The putative homodimerization or the heterodimerization of the opioid receptors have been suggested with the co-immunoprecipitation, fluorescence energy transfer (FRET), and bioluminescence energy transfer (BRET) experiments. The possibility that opioid receptor could heterodimerize provides a mechanistic explanation for the observed delta-opioid receptor modulation of mu-opioid receptor tolerance in the presence of delta-opioid antagonists and in the delta-opioid receptor null mice. Hence, we hypothesize that the oligomerization of the mu and delta-opioid receptor results in the modulation of the mu-opioid receptor chronic responses, i.e., the reported delta-opioid antagonist inhibition of morphine tolerance is not a result of neural circuitry, but rather is a result of receptor-receptor interaction. In order to demonstrate this hypothesis, we will control the delta-opioid receptor expression using the ecdysone-inducible mammalian expression system in EcR293 cells stably expressing the mu-opioid receptor. The activities of the mu-opioid receptors in these cells will be examined at different levels of the delta-opioid receptor being induced. The ability of mu-opioid agonists to regulate the adenylyl cyclase or the ERK1/2 activities will be determined when the induced delta-opioid receptors are being activated or inactivated. The ability of mu-opioid agonists to elicit cellular adpatational responses such as desensitization, or receptor internalization at different levels of induced delta-opioid receptor will be examined. In addition to the conventional mu- and delta-opioid receptor selective ligands, the bivalent ligands developed in Component #1 of this program project grant will be used also. The bridging of two receptor binding sites within the oligomers, by the bivalent ligands with pharmacophores selective of respective receptors will allow us to examine the role of the receptor oligomers on these cellular processes. The effects of the bivalent ligands on the mu-opioid receptor activities, acute and chronic, will be examined in different levels of delta-opioid receptor being induced. These effects will be compared to those obtained with the monovalent ligands having the identical spacers. Our hypothesis will predict the existence of a bivalent ligand having mu-opioid agonist and delta-opioid antagonist pharmacophores that will not induce chronic responses in the mu-opioid receptor activation in the presence of induced delta-opioid receptor. The use of these bivalent ligands and the inducible receptor system will demonstrate further any pharmacological significance of probable opioid receptor oligomerization.
