Characterization of the Endogenous Opioid and its Receptor in Tetrahymena: A Model System for the Evolution of ChemicalSignaling

点击翻译按钮获取中文摘要

The long term goal of this project is to elucidate the nature of the signal transduction mechanisms in the protozoan ciliate Tetrahymena. This is an important question from the point of view of evolution because it seems likely that the mechanisms of signal transduction in metazoa originated from autocrine and paracrine mechanisms in unicellular eukaryotes. There is evidence that opioid mechanisms may play an important role in the modulation of phagocytosis in the protozoan ciliate Tetrahymena. In particular, a beta-endorphin-like peptide has been reported in this organism, and mammalian beta-endorphin inhibits phagocytosis in these cells with a high potency and efficacy. The proposed research will characterize two important elements of the opioid mechanism in this organism, the endogenous opioid and the receptor. This laboratory has confirmed by means of a radioimmunoassay the presence of a beta-endorphin-like peptide in Tetrahymena. The next goals are to establish the optimal conditions for the maximal expression of the peptide by studying how different factors, both environmental and biochemical, modulate the level of the peptide in the cell and in the culture medium, and then to purify the peptide from cell extracts. The effect of the purified peptide on phagocytosis in Tetrahymena and in metazoan models will be compared to that of mammalian beta-endorphin. The primary sequence of the purified peptide will be studied to determine its degree of homology with mammalian beta-endorphin or with other peptides that have been reported to have opioid-like activities. Studies on the putative beta-endorphin gene in tetrahymena will also be initiated, using standard recombinant DNA techniques. This will be done by probing a cDNA library with either a pro-opiomelanocortin (POMC) probe or an oligonucleotide probe based on the amino acid sequence. This will determine whether the putative beta-endorphin gene in Tetrahymena is coded independently or is part of a POMC gene, as in vertebrates. Studies of the opioid receptor will also be initiated to establish whether there is a correlation between the potency of opioid agonists and antagonists on phagocytosis, and their potency in radioligand displacement assays in membrane fractions. This work will also give information about the degree of opioid receptor microheterogeneity in Tetrahymena. In addition, studies will be done to determine how the properties of the receptor, including desensitization, are affected by changes in the ionic environment and by G-proteins. This will be done to compare the characteristics of the opioid receptor in Tetrahymena with other described subtypes. The results of this research will contribute to our understanding of the origins and mechanisms of chemical signaling. In particular, it will provide new information on the phylogenetic origin of beta-endorphin-like peptides and opioid receptors. These studies of the mechanisms by which such peptide mediators affect a ciliated protozoan may also shed new light on their mechanisms of action on the cells of higher animals.