The long term objective of this project is to determine how activation of mu and delta-opioid receptors leads to distinct intracellular signals. Although mu- and delta-opioid receptors may relay intracellular messages in a similar fashion, a clear distinction in the abuse and dependence producing potential of opioids suggests significant differences are yet to be discovered. For this project, a cellular model has been developed in which cloned mu and delta-opioid receptors in stably transfected GH3 cells interact differently with two intracellular effectors, adenylyl cyclase and Ca++ channels. This unique and important model will be used to identify fundamental differences between mu- and delta-opioid signal transduction cascades. This will be accomplished by the systematic analysis of the interactions between opioid receptors, G proteins and effectors in these unique clones. First, the effect of receptor density on mu- and delta-coupling to effectors will be determined. Second, signal transduction in clones expressing only mu-, only delta, or both mu and delta-opioid receptors will be examined. Third, the association of opioid receptors with G proteins will be determined by purification of agonist-stimulated receptor-G protein complexes. Fourth, the activation of G proteins by opioid receptors will be studied using agonist-induced incorporation of [32P]azidoanilido-GTP into G-alpha subunits. Fifth, the composition of the heterotrimeric G proteins (G-alpha, G-beta- and G-gamma subunits) responsible for coupling mu and delta- receptors to effectors will be confirmed by the use of antisense oligonucleotides targeting specific G protein subunits. Finally, the association of Ca2+ channels with G alpha and/or G-beta,gamma subunits will be assessed after the immunoprecipitation of G protein/Ca2+ channel complexes. Understanding basic differences in the way mu and delta-opioid receptors relay information intracellularly could lead to the development of new methods for the treatment of opioid abuse and pain management.
