Agonist-directed signaling of the serotonin 2A receptor depends on beta-arrestin-2 interactions in vivo

Abstract

Visual and auditory hallucinations accompany certain neuropsychiatric disorders, such as schizophrenia, and they also can be induced by the use or abuse of certain drugs. The heptahelical serotonin 2A receptors (5-HT2ARs) are molecular targets for drug-induced hallucinations. However, the cellular mechanisms by which the 5-HT2AR mediates these effects are not well understood. Drugs acting at the 5-HT2AR can trigger diverse signaling pathways that may be directed by the chemical properties of the drug. beta-arrestins are intracellular proteins that bind to heptahelical receptors and represent a point where such divergences in ligand-directed functional signaling could occur. Here we compare the endogenous agonist, serotonin, to a synthetic 5-HT2AR hallucinogenic agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), in mice lacking beta-arrestin-2, as well as in cells lacking beta-arrestins. In mice, we find that serotonin induces a head twitch response by a beta-arrestin-2-dependent mechanism. However, DOI invokes the behavior independent of beta-arrestin-2. The two structurally distinct agonists elicit different signal transduction and trafficking patterns upon activation of 5-HT2AR, which hinge on the presence of beta-arrestins. Our study suggests that the 5-HT2AR-beta-arrestin interaction may be particularly important in receptor function in response to endogenous serotonin levels, which could have major implications in drug development for treating neuropsychiatric disorders such as depression and schizophrenia.

Fig. 1.

Head twitch response to serotonergics in WT and β-arrestin-2-KO (βarr2-KO) mice. (A) The serotonin precursor, 5-HTP (100 mg/kg, i.p), does not induce a head twitch response in βarr2-KO mice. Head twitches were counted every 5 min over 30 min after drug treatment. WT mice experience significantly more head twitches than βarr2-KO mice (two-way ANOVA for genotype: WT vs. KO, F_{(1, 126)} = 33.38, P < 0.0001; WT vs. HT, F_{(1, 140)} = 6.63, P = 0.0111; HT vs. KO, F_{(1, 112)} = 25.54, P < 0.0001; n = 12 WT, 10 βarr2-HT, 8 βarr2-KO). (B) Change in body temperature 30 min after administration of 5-HTP. Both genotypes exhibited a similar extent of hypothermia (P = 0.7421, Student's t test) after drug treatment. (C) Severity of diarrhea was scored during the observance of the head twitch response after 5-HTP treatment in the same animals analyzed previously. Both genotypes experienced the effects of 5-HTP on gastrointestinal function to a similar extent (two-way ANOVA, F_{(1, 108)} = 0.01, P = 0.9094; n = 12 WT, 8 βarr2-KO). The means ± SEM are shown. (D) DOI (1 mg/kg, i.p.) induced equivalent head twitch responses in the WT and βarr2-KO mice. Head twitches were counted every 10 min over 60 min after drug treatment (two-way ANOVA for genotype, F_{(1, 66)} = 0.71, P = 0.4023; n = 8 WT, 5 βarr2-KO). The means ± SEM are shown. (E and F) Sum number of head twitches produced over the testing period. (E) 5-HTP-induced head twitches are significantly inhibited by the 5HT2A receptor-selective antagonist, M100907 (M100, 0.05 mg/kg, i.p.), in the WT mice. One-way ANOVA with Bonferroni post hoc analysis reveals: WT vs. KO, **, P < 0.01; WT vs. WT plus M100, **, P < 0.01; KO vs. KO plus M100, P > 0.05 (n = 5–12). (F) DOI-induced head twitches are significantly inhibited by M100 (0.05 mg/kg, i.p.) in both genotypes. One-way ANOVA with Bonferroni post hoc analysis reveals: WT vs. KO, P > 0.05; WT vs. WT plus M100, **, P < 0.001; KO vs. KO plus M100, P < 0.01; n = 5–9.

Fig. 2.

5-HT2AR localization in WT and β-arrestin-2-KO cortical neurons. (A) WT neurons. (B). β-arrestin-2-KO neurons. (Upper) Endogenous 5-HT2AR staining (Left, red) and MAP2 neuronal marker staining (Right, green). (Lower) Live cell HA-594 Alexa Fluor antibody staining of neurons transfected with an N-terminally tagged HA-5-HT2AR. Expression profiles were quantified by counting neurons based on robust, weak, or absent membrane staining. WT: 55 of 369 had weak staining, 2 of 369 had robust surface staining, and 312 of 369 had no discernable surface staining. KO: 57 of 411 had weak staining, 333 of 411 had robust surface staining, and 21 of 411 had no discernable surface staining. (C) β-arrestin-2-KO neurons were transfected with β-arrestin-2-YFP (βarr2-YFP) and stained for endogenous 5-HT2AR [shown as βarr2-YFP (Left, green), 5-HT2AR (Center, red), and merged image (Right)]. Note the localization of the endogenous 5-HT2AR on the cell surface of the nontransfected neuron, compared with the internalized receptors in the neuron expressing β-arrestin-2-YFP as indicated.

Fig. 3.

Agonist-induced internalization of 5-HT2AR-YFP expressed in WT and β-arrestin-1- and β-arrestin-2-KO MEFs. (A) (Upper) WT cells incubated in complete media have mostly internalized 5-HT2AR-YFP (Left, DIC light image to show cell body outline). Serum removal (serum-free for 2 h) returns receptors to cell surface. (Lower) The 5-HT2AR-YFP is on the cell surface of β-arrestin-1- and β-arrestin-2-KO (βarr1&2-KO) MEFs regardless of serum content. Addition of 1 μM serotonin (5-HT) for 30 min internalizes the 5-HT2AR-YFP in WT, but not βarr1&2-KO MEFs. DOI (1 μM, 30 min) internalizes 5-HT2AR-YFP in both cell types. (B) Internalization of HA-5-HT2AR as determined by cell surface biotinylation assay. Cell surface proteins were biotinylated; cells were then treated with 1 μM drug or vehicle for 1 h. (Left) In the representative 5-HT2AR immunoblot, 100% represents surface biotinylation without glutathione stripping, and strip represents cells that were treated with glutathione yet did not undergo vehicle or drug treatment incubation. The 75-kDa molecular weight marker is indicated. (Right) Densitometric analysis of multiple experiments is presented with statistical analysis. One-way ANOVA was performed on each genotype for drug effect, followed by Bonferroni post hoc analysis. WT: treated vs. vehicle, ***, P < 0.001; βarr1&2-KO: treated vs. vehicle, **, P < 0.01. WT plus DOI vs. βarr1&2-KO plus DOI did not significantly differ (P > 0.05; n = 9–10 WT treatments in five separate experiments; n = 4–8 KO in three separate experiments).

Fig. 4.

Agonist-induced ERK1/2 phosphorylation in WT and β-arrestin-1- and β-arrestin-2-KO (βarr1&2-KO) MEFs expressing HA-5-HT2AR. ERK1/2 phosphorylation was assessed by Western blot and densitometric analysis. P-ERK1/2 levels were normalized first to T-ERK1/2 levels, and then drug stimulation was normalized to the vehicle (1% ascorbate in saline) and expressed as fold stimulation over control. The means ± SEM are shown. (A) Addition of 1 μM serotonin (5-HT) and 1 μM DOI for 10 min stimulates ERK1/2 to a greater extent in WT than in βarr1&2-KO MEFs. WT vehicle versus WT plus drug, ***, P < 0.0001; KO vehicle versus KO plus drug, ***, P < 0.0001, **, P < 0.001; WT plus 5-HT versus KO plus 5-HT, ##, P < 0.001; WT plus DOI vs. KO plus DOI, #, P < 0.01, Student's t test (n = 12–16; four independent transfections, with each treatment performed in two to four replicates). (B and C) A 1 μM U73122 (U7) 30-min pretreatment was used to inhibit PLC activation of ERK in WT and KO MEFs. (B) WT MEFs: vehicle versus 5-HT, ***, P < 0.001; 5HT versus 5HT plus U73122, ***, P < 0.001; vehicle versus 5HT plus U73122, ***, P < 0.001; vehicle versus DOI, ***, P < 0.001; DOI versus DOI plus U73122, ***, P < 0.001. (C) β-arr-1&2-KO MEFs: vehicle versus 5-HT, ***, P < 0.001; 5HT versus 5HT plus U73122, ***, P < 0.001; vehicle versus DOI, ***, P < 0.001; DOI versus DOI plus U73122, **, P < 0.01. Drug plus U73122 did not differ from vehicle treatment (P > 0.05). One-way ANOVA, followed by Bonferroni post hoc comparison of treatments (n = 5–6 for three independent transfections, with each treatment performed in duplicate or triplicate). (D) Representative Western blot of P-ERK and T-ERK is shown for A–C.

Fig. 5.

Agonist-induced ERK1/2 phosphorylation in frontal cortex of WT and β-arrestin-2-KO mice. Frontal cortex was dissected 15 min after vehicle (saline), 5-HTP (100 mg/kg, i.p.) or DOI (1 mg/kg, i.p.) DOI treatment, as described in Fig. 1. Brain lysates were resolved and analyzed by Western blot and densitometry as described in Fig. 4. The serotonin precursor (5-HTP) significantly stimulated ERK1/2 in the frontal cortex of WT, but not β-arrestin-2-KO mice; DOI stimulated ERK1/2 phosphorylation in both genotypes (saline vs. drug, **, P < 0.01; *, P < 0.05). One-way ANOVA performed within each genotype, followed by Bonferroni post hoc analysis. Data are the mean ± SEM (n = 9–13 mice per genotype per treatment). A representative blot of P-ERK and T-ERK is shown.