AN ANALYSIS OF THE MU OPIOID RECEPTOR AND GPR177 INTERACTION: IMPLICATIONS FOR OPIOID ADDICTION
Open Access
- Author:
- Jin, Jay
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 07, 2010
- Committee Members:
- Robert G Levenson, Dissertation Advisor/Co-Advisor
Robert G Levenson, Committee Chair/Co-Chair
Victor Alan Canfield, Committee Member
Patricia Grigson, Committee Member
Victor J Ruiz Velasco, Committee Member - Keywords:
- Wnt secretion
Wntless
GPR177
mu-opioid receptor
protein-protein interaction - Abstract:
- Opioid drugs are powerful analgesic agents that lead to the development of tolerance, physical dependence and addiction with prolonged use or abuse. Chronic exposure also appears to cause alterations in the neuronal morphology of key brain regions implicated in the development of opioid dependence and addiction. Many of these changes reflect inhibitory effects of morphine and other opioid receptor agonists on neuron size, neurite outgrowth, dendritic arborization, and neurogenesis that occur in brain regions important for reward processing, learning, and memory. The analgesic and addictive properties of opioid drugs are mediated primarily via the µ-opioid receptor (MOR). However, the precise role of MORs in the development of these neuronal alterations remains elusive. A growing body of evidence indicates that G-protein-coupled receptor (GPCR) signaling is modulated by proteins that bind to the GPCR and form multiprotein signaling complexes or signalplexes. A number of proteins that interact directly with the MOR have recently been identified and shown to affect MOR biogenesis, trafficking, and signaling. We hypothesize that identifying and characterizing novel MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms involved in the development of opioid addiction. Using a modified split-ubiquitin yeast two-hybrid screening method, we identified GPR177 as a candidate MORIP. Several recent studies have shown that GPR177 is the mammalian ortholog of Drosophila Wntless/Evenness Interrupted/Sprinter, an evolutionarily conserved protein that plays an important role in the secretion of Wnt proteins from Wnt-producing cells. Wnt signaling mechanisms are of particular interest due to their effects on neuronal development, which include promoting neuron growth, increasing dendritic arborization and regulating neurogenesis. The interaction between the MOR and GPR177 was validated using GST-pulldown and co-immunoprecipitation experiments. These two proteins were also found to co-localize within a subset of striatal neurons. Western blot analysis of various rodent brain regions and organ tissues showed that GPR177 is ubiquitously expressed throughout the body. In developing zebrafish, morpholino-induced knockdown of GPR177 expression led to disorganization of brain tissue, loss of semicircular canal formation in the ear, and abnormal curvature of the tail. In MOR-expressing Human Embryonic Kidney 293 cells, morphine treatment promoted formation of MOR/GPR177 complexes, and resulted in the inhibition of Wnt protein secretion in a cell-based assay. This effect on Wnt secretion was blocked by pre-treatment with MOR antagonists, which suggests that the inhibition of Wnt protein secretion is mediated via the MOR and its interaction with GPR177. We thus propose a novel mechanism whereby the inhibition of Wnt secretion and signaling mediated by the MOR/GPR177 interaction may be involved in the development of opioid addiction. Our studies suggest that the MOR/GPR177 interaction could serve as a target for a new therapeutic agent aimed at counteracting the addictive properties of opioid analgesics.