The purpose of the current proposal is to clone a non-opioid binding protein. The endogenous opioid peptide dynorphinA-(1-17) and its analog dynorphinA-(2-17) have high affinity for kappa opioid receptors but considerable evidence indicates that they also bind to a non-opioid protein. Identification of this protein has been hindered by several features of dynorphin, including its high non-specific binding to biological tissue, its sensitivity to proteolytic degradation and a lack of knowledge of its primary structure. Two complementary approaches are proposed here to circumvent these problems, both being applied to dynorphinA-(2-17), which does not bind to opioid receptors and would be a more selective ligand for the putative non-opioid protein. First, the yeast two-hybrid system will be used to identify any molecules interacting with DynA-(2-17). Second, expression cloning and sib selection will be used to identify clones that bind DynA-(2-17). DynA-(1-17) and its analog dynA- (2-17) are not analgesic in the brain but can enhance morphine antinociception and suppress withdrawal symptoms in morphine tolerant/dependent animals. Thus, it has great clinical promise in improving the efficacy of morphine in patients who require the drug chronically, as well as a treatment for opioid addiction. Identification of the binding protein mediating these effects is critical to these advances.
