Acute ethanol exposure reduces serotonin receptor 1A internalization by increasing ubiquitination and degradation of β-arrestin2

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. ␤-Arrestin is a key player involved in the regulation of GPCR internalization and thus controls the magnitude and duration of GPCR signaling. Although ␤-arrestin levels are influenced by various drugs of abuse, the effect of alcohol exposure on ␤-arrestin expression and ␤-arrestin-mediated GPCR trafficking is poorly understood. Here, we found that acute ethanol exposure increases ␤-arrestin2 degradation via its increased ubiquitination in neuroblastoma-2a (N2A) cells and rat prefrontal cortex (PFC). ␤-Arrestin2 ubiquitination was likely mediated by the E3 ligase MDM2 homolog (MDM2), indicated by an increased coupling between ␤-arrestin2 and MDM2 in response to acute ethanol exposure in both N2A cells and rat PFC homogenates. Importantly, ethanol-induced ␤-arrestin2 reduction was reversed by siRNA-mediated MDM2 knockdown or proteasome inhibition in N2A cells, suggesting ␤-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 ␤-arrestin2 levels and delayed agonist-induced ␤-arrestin2 recruitment to the membrane. We conclude that ␤-arrestin2 dysregulation by ethanol impairs 5-HT1AR trafficking. Our findings reveal a critical molecular mechanism underlying ethanol-induced alterations in GPCR internalization and implicate ␤-arrestin as a potential player mediating behavioral responses to acute alcohol exposure. Acute alcohol exposure produces a wide range of behavioral and neurobiological effects, including feelings of euphoria, loss of coordination, and cognitive impairment, which are thought to be key predictors of vulnerability to alcohol abuse in humans (1-3). This notion is further supported by a recent longitudinal study showing that individuals who exhibit higher sensitivity to the rewarding effect and lower sensitivity to the sedative effect of an initial acute alcohol exposure were more likely to develop symptoms of alcohol use disorders and escalate their alcohol consumption over time (4). Many aspects of behavioral responses to alcohol are mediated by various G-proteincoupled receptors (GPCRs). 2 For example, ethanol-induced locomotor stimulation is mediated by serotonin 5-HT 1A receptors (5-HT 1A Rs) because treatment with 5-HT 1A R agonist (8-OH-DPAT) reduced locomotor activity in response to an acute ethanol exposure (2.5 g/kg, i.p.) (5). Moreover, the sedative effect of ethanol is regulated by metabotropic glutamate receptor 5 (mGluR5) as genetic deletion of mGluR5 or treatment with the mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine, increases the duration of loss of righting reflex in mice a...

Prolonged ethanol exposure modulates constitutive internalization and recycling of 5‐HT1A receptors

“…To maintain stable ethanol levels during the treatment, cell plates were placed within a closed polypropylene box alongside an open culture dish of a matching ethanol concentration. We previously reported a minimal loss of ethanol (3.13 ± 0.85%) over 18 h under this treatment condition (Luessen et al, 2019).…”

“…We previously reported a minimal loss of ethanol (3.13 ± 0.85%) over 18 h under this treatment condition (Luessen et al, 2019).…”

“…N2A cells stably expressing 5‐HT1ARs with an hemagglutinin (HA)‐tag at the N‐terminus (N2A‐5‐HT1ARs) were generated in our laboratory as described previously (Luessen et al, 2019). This cell line is not commonly misidentified by the International Cell Line Authentication Committee.…”

“…Previously, we reported that a single binge-like alcohol exposure to Sprague-Dawley rats (12 h ethanol vapor exposure without withdrawal, ~200 mg/dl blood concentration levels) produced some aspects of changes that were also observed in neuroblastoma 2a (N2A) cells treated with ethanol (15-75 mM) for 18 h (Luessen et al, 2019).…”

AMPK protects against alcohol-induced liver injury through UQCRC2 to up-regulate mitophagy

“…We observed that the mRNA of Uqcrc 2 was unchanged in ethanol-induced injury livers, while UQCRC2 protein levels decreased in injured livers. UQCRC2 can be degraded by ubiquitylation [ 35 , 36 ], and ethanol can cause a high ubiquitylation level of protein [ 37 ]. The qRT-PCR results showed that AMPK significantly enhanced the Uqcrc2 fold change numbers.…”