Ethanol Dose‐Dependently Changes 5HT 1 A Receptor Trafficking in Neuroblastoma 2A Cells

Ethanol 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‐HT 1A receptors (5‐HT 1A Rs) as a model system, the goal of this study was to examine the effect and mechanism of acute ethanol exposure on constitutive 5‐HT 1A R internalization and recycling. The Rab family of proteins has been implicated as critical modulators of endosomal trafficking involved in receptor internalization and recycling. More specifically, Rab5 is implicated in GPCR internalization and commonly used as a protein marker for internalization into the early endosomes whereas Rab11 is associated with GPCR recycling. Thus, the second goal was to investigate the molecular mechanism underlying ethanol‐induced alterations in constitutive 5‐HT 1A R internalization and recycling by determining the endocytic pathway of 5‐HT 1A R. Methods In the present study, we used a neuroblastoma 2a cell line stably expressing 5‐HT 1A Rs (N2A‐5HT 1A Rs). Cells were cultured in growth medium containing ethanol (15–75 mM) for 18 hrs prior to experiments. Immunocytochemistry and confocal microscopy were utilized to examine 5‐HT 1A R surface expression, constitutive 5‐HT 1A R internalization and recycling, and co‐localization of 5‐HT 1A R with Rab proteins. Western blotting was carried out to measure HA‐tagged 5‐HT 1A R, Rab5 and Rab11 protein levels in ethanol‐ and vehicle‐treated cells. Results We found that N2A‐5HT 1A R cells treated with 30 or 75 mM of ethanol for 18 hrs significantly reduced 5‐HT 1A R surface expression compared to vehicle treatment without altering 5‐HT 1A R total protein levels in N2A‐5HT 1A R cells. To determine the mechanism underlying reduced surface 5‐HT 1A R expression, the effect of acute ethanol exposure on constitutive 5‐HT 1A R internalization and recycling was determined. Additionally, the endocytic fate of 5‐HT 1A R in Rab5 or Rab11‐positive endosomes were assessed to understand the molecular mechanisms governing ethanol‐induced alteration in trafficking of 5‐HT 1A R. This study demonstrates for the first time that acute ethanol exposure alters the constitutive trafficking of 5‐HT 1A Rs which may be mediated by Rab proteins. Alcohol‐associated alterations in GPCR trafficking may contribute to alcohol tolerance and dependence. Support or Funding Information This work is supported by NIH R01DA042862, P50DA006634, F31AA025532 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .