Deorphanization of biogenic amine-responsive G protein-coupled receptors in various model protozoa and platyhelminthes

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2012-01-01
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Agbedanu, Prince
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Steve A Carlson
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Biomedical Sciences
Abstract

This dissertation explores the relevance of G protein-coupled receptors in disease causing protozoans and free living flatworms. It explored the biological importance of a catecholamine responsive GPCR in Tetrahymena and the physiological importance of serotonergic receptors in the locomotory events of two species of flatworms; Girardia tigrina and Schmidtea mediterranea. During the preliminary studies, a protozoan GPCR was obtained by genome mining, cloned and heterologously expressed in a yeast expression system, subjected to agonist treatment, and hence, de-orphanized. The serotonin GPCRs in both Schmidtea mediterranea and Girardia tigrina were cloned into Gateway vector with which double stranded RNA production was induced in an RNAse III deficient bacterial strain for RNAi studies. We demonstrated the biological relevance of a novel Tetrahymena receptor (TetEPI-1) as related to bacterial engulfment. Further, in the presence of serotonin, TetEPI-1 was shown to have been stabilized in inactive state, giving the course to suspect serotonin as its potential inverse agonist or antagonist. TetEPI-1 is a potential therapeutic target for selective manipulation of related pathogenic protozoa, especially considering the inverse agonism is the paradigm for antihistamine drugs that stabilize H1 receptors in the absence of histamine during chronic allergic responses of atopy. By means of alternative loss-of-function technique, this dissertation also described the successful characterization of two novel serotonergic GPCR mediating locomotory events in two species of flatworms, Girardia tigrina (Dtig.ser-85) and Schmidtea mediterranea (smed-ser85, smed-ser39). Serotonin, by virtue of its dual effects, depending on its targets, demonstrated both cilio-excitatory and moderate cilio-inhibitory effect in these organisms. In the process of characterizing these receptors, this work established flatworm species differences in their response to serotonin. The suppression of these GPCRs demonstrated significant impairment in the motility of S. mediterranea while a decrease in cAMP also lessens the moderate inhibitory effects of serotonin on G. tigrina locomotion, a clear indication of the relevance of these receptors to these worms. These putative GPCRs compare with characterized 5HT4 receptors, hence, could be classified as planarian 5HT4 GPCRs, namely Dtig-ser85, Smed-ser85 and Smed-ser39. The homologs of these GPCRs will be pursued in the human parasite Schistosoma mansoni.

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Sun Jan 01 00:00:00 UTC 2012