• Energy Research

AbstractAlcohol exposure alters the signaling of the serotoninergic system, which is involved in alcohol consumption, reward, and dependence. In particular, dysregulation of serotonin receptor type 1A (5‐HT1AR) is associated with alcohol intake and withdrawal‐induced anxiety‐like behavior in rodents. However, how ethanol regulates 5‐HT1AR activity and cell surface availability remains elusive. Using neuroblastoma 2a cells stably expressing human 5‐HT1ARs tagged with hemagglutinin at the N‐terminus, we found that prolonged ethanol exposure (18 h) reduced the basal surface levels of 5‐HT1ARs in a concentration‐dependent manner. This reduction is attributed to both enhanced receptor internalization and attenuated receptor recycling. Moreover, constitutive 5‐HT1AR internalization in ethanol naïve cells was blocked by concanavalin A (ConA) but not nystatin, suggesting clathrin‐dependent 5‐HT1AR internalization. In contrast, constitutive 5‐HT1AR internalization in ethanol‐treated cells was blocked by nystatin but not by ConA, indicating that constitutive 5‐HT1AR internalization switched from a clathrin‐ to a caveolin‐dependent pathway. Dynasore, an inhibitor of dynamin, blocked 5‐HT1AR internalization in both vehicle‐ and ethanol‐treated cells. Furthermore, ethanol exposure enhanced the activity of dynamin I via dephosphorylation and reduced myosin Va levels, which may contribute to increased internalization and reduced recycling of 5‐HT1ARs, respectively. Our findings suggest that prolonged ethanol exposure not only alters the endocytic trafficking of 5‐HT1ARs but also the mechanism by which constitutive 5‐HT1AR internalization occurs. image

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.