An Investigation of Mu-Opioid Receptor Interacting Proteins Within the Mu-Opioid Receptor Signaling Complex

Open Access
Author:
Justice-bitner, Stephanie Lynn
Graduate Program:
Genetics
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 15, 2012
Committee Members:
  • Robert G Levenson, Dissertation Advisor
  • Robert G Levenson, Committee Chair
  • Victor J Ruiz Velasco, Committee Chair
  • Laura Carrel, Committee Member
  • Vincent Chau, Committee Member
Keywords:
  • mu-opioid receptor
  • ubiquitin
  • G-protein
  • kappa-opioid receptor
  • delta-opioid receptor
  • yeast two-hybrid
  • SIAH1
  • SIAH2
Abstract:
Opioids are powerful analgesic drugs that lead to the development of tolerance, physical dependence, and addiction with prolonged use or abuse. Chronic exposure appears to cause alterations in the neuronal morphology of brain regions implicated in opioid dependence and addiction. Many of these changes reflect the inhibitory effects of these drugs 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 mu-opioid receptor (MOR) is the G-protein coupled receptor (GPCR) primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. However, the precise role of the MOR in the development of these neuronal alterations remains elusive. A growing body of evidence suggests that G-protein-coupled receptor (GPCR) signaling is modulated by proteins that bind GPCRs and form multiprotein signaling complexes. A number of proteins that interact with the MOR have recently been identified and have been shown to affect MOR biogenesis, trafficking, and signaling. Therefore, identifying and characterizing MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms of opioid addiction. Using a combination of conventional and modified membrane yeast two-hybrid (MYTH) screening methods, a cohort of novel MOR interacting proteins (MORIPs) was identified. The interaction between the MOR and a subset of MORIPs was validated in pull-down, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta (DOR) and kappa (KOR) opioid receptors. Finally, three of these proteins showed altered expression in the brains of morphine treated mice. The ubiquitin pathway has been shown to be altered in the addiction process, therefore two novel MORIPs, seven in absentia 1(SIAH1) and 2 (SIAH2), were of particular interest due to their known function as E3 ligases in the ubiquitin pathway. The effects of altering SIAH1 and/or SIAH2 protein expression on opioid receptor ubiquitination and protein levels was investigated using human embryonic kidney (HEK) stably transfected with MOR, delta opioid receptor (DOR), or kappa opioid receptor (KOR). These experiments suggest that SIAH2 is an E3 ligase for the MOR, while both SIAH1 and SIAH2 may conjugate ubiquitin to DOR and KOR. The ubiquitination by SIAH2 appears to signal for degradation of the MOR, whereas SIAH1 seems to act as a shuttle protein or chaperone assisting in degradation. In contrast, the ubiquitination of DOR and KOR may be a signal for degradation or trafficking of receptors. Additionally, opioid agonist treatment of neuroblastoma cells promoted the formation of MOR/SIAH1 complexes. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.