There is burgeoning evidence that at least some of the pharmacological effects of opioids are mediated via opioid receptors that are organized as dimers or oligomers. Moreover, it is now established that in some cases different types opioid receptors can associate with one another to form heterodimers. The possibility that heterodimers may modulate signal transduction pathways that are not identical to those mediated by monomeric or homodimeric opioid receptors is an intriguing possibility that may have a bearing on tolerance and physical dependence. For these reasons, the broad, long-term objective of this project is to investigate the role of opioid receptor dimerization through a multidisciplinary, coordinated approach. In project 1, pharmacologic tools will be designed and synthesized to identify the presence of mu-delta opioid receptor dimers. The design approach involves the simultaneous occupation of neighboring mu and delta recognition sites in a heterodimer by a single bivalent ligand that contains mu agonist and delta antagonist pharmacophores. Optimization of binding and function will be accomplished by varying the length of the spacer that links the pharmacophores. In project 2, the interaction between mu and delta opioid receptors will be investigated in cultured cells. The activities of the mu receptors will be examined at different levels of expressed delta receptors. The ability of mu-selective opioid agonists to inhibit the production of intracellular CAMP or stimulation of the ERK1/2 activities will be determined when the expressed delta opioid receptors are being activated or inactivated. The ability of mu opioid agonists to elicit cellular adaptive responses such as desensitization or receptor internalization during mu opioid receptor activation or inactivation will be examined. Project 3 involves the molecular simulation of different dimer structures in a variety of dimer interfaces. Each will be built and evaluated according to theoretical scoring functions taken from protein homology and long time-scale molecular dynamics calculations. Bivalent ligands that have been found experimentally to bridge the opioid recognition sites in opioid receptor dimers will be employed as """"""""molecular rulers"""""""" to provide information on the distance between binding sites. Chronic testing of the target compounds will be carried out in mice to determine if there are in vivo correlates with the in vitro data obtained in projects 1 and 2.
