Acute ethanol exposure reduces the total protein level of β-arrestin2 in N2A-5HT_1AR cells and rat PFC

We examined the effect of acute ethanol exposure on β-arrestin1/2 expression in both N2A cells and rat PFC. In N2A cells, β-arrestin1/2 levels were determined following the treatment with ethanol (15–75 mm) or vehicle (media) for 18 h without withdrawal (Fig. 1A). Data were normalized to the protein level of β-actin, which was not altered by ethanol treatment. A one-way ANOVA revealed a significant dose effect of ethanol on β-arrestin2 protein level, F(3,20) = 5.796, p < 0.01. Bonferroni post hoc analysis showed that 30 and 75 mm ethanol treatment significantly reduced β-arrestin2 levels by 44.8 ± 2.0 and 60.3 ± 1.7%, respectively, when compared with the vehicle treatment (Fig. 1A, p < 0.01). The β-arrestin1 protein levels were not detectable in N2A cells with or without ethanol treatment. The reduced β-arrestin2 level was not due to toxicity induced by ethanol because the selected ethanol doses did not alter cell viability and membrane permeability measured by trypan blue assay (Fig. S2A) and immunocytochemistry, respectively (Fig. S2, B and C).

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Figure 1.

Acute ethanol treatment reduces the total level of β-arrestin2 protein in N2A-5HT1AR cells and rat PFC. A, representative Western blots and quantification of β-arrestin2 (β-arr2) protein levels in N2A-5HT1AR cells. Cells were acutely treated with ethanol (15–75 mm) or vehicle (Veh; media) for 18 h. The 30 and 75 mm ethanol treatment significantly reduced β-arrestin2 protein levels in N2A-5HT1AR cells (n = 6, **, p < 0.01 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test). B, representative Western blots and quantification of β-arrestin 1/2 protein levels in rat PFC. Rats were acutely exposed to ethanol vapor (EtOH) or control (Air) for 12 h followed by immediate sacrifice. Ethanol exposure significantly reduced β-arrestin2 protein level in rat PFC (n = 6, *, p < 0.05 versus air, unpaired Student's t test).

A similar effect was observed in the PFC of rats acutely exposed to ethanol vapor for 12 h without withdrawal (Fig. 1B). Ethanol exposure significantly reduced β-arrestin2 protein levels in the PFC by 32.4 ± 2.0% compared with the air exposure (Fig. 1B, unpaired Student's t test, p < 0.05). However, β-arrestin1 protein levels did not significantly differ between ethanol- and air-treated groups.

Acute ethanol exposure increases ubiquitination of β-arrestin2 in N2A-5HT_1AR cells and rat PFC

We further examined whether ethanol-induced reduction in β-arrestin2 levels was due to an increase in ubiquitination and subsequent degradation. First, immunocytochemistry was performed to measure the co-localization between fluorescently labeled β-arrestin2 (green) and ubiquitin (red) in N2A cells following 18 h of ethanol (15–75 mm) or vehicle (media) treatment (Fig. 2A). Co-localization was calculated as the number of spatially overlapped pixels (Fig. 2A, yellow) between β-arrestin2 (green) and ubiquitin (red) followed by conversion into a co-localization coefficient using FIJI software (National Institutes of Health). A one-way ANOVA indicated a significant effect of ethanol treatment on the co-localization between β-arrestin2 and ubiquitin, F(3,116) = 14.87, p < 0.01. Bonferroni post hoc analysis revealed that 30 and 75 mm ethanol treatment significantly increased this co-localization by 87.6 ± 2.1% (30 mm) and 201.9 ± 7.5% (75 mm) when compared with the vehicle treatment (Fig. 2B, p < 0.01).

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Figure 2.

Acute ethanol exposure increases ubiquitination of β-arrestin2 in N2A-5HT1AR cells and rat PFC. A, representative raw and binary fluorescent images of β-arrestin2 (green) and ubiquitin (red) and co-localization (Co-loc) in N2A-5HT1AR cells treated with ethanol (15–75 mm) or vehicle (Veh) (media) for 18 h. B, ethanol exposure dose-dependently increased β-arrestin2 co-localization with ubiquitin (n = 29–30 cells/group, six replicates, **, p < 0.01 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test). C, validation of the rabbit anti-β-arrestin2 antibody (LSBio; LS-B15546) was performed using a custom-made blocking peptide (GenScript; sequence DDIVFEDFARLRLK). Immunoprecipitation (IP) was performed using lysates from drug-naïve N2A-5HT1AR cells and rat PFC tissue in the presence and absence of the corresponding β-arrestin2 (β-arr)-blocking peptide (10 μg). Immunoprecipitates and N2A cell lysates were immunoblotted (IB) for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008). β-Arrestin2–blocking peptide abolished immunoreactive bands detected in samples without the presence of this blocking peptide. D, representative blots for immunoprecipitated β-arrestin2, ubiquitin, and MDM2 from N2A-5HT1AR cell lysates. Cells were treated with ethanol (30 mm, 18 h) or vehicle (Veh) (media), and cell lysates were immunoprecipitated overnight with rabbit anti-β-arrestin2 antibody. Immunoprecipitates and cell lysates were immunoblotted with mouse anti-ubiquitin, mouse anti-MDM2, rabbit anti-NEDD4, and mouse anti-β-arrestin2 antibodies. E–H, quantification of β-arrestin2 mono- and poly-ubiquitination, MDM2, and NEDD4 (n = 5, **, p < 0.01 versus vehicle, unpaired Student's t test). I, representative blots for immunoprecipitated β-arrestin2, ubiquitin, and MDM2 in rat PFC lysates. Samples from ethanol (EtOH) and control (Air) exposed rats were immunoprecipitated with rabbit anti-β-arrestin2 overnight. Immunoprecipitates and tissue lysates were immunoblotted with the antibodies described above. J–M, quantification of immunoprecipitated β-arrestin2, ubiquitin, MDM2, and NEDD4 (n = 5, *, p < 0.05 versus vehicle, unpaired Student's t test).

We also performed immunoprecipitation assays as a complementary approach to immunocytochemistry to examine whether there was an increased coupling between ubiquitin and β-arrestin2 following ethanol exposure (30 mm; 18 h). First, to verify the specificity of the β-arrestin2 antibody used for immunoprecipitation, we immunoprecipitated β-arrestin2 in the presence or absence of a custom-made β-arrestin2–blocking peptide in drug-naïve N2A cells and rat PFC tissue. The blocking peptide completely eliminated signals produced by antibodies used to immunoblot for ubiquitin and β-arrestin2 from immunoprecipitated β-arrestin2 samples (Fig. 2C), suggesting the specificity of this β-arrestin2 antibody for immunoprecipitation. Then, we used this antibody to immunoprecipitate β-arrestin2 followed by immunoblotting for ubiquitin, which resulted in distinct mono-ubiquitination and poly-ubiquitination signals (Fig. 2D). We selected the ~60-kDa band as mono-ubiquitinated β-arrestin2 based on the presumed ~8-kDa upward shift in the molecular mass of β-arrestin2 caused by the addition of one ubiquitin chain. The poly-ubiquitination signal for β-arrestin2 present at a molecular mass of >80 kDa was consistent with reports from the literature (22, 29, 30). In N2A cells, we found that ethanol treatment increased both mono-ubiquitination and poly-ubiquitination of β-arrestin2 by 83.2 ± 1.7 and 83.6 ± 2.7%, respectively, compared with the vehicle treatment (Fig. 2, E and F, unpaired Student's t test, p < 0.01).

Similar to our observation in N2A cells, immunoprecipitation of β-arrestin2 in rat PFC homogenates showed that ethanol exposure significantly increased mono-ubiquitination by 46.8 ± 1.5% and poly-ubiquitination of β-arrestin2 by 50.9 ± 2.2% compared with the air treatment (Fig. 2, I–K), unpaired Student's t test, p < 0.05. Because the interaction between the protein substrate and E3 ligases facilitates protein ubiquitination, we further assessed the strength of β-arrestin2 coupling to MDM2 in response to ethanol exposure. Ethanol exposure increased the association of MDM2 with immunoprecipitated β-arrestin2 by 91.1 ± 2.2 and 32.6 ± 1.3% in N2A cells and rat PFC, respectively, when compared with their corresponding control treatments (Fig. 2, G and L, unpaired Student's t test, p < 0.05). In contrast, ethanol exposure had no effect on the association of immunoprecipitated β-arrestin2 with another E3 ligase, NEDD4, in either model system (Fig. 2, H and M). It is also worth noting that the relative intensities of bands between mono- and poly-ubiquitinated β-arrestin2 in N2A cells were notably different from those in rat PFC tissues. This is likely attributed to differential expressions of various E2 and E3 ligases involved in ubiquitination of β-arrestin2 in each model. Additionally, bands located between mono- (~60 kDa) and poly-ubiquitinated β-arrestin2 (>80 kDa) likely reflect a spectrum of different ubiquitination states (bi-, tri-, tetra-ubiquitination, etc.) on individual lysine residues, as described previously (31). Because the role of these intermediate ubiquitination states is currently unclear, this study focused on mono- and poly-ubiquitination of β-arrestin2.

Acute ethanol exposure reduces β-arrestin2 protein expression via MDM2-mediated proteasomal degradation

Because there was an increased association of β-arrestin2 with MDM2 following ethanol exposure, we further determined whether MDM2 was involved in ethanol-induced degradation of β-arrestin2. To this end, MDM2 was knocked down via siRNA in N2A-5HT_1AR cells, and β-arrestin2 protein levels were measured after ethanol treatment (30–75 mm, 18 h) (Fig. 3B). MDM2 siRNA treatment reduced the MDM2 protein level by 57.8 ± 1.4% (Fig. 3A, unpaired Student's t test, p < 0.05) when compared with scrambled siRNA treatment. Furthermore, a two-way ANOVA revealed significant main effects of knockdown, F(1,24) = 18.24, p < 0.01; ethanol exposure, F(2,24) = 7.70, p < 0.01; and interaction, F(2,24) = 4.67, p < 0.05. Bonferroni post hoc analysis showed that MDM2 knockdown completely blocked ethanol-induced β-arrestin2 degradation when compared with scrambled siRNA treatment (Fig. 3, B and C, p < 0.05).

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Figure 3.

Acute ethanol exposure reduces β-arrestin2 protein expression in an MDM2- and proteasome-dependent manner in N2A-5HT1AR cells. A, verification of MDM2 protein knockdown via siRNA. N2A-5HT1AR cells were transfected with MDM2 siRNA or a corresponding scramble siRNA. MDM2 protein levels were measured 48 h after transfection by Western blotting. MDM2 siRNA treatment significantly reduced the endogenous MDM2 protein level when compared with scrambled siRNA-treated control cells (n = 5, **, p < 0.01 versus scramble, unpaired Student's t test). B, representative Western blotting of β-arrestin2 (β-arr2) protein level in scramble and MDM2 siRNA-treated cells. N2A-5HT1AR cells were transfected with MDM2 siRNA or a corresponding scramble siRNA and then treated with ethanol (30–75 mm) or vehicle (Veh) (media) for 18 h. Cell lysates were immunoblotted with rabbit anti-β-arrestin2 and goat anti-β-actin antibodies. C, quantification of β-arrestin2 levels in scramble and MDM2 siRNA-treated cells. MDM2 knockdown prevented ethanol-induced degradation of β-arrestin2 (n = 6, **, p < 0.01 versus vehicle, a two-way ANOVA followed by Bonferroni post hoc test). D, schematic illustration of spatial overlap analysis for quantitation of ubiquitin, β-arrestin2, and P19S co-localization. Co-localization between β-arrestin2 (green) and ubiquitin (red) was first determined as yellow overlapped pixels (left, middle). These co-localized pixels (yellow; left, bottom) were then isolated, and triple co-localization with P19S (blue; right, top) was determined as white overlapped pixels (right, bottom). Data were reported as a percentage of co-localization by overlapping pixel counts, y/x + y + z (ubiquitin/β-arrestin2 co-localization with respect to P19S). E, representative confocal images of ubiquitin (red), β-arrestin2 (green), and P19S (blue) in N2A-5HT1AR cells after acute treatment with ethanol (15–75 mm,18 h) or vehicle (media). Scale bars, 10 μm. F, quantification of ubiquitin, β-arrestin2, and P19S co-localization. The co-localization of the ubiquitin, β-arrestin2, and P19S significantly increased after ethanol treatment (30–75 mm) (n = 25–28 cells/group, five replicates, **, p < 0.01 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test). G, representative Western blotting of β-arrestin2 levels in N2A-5HT1AR incubated with or without MG132 (5 μm) during treatment with ethanol (30–75 mm, 18 h) or vehicle (media). H, quantification of β-arrestin2 protein levels. Treatment with MG132 prevented acute ethanol-induced (30 and 75 mm) reduction in β-arrestin2 protein levels in N2A-5HT1AR cells (n = 5, **, p < 0.01 versus vehicle, a two-way ANOVA followed by Bonferroni post hoc test). I, representative confocal images of LAMP1 (red) and β-arrestin2 (green) in N2A-5HT1AR cells after acute treatment with ethanol (30–75 mm, 18 h) or vehicle (media). Scale bars, 10 μm. J, quantification of β-arrestin2 and LAMP1 co-localization. β-Arrestin2 co-localization with LAMP1 significantly increased after a high-dose ethanol treatment (75 mm) compared with vehicle treatment (n = 35 cells/group, five replicates, *, p < 0.05 versus vehicle, a one-way ANOVA followed by Bonferroni post hoc test).

Previous studies showed that ubiquitination primarily directs β-arrestin2 for proteasome-dependent degradation in C6 glioma and PC12 cells (21, 22). Therefore, we performed immunocytochemistry to examine the co-localization between fluorescently labeled β-arrestin2, ubiquitin, and proteasome marker P19S in N2A-5HT_1AR cells (Fig. 3, D–F) following treatment with ethanol (30–75 mm, 18 h) or vehicle (media). Briefly, co-localization between β-arrestin2 (Fig. 3, D–F, green) and ubiquitin (red) was first determined as yellow overlapped pixels. Then the co-localization of β-arrestin2, ubiquitin, and P19S was quantified as the spatial overlap between co-localized pixels (yellow) and P19S (blue), which was represented by white overlapped pixels (Fig. 3D). A one-way ANOVA indicated a significant effect of ethanol exposure on ubiquitin, β-arrestin2, and P19S co-localization, F(2,12) = 54.48, p < 0.01. Bonferroni post hoc analysis showed that 30 and 75 mm ethanol treatment significantly increased the co-localization of β-arrestin2, ubiquitin, and P19S by 44.5 ± 2.8% (30 mm) and 58.3 ± 2.5% (75 mm), respectively, when compared with the vehicle treatment (Fig. 3F, p < 0.01). To test whether the proteasome-dependent degradation pathway is the primary mechanism for ethanol-induced β-arrestin2 reduction, N2A-5HT_1AR cells were treated with the proteasome inhibitor MG132 (5 μm) or vehicle (media) in the presence or absence of ethanol (30–75 mm; 18 h) followed by measurement of β-arrestin2 levels (Fig. 3G). A two-way ANOVA revealed significant main effects of ethanol exposure, F(2,24) = 19.64, p < 0.01; proteasome inhibition, F(1,24) = 19.19, p < 0.01; and interaction, F(2,24) = 9.70, p < 0.01. Bonferroni post hoc analysis showed that MG132 treatment blocked β-arrestin2 degradation induced by ethanol exposure (30–75 mm) when compared with the vehicle treatment (Fig. 3H, p < 0.01).

To assess whether acute ethanol exposure also directed β-arrestin2 into lysosomes for degradation, we measured the co-localization between fluorescently labeled β-arrestin2 and a lysosome marker, LAMP1, following treatment with ethanol (30–75 mm, 18 h) or vehicle (media) (Fig. 3I). A one-way ANOVA showed a significant effect of ethanol exposure on the co-localization between β-arrestin2 and LAMP1, F(2,102) = 4.87, p < 0.01. Bonferroni post hoc analysis showed that only the high-dose ethanol treatment (75 mm) produced a small but significant increase in the co-localization of β-arrestin2 with LAMP1 by 14.0 ± 2.6% (Fig. 3J, p < 0.05). Collectively, these data suggest that acute ethanol exposure primarily directs β-arrestin2 to undergo the proteasome-dependent degradation; however, a small pool of β-arrestin2 can also be routed to lysosomes under specific conditions such as high doses of ethanol exposure.

Acute ethanol exposure inhibits agonist-induced 5-HT_1AR internalization

Because β-arrestin regulates internalization of many GPCRs, including 5-HT_1AR (32, 33), the functional consequence of ethanol-induced β-arrestin2 reduction on GPCR internalization was examined using 5-HT_1AR as a receptor model system. The internalization of 5-HT_1AR was measured by immunocytochemistry using an antibody-feeding protocol described previously (34). We first assessed the kinetics of 8-OH-DPAT–stimulated 5-HT_1AR internalization. It has been shown previously that 5-HT_1AR internalization occurs in a clathrin- and β-arrestin-dependent manner in LLC-PK1 cells (35). In N2A-5HT_1AR cells, pretreatment with dynasore or concanavalin A, inhibitors of dynamin- and clathrin-mediated internalization, respectively, completely inhibited internalization of the receptor (Fig. S3), indicating that 5-HT_1AR internalization is clathrin- and dynamin-dependent. Next, the effect of ethanol treatment (15–75 mm; 18 h) on 8-OH-DPAT–stimulated 5-HT_1AR internalization was determined (Fig. 4A). A two-way ANOVA revealed significant main effects of ethanol treatment, F(3,577) = 79.36, p < 0.01; time, F(4,577) = 311.7, p < 0.01; and interaction, F(12,577) = 14.61, p < 0.01. Although 8-OH-DPAT treatment (1 μm) caused a time-dependent 5-HT_1AR internalization in both vehicle- and ethanol-treated cells, Bonferroni post hoc analysis showed that treatment with 30 or 75 mm ethanol significantly inhibited 8-OH-DPAT–stimulated 5-HT_1AR internalization at the indicated time points when compared with the vehicle treatment (Fig. 4B, p < 0.01). Because there was an increase in MDM2 association with β-arrestin2 in response to acute ethanol exposure (Fig. 2), we examined whether dysregulated 5-HT_1AR internalization was mediated by increased β-arrestin2 ubiquitination and degradation by knocking down MDM2. A two-way ANOVA revealed significant main effects of knockdown, F(1,214) = 11.62, p < 0.01; ethanol treatment, F(2,214) = 18.66, p < 0.01; and interaction, F(2,214) = 7.97, p < 0.01. Bonferroni post hoc analysis showed that MDM2 siRNA knockdown abolished ethanol-induced attenuation of 5-HT_1AR internalization (Fig. 4C, p < 0.01), suggesting that MDM2-mediated degradation of β-arrestin2 directly impacts 5-HT_1AR internalization.

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Figure 4.

Acute ethanol exposure inhibits agonist-induced 5-HT1AR internalization. N2A-5HT1AR cells were acutely treated with ethanol (15–75 mm, 18 h) or vehicle (Veh) (media) followed by labeling the surface 5-HT1ARs with mouse anti-HA antibody. Then cells were warmed to 37 °C and treated with 8-OH-DPAT (1 μm) for the indicated time points to induce internalization. At the end of each time point, the remaining primary antibody-bound surface 5-HT1ARs that did not internalize were labeled with goat anti-mouse Alexa 647 (red). Internalized intracellular 5-HT1ARs were identified by labeling with goat anti-mouse Alexa 405 (blue, pseudo-colored to green). A, representative confocal images of surface and internalized 5-HT1ARs from vehicle- and ethanol-treated cells (15–75 mm). Scale bar, 10 μm. B, quantification of 8-OH-DPAT–stimulated 5-HT1AR internalization. Internalized 5-HT1ARs were calculated as percent of their own total surface 5-HT1AR intensity. Receptor internalization occurred in a time-dependent manner in both vehicle- and ethanol-treated cells (n = 47–50 cells/group, five replicates, **, p < 0.01 versus vehicle, a two-way ANOVA followed by Bonferroni post hoc test). Ethanol treatment (30–75 mm) significantly inhibited 8-OH-DPAT–stimulated 5-HT1AR internalization compared with the vehicle treatment. Data are normalized and expressed relative to vehicle. C, MDM2 siRNA knockdown blocked the inhibition of 5-HT1AR internalization after ethanol (30–75 mm, 18 h) or vehicle (media) treatment (n = 35–38 cells/group, six replicates, **, p < 0.01 versus scramble, a two-way ANOVA followed by Bonferroni post hoc test).

Generation of N2A-5-HT_1AR stable cell line

Parental N2A cells (ATCC) were grown in Opti-MEM media supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. To generate a cell line stably expressing HA-5HT_1AR, N2A cells were transfected with human 5-HT_1AR cDNA with an HA-tag at the N terminus (HA-5-HT_1AR; Missouri S&T cDNA Resource Center) using Lipofectamine 2000 (Invitrogen) followed by an antibiotic selection with geneticin G418 (400 μg/ml). The expression of HA-5-HT_1ARs was confirmed by the mRNA level and the protein presence using quantitative PCR and immunocytochemistry, respectively.

N2A-5HT_1AR cells were subject to fluorescence-activated cell sorting (FACS) to isolate cells with a high expression of HA-tagged 5HT_1AR. Briefly, cells suspended in ice-cold PBS containing 3% BSA, at ~5 × 10⁶ viable cells/ml, were incubated with HA-tag DyLight488-conjugated antibody (Columbia Biosciences; D8–1830) on ice for 1 h. Then cells were washed three times by centrifugation at 400 × g for 5 min. Cells were resuspended in ice-cold PBS, 10% fetal bovine serum and sorted using a FACS Aria cell sorter (BD Biosciences). Live cells were gated based on forward/side scatter, and events in the top 3–4% of 488 nm fluorescence were selected, cultured, and expanded in medium containing geneticin (400 μg/ml).

Acute ethanol treatment to N2A-5-HT_1AR cells

N2A-5HT_1AR cells were treated with ethanol (15, 30, or 75 mm) or vehicle (media) for 18 h. Ethanol-treated cell culture plates were placed in a closed but not sealed polystyrene box with an open dish containing the corresponding concentration of ethanol. As shown previously, this procedure results in stable ethanol levels for several days (55, 56). The concentration of ethanol in the culture medium was determined using a colorimetric ethanol assay (Bioassay Systems; ECET100), which was performed at the end of the 18-h treatment period. Under these growing conditions, the average reduction in ethanol concentration after 18 h for each dose of ethanol was 3.13 ± 0.85% compared with initial ethanol concentrations at the time of treatment (not significant, p = 0.24 versus initial treatment, paired Student's t test, n = 6).

Validation of a β-arrestin2 antibody for Western blotting and immunocytochemistry

We verified the specificity of a commercially available β-arrestin2 antibody (LSBio; LS-B15546) in cells treated with β-arrestin2 siRNA. N2A-5HT_1AR cells were transiently transfected with 5 nm β-arrestin2 siRNA (Santa Cruz Biotechnology; sc-29743) or scrambled siRNA (Santa Cruz Biotechnology; sc-37007). Cells were lysed 48 h after transfection, and the protein concentrations were determined using BCA protein assay (Thermo Fisher Scientific). Protein (20 μg) was loaded on 10% Tris-glycine gels for SDS-PAGE and then was transferred to nitrocellulose membranes. β-Arrestin2 proteins were probed with β-arrestin2 antibody (LSBio; LS-B15546) followed by goat anti-rabbit IgG HRP (Santa Cruz Biotechnology; sc-2005). β-Actin (Santa Cruz Biotechnology; sc-1616) was used as an internal control. Bands were visualized using the chemiluminescence detection method (Pierce; catalog no. 32106). The intensity of β-arrestin2 immunoreactive bands was normalized to β-actin and expressed as relative to the scrambled siRNA control.

Additionally, β-arrestin2 siRNA knockdown and scrambled siRNA-treated control cells were analyzed by immunocytochemistry. Cells were fixed with 4% formaldehyde for 15 min and permeabilized with the blocking buffer containing 6% normal horse serum and 0.01% Triton X-100 for 10 min. Then cells were incubated with the rabbit anti-β-arrestin2 antibody (LS-B15546) followed by goat anti-rabbit Alexa 488 antibody. Coverslips were mounted using Prolong Gold Antifade reagent with 4,6-diamidino-2-phenylindole (Invitrogen; P36931). Fluorescence was imaged by a Zeiss LSM 880 confocal microscope.

MDM2 protein knockdown via siRNA

N2A-5HT_1AR cells were transiently transfected with a mouse MDM2 siRNA (Mm01_00139357) or a corresponding scrambled siRNA obtained from Sigma. Cells were lysed in solubilization buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 1% Triton X-100. Western blotting was performed to determine MDM2 levels using mouse anti-MDM2 antibody (Millipore; OP46) followed by a horse anti-mouse HRP-conjugated secondary antibody (Cell Signaling Technology, catalog no. 7076). Bands were visualized using chemiluminescence detection method (Pierce; catalog no. 32106). Quantification of the bands was performed using ImageJ (National Institutes of Health). MDM2 immunoreactive bands were normalized to β-actin (Santa Cruz Biotechnology) and expressed as relative to the scrambled siRNA control.

Confocal microscopy

Fluorescent images were acquired with Zeiss LSM 710 and 880 laser-scanning confocal microscopes. Confocal image planes were acquired with a ×63/NA 1.4 PlanApo oil-immersion objective. Alexa 405, Alexa 555, and Alexa 647 signals were excited at 405, 561, and 633 nm with diode-pumped solid-state (DPSS), and HeNe lasers, respectively. Fluorescent channels were acquired sequentially to prevent cross-excitation of laser signal and to minimize bleed-through. All images were acquired with identical settings for excitation intensity, detector sensitivity, and pinhole to allow intensity comparison between samples. Images for analysis were obtained by taking a z axis stack of image planes (1024 × 1024 pixels) with 0.2-μm steps encompassing the entire cell structure. Fluorescent intensity was quantitated for each channel using ImageJ software (National Institutes of Health). All images were adjusted with Gaussian blur for presentation purposes only. Alexa 405 emission was pseudocolored as green upon acquisition for optimal presentation appearance. All experiments were performed independently at least four times. For each group, ~25–35 cells were selected for analysis from each experiment.

Immunocytochemistry

Stimulated 5-HT_1AR internalization following treatment with ethanol (15–75 mm) or vehicle (media) for 18 h in N2A-5HT_1AR cells was measured using an antibody feeding technique, as described previously (34). N2A-5HT_1AR cells were washed with PBS and blocked with 6% normal horse serum in PBS at 4 °C for 30 min. Surface 5-HT_1ARs were then labeled with mouse anti-HA tag (Sigma; H6958) for 45 min at 4 °C. Cells were warmed to 37 °C and stimulated with 8-OH-DPAT (1 μm) for 5, 15, 30, or 60 min. Internalization was terminated by washing cells with cold PBS and placing coverslips on ice. The remaining primary antibody-bound surface 5-HT_1ARs were incubated with rabbit anti-mouse Alexa 647 to determine the pool of surface 5-HT_1ARs that were not internalized. Next, cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.1% Triton X-100 for 10 min. Internalized 5-HT_1ARs bound with HA-tag primary antibody were labeled with goat anti-mouse Alexa 405 for quantification of internalized 5-HT_1ARs. Stimulated 5-HT_1AR internalization was calculated as the percent fluorescent intensity of Alexa 405 (internalized 5-HT_1ARs) of the total (Alexa 647 + Alexa 405) (total surface 5-HT_1ARs before internalization). Data were presented as relative to the baseline level of 5-HT_1AR internalization in vehicle-treated cells.

We examined the co-localization of β-arrestin2, ubiquitin, and the proteasome marker P19S. Briefly, cells were pretreated with ethanol (15–75 mm) or vehicle (media) for 18 h, then washed with cold PBS prior to fixation with 4% formaldehyde for 15 min, and permeabilized with 0.1% Triton X-100 in blocking buffer containing 6% normal horse serum for 10 min. After blocking with 6% normal horse serum in PBS for 20 min, cells were incubated with the following antibodies: rabbit anti-β-arrestin2 (LSBio; LS-B15546), sheep anti-ubiquitin (LSBio; LS-C76671), and goat anti-P19S (Novus Biologicals; NB100-1483) for 1 h followed by incubation with the secondary antibodies donkey anti-goat Alexa 633, donkey anti-sheep Alexa 555, and goat anti-rabbit Alexa 405, respectively. All fluorescent secondary antibodies are from Invitrogen. Coverslips were mounted using Prolong Gold mounting reagent (Invitrogen, P36930) and imaged via confocal microscopy. Images were processed, and spatial overlap was calculated using FIJI and Photoshop. To directly assess spatial overlap of these proteins, background was removed, and variable intensity information from each image was done by thresholding. This method allows each pixel to provide binary information for the presence or absence of each protein and to provide specific quantitation and display of positional overlap. The co-localization between β-arrestin2 (green) and ubiquitin (red) was first determined as yellow overlapped pixels. These co-localized pixels (yellow) were then isolated, and triple co-localization with P19S (blue) was determined as white overlapped pixels. Data were reported as a percentage of co-localization by overlapping pixel counts, y/x + y + z, (ubiquitin/β-arrestin2 co-localization with respect to P19S).

Western blotting

β-Arrestin levels were measured in N2A-5HT_1AR cells and rat PFC tissue, and GRK2 and HA-tagged 5-HT_1AR levels were measured in N2A-5HT_1AR cells only. N2A-5HT_1AR cells were lysed in solubilization buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, and 1% Triton X-100), and rat PFC tissue was homogenized in RIPA buffer containing 25 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100. Lysates were resolved by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were immunoblotted with rabbit anti-β-arrestin antibody (LSBio; LS-B15546), mouse anti-GRK2 (Santa Cruz Biotechnology; sc-13143), or mouse anti-HA tag (GenScript, A01244) followed by a goat anti-rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology; sc-2005) or horse anti-mouse HRP-conjugated secondary antibody (Cell Signaling Technology, catalog no. 7076), respectively. β-Actin (Santa Cruz Biotechnology; sc-1616) was used as an internal control. Bands were visualized using chemiluminescence detection method (Pierce; catalog no. 32106). Quantification of the bands was performed using ImageJ (National Institutes of Health). The intensity of β-arrestin immunoreactive bands was normalized to that of β-actin and expressed as relative to the control.

To evaluate whether ethanol-induced β-arrestin2 degradation was proteasome-dependent, cells were incubated with or without MG132 (5 μm), a proteasome inhibitor as shown previously (57), during treatment with ethanol (30–75 mm) or vehicle for 18 h. Western blotting was performed to measure β-arrestin2 protein levels.

Immunoprecipitation

To examine β-arrestin2 ubiquitination, N2A-5HT_1AR cells and rat PFC tissue were solubilized in a lysis buffer containing 50 mm NaCl, 50 mm HEPES (pH 7.2), 10% glycerol, 1 mm EGTA, 1 mm EDTA, 10 mg/ml iodoacetamide, 1% Triton X-100, or RIPA buffer containing 25 mm Tris-HCl (pH 7.4), 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% Triton X-100, respectively, supplemented with 10 mm N-ethylmaleimide for 1 h on ice. Samples were incubated with rabbit anti-β-arrestin2 antibody (LSBio; LS-B15546), conjugated with protein A/G beads (Santa Cruz Biotechnology) at 4 °C overnight. Samples were washed, and the immunoprecipitated proteins were eluted with the sample buffer containing dithiothreitol (DTT). The proteins were separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes for sequential Western blotting with mouse anti-MDM2 (Millipore; OP46), mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017), and then mouse anti-β-arrestin2 (LSBio; LS-B6008). In an additional set of experiments, nitrocellulose membranes were cut according to molecular weight and Western-blotted with rabbit anti-NEDD4 (Cell Signaling Technology; catalog no. 3607S) and mouse anti-β-arrestin2 (LSBio; LS-B6008).

To confirm the specificity of β-arrestin2 immunoprecipitation by rabbit anti-β-arrestin2 antibody (LSBio; LS-B15546) in drug-naïve N2A cells and rat PFC tissue, a corresponding blocking peptide (GenScript: sequence DDIVFEDFARLRLK) was used. Briefly, immunoprecipitation of β-arrestin2 was performed as described above in the presence or absence of the β-arrestin2–blocking peptide (10 μg). Immunoprecipitates and whole-cell lysate were immunoblotted for mouse anti-ubiquitin (Santa Cruz Biotechnology; sc8017) and mouse anti-β-arrestin2 (LSBio; LS-B6008).

We thank Dr. Allyn Howlett for helpful comments during the preparation of this manuscript. Additionally, we thank the Wake Forest Center for Molecular Signaling and Wake Forest Microscopic Imaging Core Facility.