This proposal seeks support to study kappa opioid receptors at the molecular, cellular, anatomical and pharmacological level. These receptors are part of the complex system of endogenous opioids which modulates numerous functions including pain regulation, and drug abuse. The kappa receptors are highly unusual since they mediate pain relief without producing drug dependence. Their activation is responsible for a vast array of effects, ranging from basic physiological functions (such as water balance), to more complex brain functions such as changes in affect or perception. While the existence of these receptors has been shown on the basis of pharmacological data, their molecular structure had not been elucidated. We have recently cloned a member of this kappa family from a rat brain library, based on homology to the newly cloned delta opioid receptor. We have shown that this clone has a seven transmembrane structure typical of the G-protein coupled receptors, and that it binds with high affinity a number of classical kappa ligands and to the products of the prodynorphin precursor. Based on its profile, we have classified it as kappa 1 and shown that its activation by agonists leads to a decrease in forskolin-stimulated cyclic AMP levels. We have demonstrated that this clone has a pattern of tissue expression in the brain characteristic of the kappa 1 site. We have also obtained two related clones from guinea pig, which we tentatively classify as kappa. The proposed project is aimed at completing the task of cloning and characterizing members of the kappa receptor family. Kappa receptor multiplicity is well established, although the exact number of sites is not clear, and the differences across species are very striking. Consequently, a major aim of this proposal is to determine the extent of kappa receptor heterogeneity both within and across species. A related purpose is to fully characterize these receptors in terms of their pharmacological profile, their interactions with endogenous ligands, and their coupling to various signal transduction pathways. Of particular interest is the structure-function determinants of these receptors, both in terms of binding selectivity and coupling mechanisms. This will be studied with molecular techniques used to generate specific mutants and chimeras. The tissue-specific expression of the cloned kappa receptors will be examined in detail, with particular attention to the interface between these receptors and the endogenous ligands. Finally, the last aim is focused on the modulation of these receptors by long-term exposure to opiate agonists and antagonists, be they kappa or mu ligands. These studies should improve our knowledge of these unique receptors and shed light on a number of key mechanisms in which they are involved including pain control, reward and aversiveness, and drug abuse.
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