction would prevent the important Ul snRNA/5' splice-site interaction occurring.

In endocrinology dynamic testing forms an essential part of investigation. To assess the central capacity of the GH-lGF-1 axis there is extensive experience in paediatric and adult populations of numerous physiological and pharmacological tests. However tests to assess the peripheral capacity of the GH-IGF-1 axis beyond a simple baseline lGF-1 level are limited to the IGF-1 generation test (IGFGT).

The ghrelin receptor (GRLN-R) was cloned in 1996 after the discovery that a series of synthetic growth hormone-releasing compounds (the growth hormone secretagogues) acted through a receptor distinct from the growth hormone-releasing hormone receptor. In 1999 the endogenous ligand of the receptor, ghrelin, was discovered. As well as stimulating growth hormone release, ghrelin has been shown to be involved in many other processes such as appetite stimulation and the regulation of energy homeostasis, making the ghrelin/GRLN-R system an attractive pharmaceutical target for the treatment of disorders such as growth hormone deficiency, cachexia and obesity. The GRLN-R displays a high level of ligand-independent (constitutive) activity and has been suggested to couple to Gaq/11, Gai/o, Gas and Ga12/13 G protein pathways, although little is known about the signalling of ghrelin and the growth hormone secretagogues in all but the Gaq/11 pathway. Two of the growth hormone secretagogues, GHRP-6 and L-692,429, have been described as ‘ago-allosteric modulators’ of the GRLN-R as, when co-administered with ghrelin, GHRP-6 and L-692,429 were reported to act both as co-agonists (increasing the efficacy of the ghrelin response) and as negative or positive (respectively) regulators of the potency of ghrelin. This study sought to investigate the pharmacology of the GRLN-R through the Gai/o pathway. [35S]GTPyS binding assays were used to measure activation of the Gai/o pathway, demonstrating that GHRP-6, L-692,585 (a commercially available analogue of L-692,429) and a third growth hormone secretagogues, MK-677, acted with higher efficacy than ghrelin. At least in the system tested, upon co-administration with ghrelin each of the growth hormone secretagogues acted in a simple competitive fashion with ghrelin. Radioligand binding experiments showed that the dissociation kinetics of [His[125I]]- ghrelin from the GRLN-R were not altered by co-administration of the growth hormone secretagogues. Fitting data to a modified operational model of allosterism demonstrated that GHRP-6, L-692,585 and MK-677 were not ago-allosteric modulators of the GRLN-R but simple orthosteric agonists. In order to further examine the receptor-specific effects of ghrelin and the growth hormone secretagogues, a Flp-In™ T-REx™ HEK293 cell line expressing the GRLN-R was constructed. [35S]GTPyS binding assays confirmed that the GRLN-R was constitutively active through both the Gaq/11 and Gai/o pathways, demonstrated by an increase in [35S]GTPyS loading upon receptor expression which could be reduced by the administration of the GRLN-R inverse agonist SPA. Upon expression of the GRLN-R a considerable level of cell detachment was observed with the remaining cells appearing rounded compared to parental HEK293 cells, an affect that appeared to be mediated by the constitutive activity of the receptor. In contrast to the [35S]GTPyS assays used to measure activation of Gai/o, [35S]GTPyS assays with a Gaq-immunoprecipitation step demonstrated that the growth hormone secretagogues acted with equal efficacies to that of ghrelin, demonstrating functional selectivity at the GRLN-R. Intact cell assays were also used to measure Gaq/11 and Gai/o responses, however, a Gai/o-mediated response could not be measured in cAMP accumulation assays, suggesting that the GRLN-R could signal via activation of Gao but not Gai1-3. Although the activation of the Gas pathway by the GRLN-R remains controversial, in this study ghrelin and the growth secretagogues could evoke a Gas-mediated cAMP response (although L-692,585 acted with a lower efficacy than ghrelin). Finally, two naturally occurring missense mutations of the GRLN-R (A204E in the second extracellular loop and I134T in the third transmembrane helix) were analysed to investigate whether these mutations led to retention of the receptor within the endoplasmic reticulum and to investigate whether the mutations affected the ability of the GRLN-R to signal to the growth hormone secretagogues. The A204E mutation caused partial retention of the GRLN-R within the endoplasmic reticulum, whilst receptor that was transported to the plasma membrane did not display any measurable constitutive activity. In contrast, the I134T mutation did not alter receptor localisation, nor did it have any effect on the constitutive or ligand-induced activation of the GRLN-R, however, it appeared to lower the efficacy of the inverse agonist SPA.