Principal Investigator/Program Director: Evans, Christopher J., Ph.D., Component II Component II title: Opioid Receptor Signaling: selective mechanisms of regulation and receptor cross-talk ABSTRACT: Component II will study the mechanisms by which mu opioid agonists can trigger differential signaling and receptor trafficking. During the past funding period we have developed mouse dorsal root ganglia (DRG) primary cultures to study both native and virally expressed opioid receptors in wild-type and mutant mice (such as those generated in Component I and others available through the Mutant Mouse Core). The use of real time PCR, gene arrays, flow cytometry and confocal microscopy has enabled us to fully characterize the expression of regulated mRNAs as well as protein expression, distribution, and states of phosphorylation. Using these techniques in combination with electrophysiological recording, we have developed evidence for unique trafficking and signaling profiles for morphine and DAMGO with implication for a major regulatory Vole of arrestin isoforms. Based upon these preliminary data Component II will: 1) Characterize the differential mu-receptor-mediated signaling initially observed with DAMGO and morphine and subsequently define signaling profiles of other clinically important and endogenous agonists; 2) Analyze the contributions of receptor cross-talk via alpha 2a and delta opioid receptors on mu-receptor trafficking and signaling; 3) Test the hypothesis that beta-arrestins, and the cellular targeting of c-src are critical in determining both the opioid agonist signaling-profiles and trafficking of mu opioid receptors. Together these aims will provide a foundation for understanding agonist-directed signaling via mu opioid receptors with the goal of identifying agonist properties that may differentiate the clinically useful from the detrimental effects of opioid drugs.