Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of
Deborah J Luessen1, Haiguo Sun1, Molly M McGinnis1
1Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston Salem, North Carolina 27157.
Abstract:
Acute alcohol exposure alters the trafficking and function of many G-protein-coupled receptors (GPCRs) that are associated with aberrant behavioral responses to alcohol. However, the molecular mechanisms underlying alcohol-induced changes in GPCR function remain unclear. β-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although β-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on β-arrestin expression and β-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases β-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). β-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between β-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced β-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting β-arrestin2 degradation is mediated by MDM2 through the proteasomal pathway. Using serotonin 5-HT1A receptors (5-HT1ARs) as a model receptor system, we found that ethanol dose-dependently inhibits 5-HT1AR internalization and that MDM2 knockdown reverses this effect. Moreover, ethanol both reduced β-arrestin2 levels and delayed agonist-induced β-arrestin2 recruitment to the membrane. We conclude that β-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate β-arrestin as a potential player mediating behavioral responses to acute alcohol exposure.
Insights
Acute alcohol exposure causes the degradation of beta-arrestin2, a key protein in G-protein-coupled receptor (GPCR) regulation. This impacts GPCR trafficking and may explain alcohol-related behavioral changes.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Acute alcohol exposure disrupts G-protein-coupled receptor (GPCR) trafficking and function, contributing to behavioral changes, but the underlying molecular mechanisms are unclear.
- Beta-arrestin (β-arrestin) is crucial for regulating GPCR internalization and signaling duration, and its expression is affected by drugs of abuse.
- The specific impact of alcohol on β-arrestin expression and its role in GPCR trafficking remains poorly understood.
Purpose of the Study:
- To investigate the effect of acute ethanol exposure on β-arrestin expression and β-arrestin-mediated GPCR trafficking.
- To elucidate the molecular mechanisms by which ethanol alters β-arrestin levels and function.
- To examine the role of the E3 ligase MDM2 homolog (MDM2) in ethanol-induced β-arrestin2 regulation.
Main Methods:
- Acute ethanol exposure was administered to neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC) tissue.
- β-arrestin2 ubiquitination, degradation, and interaction with MDM2 were assessed.
- Serotonin 5-HT1A receptors (5-HT1ARs) were used as a model to study ethanol's effects on GPCR internalization and β-arrestin recruitment, employing siRNA-mediated MDM2 knockdown and proteasome inhibition.
Main Results:
- Acute ethanol exposure increased β-arrestin2 degradation via enhanced ubiquitination in N2A cells and rat PFC.
- Ethanol exposure increased the coupling between β-arrestin2 and MDM2, suggesting MDM2 mediates ubiquitination.
- Ethanol-induced β-arrestin2 reduction was prevented by MDM2 knockdown or proteasome inhibition, confirming MDM2-proteasomal degradation.
- Ethanol inhibited 5-HT1AR internalization dose-dependently, an effect reversed by MDM2 knockdown.
- Ethanol reduced β-arrestin2 levels and delayed its membrane recruitment.
Conclusions:
- Acute ethanol exposure leads to β-arrestin2 degradation mediated by MDM2 through the proteasomal pathway.
- Ethanol-induced dysregulation of β-arrestin2 impairs 5-HT1A receptor trafficking.
- These findings reveal a key molecular mechanism for alcohol-induced GPCR alterations and highlight β-arrestin's role in alcohol's behavioral effects.
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