Understanding important opioid-mediated effects such as analgesia and complex behavioral effects like tolerance and dependence has been greatly facilitated by molecular cloning of opioid receptors and ligands. Evidence suggest that multiple opioid receptor forms (besides the four cloned mammalian receptors) and their endogenous ligands are involved in opioid-mediated effects. We have initiated the closing of these new opioid prototypic (polypeptide precursors) molecules and their potential receptors, initially in amphibians to take advantage of the relative high levels of expression that cacilitates closing strategies and provides cDNAs and sequence information that will facilitate their characterization in mammals. We have isolated two novel amphibian opioid-like pre-pro- hormones that are distinct from the enkephalin and prodynorphin precursors and a new opioid receptor, that may act as a cognate receptor for these new ligands. The new Xenopus receptor isolated binds to Ze3n-dorphins and to know opioid ligands. Using the amphibian receptor sequence, we have isolated the mammalian hormolog from mouse brain that belongs to this new receptor subfamily, and that is distinct from the four known mammalian receptors. The new opioid-like peptides (Xen- dorphins) bind to known opioid receptors Qld. can produce potenent opioid medicated analgesia in mice. It is our hypothesis that the mammalian homologs of these novel opioid-like prohormones and their cognate receptors play an important role in mediating opioid effects such as analgesia and tolerance. To address our hypothesis we propose the following specific aims: 1) To define the ligand specificity of the amphibian Zen-dorphin receptor; this information will facilitate our mammalian receptor characterization of the new subfamily. 2) To clone the full length form of the mammalian receptor from mouse brain using the partial receptor fragment and to define its ligand specificity and distribution 3) To further characterize the opioid mediated analgesia induced by Xen-dorphins in mice. Specifically, we will examine if the novel opioid-like peptides produce tolerance or modulate tolerance produced by morphine. characterization of this novel opioid ligand-receptor family (Xen-dorphins) will further our understanding of the opioid mediated analygesia and tolernace and in turn provide better tools for pain and drug abuse management.
