Project Summary/Abstract The goal of this study is to determine the role of Gαz signaling in modulating opioid pharmacology. To produce a wide variety of cellular responses, all G protein coupled receptors (GPCRs) couple to heterotrimeric G proteins to serve as the intermediaries between the receptor and downstream effectors. There are 16 different Gα subunits, five Gβ, and 12 Gγ subunits. Because of the number of G protein subunits, there are numerous possible trimer combinations. Gα proteins control signal duration by binding to either GDP or GTP. Opioid receptors (ORs) predominately couple to the Gαi/o class, comprised of Gαi1, Gαi2, Gαi3, GαoA, GαoB, and Gαz. Previous studies showed that mice lacking Gαz had an accelerated rate of morphine antinociceptive tolerance development. Knockout of the regulator of G protein signaling (RGS), RGSz1, increased Gαz signaling, and increased the analgesic efficacy of MOR agonists in mice and delayed the development of morphine tolerance. Collectively, enhancing signaling through Gαz increases morphine analgesic efficacy and reduces the development of morphine tolerance. Our preliminary published studies, using bioluminescence resonance energy transfer (BRET) and HEK 293T cells transfected with the κ opioid receptor (KOR), showed differential potency and efficacy of κ opioid agonists when the KOR signaled through different Gα subunits. When the KOR signaled through Gαz, opioid agonists, particularly partial agonists, were more potent and sometimes more efficacious than when the KOR signaled through other Gα subunits. Our hypothesis is that µ opioid agonists signaling through the MOR coupled to Gαz will be more potent and sometimes more efficacious than when the MOR signals through other Gαi/o subunits. By understanding how opioid pharmacology is affected by the MOR signaling through Gαz in comparison to other Gα subunits from the Gαi/o family, it may be possible to design therapeutics to increase or decrease signaling of the MOR through Gαz, thereby increasing or decreasing the potency and efficacy of µ opioid agonists. The following Specific Aims will be investigated using a BRET assay to measure receptor activation and steady-state conditions. 1) Mu opioid agonists will be tested for efficacy and potency with the MOR signaling through the Gαz subunit and results will be compared with signaling through other Gα subunits. When the MOR is signaling through Gαz, the simple classification of opioids as full or partial agonists or antagonists may be changed. 2) Studies will determine the effect of RGSZ, which is RGS20, on activation and deactivation kinetics and to determine if RGSZ affects opioid efficacy and potency when the MOR is signaling through Gαz. Collectively, these experiments will determine if MOR signaling through Gαz changes MOR pharmacology, and if increasing or decreasing Gαz activity is a potential therapeutic target.