Agonist-directed signaling of the serotonin 2A SEE COMMENTARY receptor depends on ␤-arrestin-2 interactions in vivo Cullen L. Schmid, Kirsten M. Raehal, and Laura M. Bohn* Departments of Pharmacology and Psychiatry, Ohio State University College of Medicine, Columbus, OH 43210 Edited by Robert J. Lefkowitz, Duke University Medical Center, Durham, NC, and approved November 16, 2007 (received for review September 18, 2007) Visual and auditory hallucinations accompany certain neuropsy- Regulation of GPCRs can set the tone for receptor sensitivity to chiatric disorders, such as schizophrenia, and they also can be basal levels of neurotransmitters (4, 17). Although ␤-arrestins are induced by the use or abuse of certain drugs. The heptahelical important for the regulation of many GPCRs, their role in serotonin 2A receptors (5-HT2ARs) are molecular targets for drug- 5-HT2AR regulation and signaling remains unclear. Previous work induced hallucinations. However, the cellular mechanisms by has shown that the 5-HT2AR colocalizes with ␤-arrestin-1 and Ϫ2 which the 5-HT2AR mediates these effects are not well understood. in cortical neurons, and some colocalization is apparent in intra- Drugs acting at the 5-HT2AR can trigger diverse signaling path- cellular vesicles (18). Previous studies have shown that the role of ways that may be directed by the chemical properties of the drug. ␤-arrestins in mediating 5-HT2AR internalization can vary be- ␤-arrestins are intracellular proteins that bind to heptahelical tween cell lines (19), further emphasizing the importance of eval- receptors and represent a point where such divergences in ligand- uating ␤-arrestin’s impact on 5-HT2AR function and trafficking in directed functional signaling could occur. Here we compare the vivo. In the current study, we test whether 5-HT2AR regulation by endogenous agonist, serotonin, to a synthetic 5-HT2AR hallucino- ␤-arrestins can contribute to serotonergic responsiveness in vivo by genic agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), in mice assessing behavioral responses and examining 5-HT2AR trafficking lacking ␤-arrestin-2, as well as in cells lacking ␤-arrestins. In mice, and signaling in mice that lack ␤-arrestin-2. Understanding the we find that serotonin induces a head twitch response by a functional significance of 5-HT2AR ligand-directed signaling and ␤-arrestin-2-dependent mechanism. However, DOI invokes the its impact on behavioral responsiveness in vivo may point to new behavior independent of ␤-arrestin-2. The two structurally distinct avenues in serotonergic drug development. agonists elicit different signal transduction and trafficking patterns Fig. 1A shows that treatment with the serotonin precursor, upon activation of 5-HT2AR, which hinge on the presence of L-5-hydroxytryptophan (5-HTP), produces the expected display of ␤-arrestins. Our study suggests that the 5-HT2AR–␤-arrestin inter- the head twitch response in wild-type (WT) mice (16). However, ␤ action may be particularly important in receptor function in re- this response is greatly attenuated in -arrestin-2-KO mice (Fig. ␤ sponse to endogenous serotonin levels, which could have major 1A). A gene dosage effect was seen in the -arrestin-2 heterozygous implications in drug development for treating neuropsychiatric mice because they displayed significantly fewer head twitches, ␤ disorders such as depression and schizophrenia. compared with WT mice (Fig. 1A). To determine whether -ar- restin-2-KO mice respond to any biological effects resulting from PHARMACOLOGY 5-HT2A receptor ͉ G protein-coupled receptor ͉ internalization ͉ the surge in endogenous serotonin produced by the systemic 5-HTP MAP kinase ͉ schizophrenia injection, we simultaneously assessed additional behavioral re- sponses attributed to high serotonin levels and found that hypo- thermia (Ϸ3.5°C decrease in body temperature in 30 min) and the protein-coupled receptors (GPCRs) are major drug targets, onset and severity of diarrhea did not differ between the two Gyet different compounds acting at a given receptor can elicit genotypes (Fig. 1 B and C). These physiological responses are substantially different biological responses. The growing body of generally attributed to actions of other serotonin receptor subtypes evidence supports a model wherein GPCR regulation and subse- and not the 5-HT2AR (20, 21). Surprisingly, treatment with the quent signaling are determined by proteins that interact with the hallucinogenic drug, 2,5-dimethoxy-4-iodoamphetamine (DOI) receptor within distinct cellular environments (1, 2). Moreover, the (22), produced head twitch responses of equal magnitude in both chemical nature of the ligand can dictate the receptor’s ability to genotypes (Fig. 1D). Taken together, these findings suggest that recruit and interact with such proteins and can thereby determine ␤-arrestin-2 mediates 5-HTP-induced head twitches, whereas DOI the extent of overall drug responsiveness. Proteins that regulate ␤ ␤ produces this behavior in a -arrestin-2-independent manner. The GPCR signaling, such as GPCR kinases and -arrestins, have been 5-HT2AR-selective antagonist, R(ϩ)-␣-(2,3-dimethoxyphenyl)-1- shown to define receptor responsiveness to drugs and endogenous [2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907) (23), ␤ neurotransmitters in vivo (3–9). Because -arrestins can both blocked the head twitch response in the WT mice after both drug desensitize and promote GPCR signaling, they are particularly well treatments, as well as in the ␤-arrestin-2-KO mice after DOI positioned to play a significant role in ligand-directed functional treatment, which further indicates that this behavior is due to signaling (2). 5-HT2AR activation (Fig. 1 E and F). The heptahelical serotonin 2A receptor (5-HT2AR) is a GPCR Classically, ␤-arrestins are known for their role in promoting that couples primarily with Gq proteins, yet several cellular studies GPCR internalization (24). Previously, Gelber et al. (18) showed have shown that this receptor can have different signaling and trafficking profiles depending on the nature of the ligand bound (1, 10–15). However, such divergences in ligand-directed 5-HT2AR Author contributions: C.L.S., K.M.R., and L.M.B. designed research, performed research, signaling have yet to be correlated with drug-induced behaviors. analyzed data, and wrote the paper. Serotonergic drugs that induce hallucinations in humans also The authors declare no conflict of interest. produce a head twitch response in mice. Extensive pharmacological This article is a PNAS Direct Submission. studies strongly implicate the 5-HT2AR in mediating this effect See Commentary on page 831. (16). Furthermore, 5-HT2AR knockout mice do not exhibit head *To whom correspondence should be addressed. E-mail: [email protected]. twitches in response to a wide panel of hallucinogenic drugs, further This article contains supporting information online at www.pnas.org/cgi/content/full/ supporting the 5-HT2AR as a principle target in drug-induced 0708862105/DC1. hallucinations (10). © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708862105 PNAS ͉ January 22, 2008 ͉ vol. 105 ͉ no. 3 ͉ 1079–1084 Downloaded by guest on September 26, 2021 Fig. 1. Head twitch response to serotonergics in WT and ␤-arrestin-2-KO (␤arr2-KO) mice. (A) The serotonin precursor, 5-HTP (100 mg/kg, i.p), does not induce a head twitch response in ␤arr2-KO mice. Head twitches were counted every 5 min over 30 min after drug treatment. WT mice experience significantly more head twitches than ␤arr2-KO mice (two-way ANOVA for genotype: WT vs. KO, F(1, 126) ϭ 33.38, P Ͻ 0.0001; WT vs. HT, F(1, 140) ϭ 6.63, P ϭ 0.0111; HT vs. KO, F(1, 112) ϭ 25.54, P Ͻ 0.0001; n ϭ 12 WT, 10 ␤arr2-HT, 8 ␤arr2-KO). (B) Change in body temperature 30 min after administration of 5-HTP. Both genotypes exhibited a similar extent of hypothermia (P ϭ 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) ϭ 0.01, P ϭ 0.9094; n ϭ 12 WT, 8 ␤arr2-KO). The means Ϯ SEM are shown. (D) DOI (1 mg/kg, i.p.) induced equivalent head twitch responses in the WT and ␤arr2-KO mice. Head twitches were counted every 10 min over 60 min after drug treatment (two-way ANOVA for genotype, F(1, 66) ϭ 0.71, P ϭ 0.4023; n ϭ 8 WT, 5 ␤arr2-KO). The means Ϯ 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 Ͻ 0.01; WT vs. WT plus M100, **, P Ͻ 0.01; KO vs. KO plus M100, P Ͼ 0.05 (n ϭ 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 Ͼ 0.05; WT vs. WT plus M100, **, P Ͻ 0.001; KO vs. KO plus M100, P Ͻ 0.01; n ϭ 5–9. that ␤-arrestin-2 and 5-HT2AR can be colocalized in intracellular mouse embryonic fibroblasts (MEFs) derived from WT and vesicles of pyramidal neurons within cortical sections from un- ␤-arrestin-1- and ␤-arrestin-2-KO embryos (26). Studies by Roth treated rats. Here we examined the endocytic profile of the and colleagues (19, 27–29) reported 5-HT2AR internalization in 5-HT2AR in primary cortical neuronal cultures isolated from WT response to both agonists and antagonists and found that such and ␤-arrestin-2-KO postnatal day 1 (P1) mice.