Acute Ethanol Exposure Enhances Ubiquitination and Degradation of β‐arrestin

Abstract

IntroductionEthanol exposure elicits robust changes in the trafficking and function of many neurotransmitter G protein‐coupled receptors (GPCRs). However, the molecular mechanisms remain elusive. Using serotonin 5‐HT1A receptors (5‐HT1ARs) as a model system this study was to examine the effect and mechanism of acute ethanol exposure on arrestin‐mediated 5‐HT1AR internalization. The second goal was to investigate the molecular mechanism underlying ethanol‐induced alterations in 5‐HT1AR trafficking by determining the endocytic recycling and degradation pathways of arrestin.MethodsIn the present study, we used prefrontal cortex (PFC) tissue from Sprague Dawley rats and a neuroblastoma 2a cell line stably expressing 5‐HT1ARs (N2A‐5HT1AR). Rats were exposed to ethanol vapor for 12 hours then sacrificed immediately and prefrontal cortex tissue was dissected. Cells were cultured in growth medium containing ethanol (15–75 mM) for 18 hours prior to experiments. Western blot experiments were carried out to measure β‐arrestin protein level. Immunoprecipitation was performed to evaluate agonist‐induced coupling between β‐arrestin, ubiquitin, and Mdm2 (an E3 ligase). Immunocytochemistry and confocal microscopy were utilized to examine agonist (8‐OH‐DPAT) stimulated 5‐HT1AR internalization and the endocytic fate of β‐arrestin in the control and ethanol‐treated cells. The Mdm2 siRNA or scrambled siRNA were transiently transfected into N2A‐5‐HT1AR cells to examine the knockdown effect on the arrestin expression and receptor internalization.ResultsWe found that 12‐hour ethanol vapor exposure significantly reduced β‐arrestin protein expression in the PFC of rats when compared to air‐exposed control animals. The reduced β‐arrestin level parallels enhanced β‐arrestin ubiquitination. We further studied the molecular mechanisms underlying ethanol‐induced changes in β‐arrestin using N2A‐5HT1AR cells. We found that acute ethanol treatment: a) causes a dose‐dependent reduction in β‐arrestin levels in parallel to our observation in ethanol‐treated rats; b) delays and reduces the recruitment of β‐arrestin to the membrane by the 5‐HT1AR agonist; c) attenuates agonist‐induced 5‐HT1AR internalization; and d) enhances ubiquitination of β‐arrestin via Mdm2, an E3 ligase. Importantly, Mdm2 siRNA knockdown reverses ethanol‐induced β‐arrestin degradation and attenuation of agonist‐mediated 5‐HT1AR internalization.The present study provides first time evidence that acute ethanol exposure abolished 5‐HT1AR internalization through enhanced Mdm2‐dependent β‐arrestin ubiquitination and proteasome‐dependent degradation. These findings provide insight into the molecular mechanisms underlying alcohol‐associated alterations in GPCR function and highlight β‐arrestin as a potential target for pharmacological intervention of alcohol abuse.Support or Funding InformationThis work is supported by NIH NIAAA training grant T32 AA007565, DA042862 and a pilot grant from Center for Molecular Communication and Signaling at Wake Forest University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.