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Serotonin receptors and G-protein coupled receptor interacting proteins: potential drug targets in clinical medicine

thesis
posted on 06.02.2017 by Chinkwo, Kenneth Anye
The experimental approaches from this study have been used to characterize and investigate the function, expression and distribution of the 5-HT4 and 5-HT7 receptor splice variants and G-protein coupled receptor interacting proteins. The results in chapter two provide knowledge of the functional response of the 5-HT4d and 5-HT4g receptor splice variants to different 5-HT receptor agonists. It was found that the ranking order of potency for the various agonists differs. Tegaserod and Y-36912 were reasonably potent similar to 5-HT, MeOT and prucalopride at the (d) and the (g) splice variants. No significant differences regarding response were observed between the 5-HT4d and 5-HT4g receptor splice variants. Indeed it has been suggested previously that, the function of serotonin receptors is enhanced by downstream G-protein coupled receptor interacting proteins (GIPs). This prompted the study of 5-HT4 receptor and GIPs distribution in the guinea pig intestine. In chapter three reverse transcriptase–polymerase chain reaction (RT-PCR) assays and western blot analysis were used to detect the presence of 5-HT4, 5-HT7 receptors and GIPs in different regions of the guinea pig intestine. There is significant evidence to suggest that the guinea pig can be used as a suitable model for biological assays and that a high density of serotonin receptors can be found in the gastrointestinal tract of all mammals. Moreover, functional gastrointestinal disorders such as irritable bowel syndrome (IBS), has been associated with 5-HT receptor function, but there is limited knowledge of GIPs association with IBS. This prompted the introduction of GIPs in chapter three and chapter four. Results showed that 5-HT4 and 5-HT7 receptors and G-protein coupled receptor kinases (GRKs) which are GRK2, GRK3, GRK5, GRK6 and PDZ domain interacting proteins which are Veli-1, Veli-2 and Veli-3 were expressed in the guinea pig intestine. Veli-1 and Veli-2 were expressed in the jejunum, ileum, proximal colon and distal colon of the guinea pig. Veli-3 was not seen in any of the regions studied. The 5-HT4 receptor and the interacting GIPs appeared to co-exist in the same tissue which has been shown in this study for the first time. In chapter four, RT-PCR was used to identify expression patterns of 5-HT4 and 5-HT7 receptor splice variants and GIPs in the human colon. The 5-HT4 and 5-HT7 receptor splice variants, GRK2, GRK3, GRK5 and GRK6 as well as Veli-1, Veli-2 are present in the different regions studied. However, some of the genes are less frequently detected in some of the areas examined. The 5-HT4d and 5-HT4g receptor splice variants were not detected in the ileum and transverse colon respectively. Veli-1 was not detected in the transverse colon while GRK6 was not detected in the ileum, transverse and sigmoid colon. This study suggests that splice variant-specific modulation of 5-HT4 receptor function might be achieved with compounds that affect the interaction of a 5-HT4 receptor with either Velis or GRKs. However, targeting a combination of specific 5-HT4 receptor splice variant and interacting Veli-1, 2 and 3 or a specific 5-HT4 receptor splice variant and interacting GRK2, 3, 5, 6 requires understanding of interactions in the same naturally occurring cell. In chapter five an attempt was made to determine if any of the 5-HT4 receptor splice variants interact with Velis in COS-7 cell lines. Using gateway cloning techniques 5-HT4a, b, d, g and Veli 1, 2 and 3 were expressed and visualized in COS-7 cells. The 5-HT4a receptor splice variant appeared to co-immunoprecipitated with Veli-3 confirming earlier reports that these two proteins are binding partners. It is possible to speculate that, increasing the expression of Veli-3 could potentially up regulate 5-HT4a receptor function towards future therapeutic gains

History

Campus location

Australia

Principal supervisor

Helen R. Irving

Additional supervisor 1

Ian Coupar

Year of Award

2012

Department, School or Centre

Medicinal Chemistry and Drug Action

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Pharmacy and Pharmaceutical Sciences

Exports