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

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