-directed signaling of the 2A SEE COMMENTARY depends on ␤-arrestin-2 interactions in vivo

Cullen L. Schmid, Kirsten M. Raehal, and Laura M. Bohn*

Departments of and Psychiatry, Ohio State University College of , 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 (4, 17). Although ␤-arrestins are induced by the use or abuse of certain . The heptahelical important for the regulation of many GPCRs, their role in serotonin 2A receptors (5-HT2ARs) are molecular targets for - 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 - 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. 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. The majority of internalization can occur in a ␤-arrestin-1-independent manner. endogenous 5-HT2AR are found within the intracellular region of Upon culturing WT MEFs transfected with a YFP C-terminally WT neurons, which is consistent with previous studies (Fig. 2A tagged 5-HT2AR in complete media containing 10% FBS, we find Upper) (18, 25). However, neurons from the ␤-arrestin-2-KO mice that the majority of the 5-HT2AR-YFP is localized intracellularly. display more prominent membrane staining of the endogenous This pattern of expression can be reversed by removal of the serum receptor (Fig. 2B Upper). To determine whether receptors are from the media for2hofincubation, implicating the presence of trafficking from the cell surface to the intracellular vesicles, we the agonist (serotonin) within the complete media in contributing transfected neurons with an N-terminally HA-tagged 5-HT2AR to receptor internalization. Adding serotonin directly to the serum- and performed live cell antibody staining. We find that WT neurons free media induces 5-HT2AR-YFP internalization in the WT internalized the cell surface-labeled receptors, whereas the ␤- MEFs (Fig. 3A). The ␤-arrestin-1- and ␤-arrestin-2-KO MEFs arrestin-2-KO neurons retained more prominent antibody staining retain 5-HT2AR-YFP surface expression regardless of serum me- on the cell surface (Fig. 2A Lower). The 5-HT2AR on the cell dia content and do not internalize the receptor upon addition of surface of ␤-arrestin-2-KO neurons could be internalized upon serotonin (Fig. 3B). DOI, however, produces 5-HT2AR-YFP in- expression of ␤-arrestin-2-YFP (Fig. 2C). Therefore, our data ternalization in both WT and ␤-arrestin-1- and ␤-arrestin-2-KO suggest that ␤-arrestin-2 plays an important role in determining MEFs (Fig. 3 A and B). Longer agonist incubations (60 or 120 min) 5-HT2AR trafficking in cortical neuron cultures. do not change receptor internalization profiles (data not shown). Serotonin is present in neuronal culturing conditions and likely To quantitatively assess the internalization profiles of the MEFs, contributes to the 5-HT2AR trafficking seen in the WT neurons. we performed cell surface biotinylation studies. Serotonin and DOI Because manipulations of neuronal culturing conditions can be both induce an increase in the amount of protected biotinylated cell detrimental to neuron survival, we further explored ␤-arrestin’s surface receptor after glutathione stripping, suggesting an increase contributions to trafficking and signaling by using transfected in receptor internalization in the WT cells. However, only DOI

1080 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708862105 Schmid et al. Downloaded by guest on September 26, 2021 SEE COMMENTARY PHARMACOLOGY

Fig. 2. 5-HT2AR localization in WT and ␤-arrestin-2-KO cortical neurons. (A) WT neurons. (B). ␤-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) ␤-arrestin-2-KO neurons were transfected with ␤-arrestin-2-YFP (␤arr2-YFP) and stained for endogenous 5-HT2AR [shown as ␤arr2-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 ␤-arrestin-2-YFP as indicated.

significantly induced receptor internalization in the ␤-arrestin-1- 5-HT2AR or respond to either agonist (SI Fig. 6B). Time course and ␤-arrestin-2-KO MEFs, which is consistent with the confocal studies in the ␤-arrestin-1- and ␤-arrestin-2-KO MEFs do not reveal microscopy studies (Fig. 3 A and B). Taken together, our findings higher ERK1/2 activation at earlier or later time points (data not indicate that DOI-induced 5-HT2AR internalization is ␤-arrestin- shown). These findings suggest that ␤-arrestins play a key role in independent, whereas serotonin-induced internalization requires serotonin-induced activation of ERK1/2 by the 5-HT2AR. ␤-arrestins. Signaling of the 5-HT2AR can transduce by multiple G protein- We next examined whether ␤-arrestins play a role in 5-HT2AR coupling pathways, including the Gq stimulation of phospholipase signaling in response to serotonin or DOI because ␤-arrestins can C (PLC) (15). Therefore, we tested the contribution of PLC mediate MAP kinase activation by GPCR stimulation (30). Sero- signaling to ERK1/2 activation with the selective inhibitor, U73122 tonin induces robust ERK1/2 phosphorylation in serum-fasted WT (15). Inhibition of PLC blocked approximately one-third of the MEFs, which is significantly greater than that seen for DOI (Fig. serotonin-mediated activation of ERK1/2 in the WT cells and also 4A). Both serotonin and DOI induce ERK1/2 phosphorylation in prevented DOI-induced activation. In the ␤-arrestin-null MEFs, the KO cells. However, the degree of 5-HT stimulation is much U73122 pretreatment prevented all ERK1/2 phosphorylation in- lower than that observed in the WT cells (Fig. 4 A and B). Overall duced by both serotonin and DOI (Fig. 4 B and C). These findings 5-HT2AR expression levels were similar for both cell lines, as demonstrate that the two diverse agonists direct differential sig- assessed by radioligand-binding assays [supporting information (SI) naling by the 5-HT2AR. When the 5-HT2AR is expressed in MEF Fig. 6A]. Furthermore, mock-transfected cells do not express cells, DOI stimulates ERK1/2 primarily through a PLC-dependent

Schmid et al. PNAS ͉ January 22, 2008 ͉ vol. 105 ͉ no. 3 ͉ 1081 Downloaded by guest on September 26, 2021 Fig. 3. Agonist-induced internalization of 5-HT2AR-YFP expressed in WT and ␤-arrestin-1- and ␤-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 ␤-arrestin-1- and ␤-arrestin-2-KO (␤arr1&2-KO) MEFs regardless of serum content. Addition of 1 ␮M serotonin (5-HT) for 30 min internalizes the 5-HT2AR-YFP in WT, but not ␤arr1&2-KO MEFs. DOI (1 ␮M, 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 ␮M 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 Ͻ 0.001; ␤arr1&2-KO: treated vs. vehicle, **, P Ͻ 0.01. WT plus DOI vs. ␤arr1&2-KO plus DOI did not significantly differ (P Ͼ 0.05; n ϭ 9–10 WT treatments in five separate experiments; n ϭ 4–8 KO in three separate experiments).

pathway, whereas serotonin activates ERK1/2 predominantly Discussion through a ␤-arrestin-dependent pathway. Our findings indicate that ␤-arrestin-2 plays a major role in deter- Behavioral responses to serotonin are greatly attenuated in the mining 5-HT2AR responsiveness in vivo. In the absence of ␤-ar- ␤-arrestin-2-KO mice. Therefore, we tested the effects of serotonin restin-2, serotonin no longer induces the head twitch response in on MAP kinase signaling in vivo. The degree of ERK1/2 phos- mice. In ␤-arrestin-2-KO cortical neurons, the 5-HT2AR becomes phorylation levels in frontal cortex lysates after 5-HTP and DOI predominantly localized to the cell surface, as opposed to the treatment were assessed in WT and ␤-arrestin-2-KO mice. Mice intracellular distributions observed in normal mice and rats (18). were treated in the same manner as described in Fig. 1. However, Finally, serotonin no longer leads to ERK activation in the frontal frontal cortex was dissected 15 min after drug treatment, when cortex when ␤-arrestin-2 is genetically ablated. These observations behavioral effects peaked. Serotonin induced ERK1/2 phosphor- suggest that ␤-arrestin-2 mediates ERK activation and intracellular ylation in the WT mice but did not induce significant ERK1/2 trafficking of the 5-HT2AR and that both cellular events may play phosphorylation over vehicle treatments in the ␤-arrestin-2-KO a role in the induction of head twitches in response to elevated mice (Fig. 5). Treatment with DOI promoted significant ERK1/2 serotonin levels. activation in both genotypes (Fig. 5). Total receptor levels did not Alternatively, we found that DOI, a synthetic 5-HT2AR agonist, differ between the genotypes as determined by radioligand-binding produces the head twitch response in mice that lack ␤-arrestin-2 to studies; phospho-ERK (P-ERK) and total ERK (T-ERK) levels the same extent as that seen in WT mice. ␤-arrestins are not also did not differ between saline-treated genotypes (SI Fig. 7). required for DOI-induced receptor internalization in MEFs, nor is These findings indicate that ␤-arrestin-2 is critical for serotonin- ␤-arrestin-2 required for DOI-induced ERK1/2 activation in the induced ERK1/2 phosphorylation in the frontal cortex, whereas frontal cortex. These findings suggest that DOI activates 5-HT2AR DOI can activate ERK1/2 in the absence of ␤-arrestins. signaling pathways that do not require ␤-arrestins and that DOI-

1082 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708862105 Schmid et al. Downloaded by guest on September 26, 2021 SEE COMMENTARY

Fig. 5. Agonist-induced ERK1/2 phosphorylation in frontal cortex of WT and ␤-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 ␤-arrestin-2-KO mice; DOI stimu- lated ERK1/2 phosphorylation in both genotypes (saline vs. drug, **, P Ͻ 0.01; , P Ͻ 0.05). One-way ANOVA performed within each genotype, followed by ␤ * Fig. 4. Agonist-induced ERK1/2 phosphorylation in WT and -arrestin-1- and Bonferroni post hoc analysis. Data are the mean Ϯ SEM (n ϭ 9–13 mice per ␤ ␤ -arrestin-2-KO ( arr1&2-KO) MEFs expressing HA-5-HT2AR. ERK1/2 phos- genotype per treatment). A representative blot of P-ERK and T-ERK is shown. phorylation 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 ex- pressed as fold stimulation over control. The means Ϯ SEM are shown. (A) determining hallucinogenic drug properties remains to be deter- Addition of 1 ␮M serotonin (5-HT) and 1 ␮M DOI for 10 min stimulates ERK1/2 mined. to a greater extent in WT than in ␤arr1&2-KO MEFs. WT vehicle versus WT plus Our data emphasize the contribution of the nature of the ligand drug, ***, P Ͻ 0.0001; KO vehicle versus KO plus drug, ***, P Ͻ 0.0001, **, P Ͻ to determining the receptor signaling pathway and, ultimately, the 0.001; WT plus 5-HT versus KO plus 5-HT, ##, P Ͻ 0.001; WT plus DOI vs. KO plus physiological responses induced by the compound. Regulation of Ͻ ϭ DOI, #, P 0.01, Student’s t test (n 12–16; four independent transfections, the 5-HT2AR in vivo may set the tone for neuronal sensitivity to with each treatment performed in two to four replicates). (B and C)A1␮M PHARMACOLOGY U73122 (U7) 30-min pretreatment was used to inhibit PLC activation of ERK in endogenous levels of serotonin, as well as the responsiveness to WT and KO MEFs. (B) WT MEFs: vehicle versus 5-HT, ***, P Ͻ 0.001; 5HT versus pharmacological agents. Moreover, drugs that disrupt the 5HT plus U73122, ***, P Ͻ 0.001; vehicle versus 5HT plus U73122, ***, P Ͻ 5-HT2AR–␤-arrestin interaction might provide a means to alter the 0.001; vehicle versus DOI, ***, P Ͻ 0.001; DOI versus DOI plus U73122, ***, P Ͻ sensitivity of the receptor to the levels of serotonin present in brain 0.001. (C) ␤-arr-1&2-KO MEFs: vehicle versus 5-HT, ***, P Ͻ 0.001; 5HT versus and maintain a desired basal serotonergic tone while eliminating Ͻ Ͻ 5HT plus U73122, ***, P 0.001; vehicle versus DOI, ***, P 0.001; DOI versus excessive receptor responsiveness to endogenous serotonin. Such DOI plus U73122, **, P Ͻ 0.01. Drug plus U73122 did not differ from vehicle treatment (P Ͼ 0.05). One-way ANOVA, followed by Bonferroni post hoc manipulations may provide an approach in drug development for comparison of treatments (n ϭ 5–6 for three independent transfections, with treating neuropsychiatric disorders. each treatment performed in duplicate or triplicate). (D) Representative West- ern blot of P-ERK and T-ERK is shown for A–C. Methods Drugs. DOI (Sigma–Aldrich) and 5-HTP (Sigma–Aldrich) were prepared in 0.9% saline. Serotonin hydrochloride [(5-HT) Sigma–Aldrich] was prepared in 20 mM induced head twitches are mediated by ␤-arrestin-2-independent ascorbate in saline. M100907 (kindly provided by Kenner Rice, National Institute pathways. on Drug Abuse/National Institutes of Health, Bethesda, MD) was prepared in ␤-arrestins have been shown to mediate ERK1/2 activation by saline plus 0.02% Tween 80. some GPCRs (2, 30). Our data suggest that the 5-HT2AR is among these receptors. Interestingly, ␤-arrestins mediate 5-HT2AR- Behavioral Experiments. Subjects used in behavioral experiments included male induced ERK1/2 activation upon binding the endogenous agonist, ␤-arrestin-2-KO, heterozygotes, and WT littermates between 3 and 6 months of serotonin, and not the synthetic hallucinogen, DOI. The normal age and were derived by heterozygous breeding (3). Mice were treated with action of serotonin in vivo may depend on the interaction between vehicle (0.9% saline) or drug (1 mg/kg DOI or 100 mg/kg 5-HTP) given i.p. at a the 5-HT2AR and ␤-arrestin-2. An increase in the association volume of 10 ␮l per gram of body weight. Immediately after the injection, each between these components could lead to a greater degree of mouse was placed individually into a Plexiglas box. The number of head twitches responsiveness to the normal levels of serotonin. Current atypical was counted by two observers in 5- or 10-min increments. Body temperature was assessed by using an electronic thermometer (TH5; Physitemp) connected to a antipsychotics, such as clozapine, act as antagonists at 5-HT2AR; rectal temperature probe (RET-3; Physitemp) before and after the 30-min drug their function may be to offset hyperserotonergic responsiveness treatment for the 5-HTP studies (3). Animals treated with 5-HTP also were scored (31). Interestingly, clozapine induces 5-HT2AR internalization; for severity of diarrhea concurrently with head twitches. Severity of diarrhea was however, it does not induce ERK activation in WT MEFs or in the scored as follows: 0, normal or no fecal boli (no diarrhea); 1, visibly wet fecal boli prefrontal cortex of WT mice (refs. 29 and 32 and C.L.S., K.M.R., (moderate); 2, liquid fecal boli lacking form (severe) (33). In some studies, 0.05 and L.M.B., unpublished data). It is attractive to speculate that mg/kg M100907 was injected i.p. 10 min before 5-HTP or DOI. In all cases, mice clozapine may act by preventing downstream ERK 1/2 activation, were used only once for any drug treatment. All experiments were performed although it does not prevent receptor internalization. However, the with the approval of the Institutional Animal Care and Use Committee of The role of 5-HT2AR internalization versus ERK 1/2 activation in Ohio State University.

Schmid et al. PNAS ͉ January 22, 2008 ͉ vol. 105 ͉ no. 3 ͉ 1083 Downloaded by guest on September 26, 2021 Neurons. Neuron cultures. Primary cortical neuronal cultures were obtained from imaging by using HA-488 Alexa Fluor antibody staining in live cells (1:100; P1 mouse pups generated from both homo- and heterozygous breeding of Molecular Probes/Invitrogen) and Western blotting (SI Fig. 6). ␤-arrestin-2 mice. Frontal cortex neuron cultures were prepared as described (34). MAP kinase assays. To assess agonist-induced ERK1/2 phosphorylation, WT and Neurons were grown on poly-L-lysine-coated glass coverslip culture plates (Mat- ␤-arrestin-1- and ␤-arrestin-2-KO MEFs were serum starved for 2 h, followed by 1 Tek) at 37°C and in 5% CO2. One day after plating, 10 ␮l of cytosine B-D ␮M drug treatment for 10 min. Pretreatment with 1 ␮M U73122 (in 0.1% DMSO arabinofuranoside (Sigma–Aldrich) was added per 1 ml of neurobasal medium. final concentration) occurred during the last 30 min of serum fasting. For studies Neurons were fixed, permeabilized, and blocked as described by Mu et al. (35). with U73122, all other treatments within the group received the same vehicle. For endogenous receptor staining, neurons were fixed 4 days after culturing. After drug treatment, cell lysates were prepared, and Western blots were per- ␤ Neurons were transfected with 500 ng of HA-5-HT2AR or 200 ng of -arrestin- formed as previously described (37). Membranes were immunoblotted for 2-YFP cDNA by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s T-ERK1/2 levels (p44/42 MAP kinase antibody; Cell Signaling Technology) and directions 3 or 4 days after culturing. Media was changed the next day, and P-ERK1/2 levels (p-ERK E-4; Santa Cruz Biotechnology). Chemiluminescence was neurons were fixed and stained 24 h after transfection. Neurons were incubated detected and quantified by using a Kodak 2000R imaging system (Eastman with 5-HT2AR polyclonal rabbit (1:100; Neuromics) and Map2 monoclonal mouse Kodak). (1:20,000; Abcam) primary antibodies overnight (Map2) or for 36 h (5-HT2AR) at Cell surface biotinylation assay. MEF WT and ␤-arrestin-1- and ␤-arrestin-2-KO cells 4°C in 5% BSA, 5% goat serum, and 0.02% sodium azide; washed with PBS; and expressing HA-5-HT2AR were serum starved for 2 h before the assay as previously incubated in Alexa Fluor goat anti-mouse 488 and goat anti-rabbit 568 (1:5,000; described (37). Anti-HA antibody-conjugated beads (Sigma–Aldrich) were used Molecular Probes/Invitrogen) secondary antibodies at room temperature for 1 h. An Olympus Fluoview 300 confocal microscope with green-helium neon and to immunoprecipitate the receptor, and immunoblotting was performed with argon lasers was used to capture images. All experiments were performed on at the 5-HT2AR polyclonal rabbit (1:500; Neuromics). least seven separate neuronal preparations of each genotype. Several neurons were imaged per preparation in each plate, and four to five plates of each Frontal Cortex Map Kinase Assays. Mice were treated with the drug as described staining/transfection condition were generated per preparation. Counting of for behavior experiments. Fifteen minutes after drug treatment, animals were neurons was performed on three separate plates from three separate neuronal killed by cervical dislocation. Frontal cortex was isolated and frozen immediately preparations. At least 100 cells were counted per plate by two separate observers, in liquid nitrogen. Tissue was homogenized in solubilization lysis buffer (37) with one of whom was blinded to the experimental conditions. a polytronic tissue grinder. To assess drug-induced effects, WT and ␤-arrestin- Neuronal receptor trafficking. For live cell antibody staining of transfected HA-5- 2-KO samples were run on independent gels. Vehicle-treated controls were run HT2AR, 24 h after transfection, neurons were incubated for 45 min in neurobasal on the same gels with drug-treated samples. Fold stimulation over vehicle control media containing anti-HA Alexa Fluor 594 conjugate (1:100; Molecular Probes/ was calculated by dividing the P-ERK intensity by the total-ERK intensity deter- Invitrogen). Plates were then washed, fixed, permeabilized, stained for Map2, mined by densitometric analysis and normalizing all values on the gel to the and imaged as described above. average of the vehicles on the same gel.

␮ MEFs. MEF trafficking. MEF cells were transiently transfected with 2–2.5 gof Statistical Analysis. Statistical analyses are indicated in the figure legends. All 5-HT2AR-YFP cDNA by using the Gene Pulser Xcell electroporation system as tests were performed by using GraphPad Prism 3.0 software. described previously (Bio-Rad) (36). Images presented are representative of at least four separate transfections and drug treatments. ACKNOWLEDGMENTS. We thank Dr. Robert J. Lefkowitz (Duke University, HA-5-HT2AR MEF stable cell lines. Stable and efficient HA-5-HT2AR expression in Durham, NC) for WT and ␤-arrestin-1- and ␤-arrestin-2-KO MEFs, as well as the ␤ ␤ WT and -arrestin-1- and -arrestin-2-KO MEFs were obtained by using murine ␤-arrestin-2 mice; Dr. Marc G. Caron (Duke University) for the ␤-arrestin-2-YFP stem cell retroviral expression vectors. To avoid posttransfection cellular adapta- cDNA construct; and Lori Hudson for mouse colony maintenance and technical tions, cells were not maintained over two passages after viral transfection. Cells assistance. This work was supported by The National Institute on Drug Abuse via were grown in complete media (DMEM plus 10% heat-inactivated FBS) at 37°C Training Fellowship F31DA219532 (to K.M.R.) and Grants K01 DA014600 and R01 under 5% CO2. Transfection efficiency was determined by live cell confocal DA18860 to (L.M.B.).

1. Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, Javitch JA, 20. Fiorica-Howells E, Hen R, Gingrich J, Li Z, Gershon MD (2002) Am J Physiol Gastrointest Roth BL, Christopoulos A, Sexton PM, et al. (2007) J Pharmacol Exp Ther 320:1–13. Liver Physiol 282:G877–G893. 2. Violin JD, Lefkowitz RJ (2007) Trends Pharmacol Sci 28:416–422. 21. Hedlund PB, Danielson PE, Thomas EA, Slanina K, Carson MJ, Sutcliffe JG (2003) Proc 3. Bohn LM, Lefkowitz RJ, Gainetdinov RR, Peppel K, Caron MG, Lin FT (1999) Science Natl Acad Sci USA 100:1375–1380. 286:2495–2498. 22. Glennon RA (1986) Life Sci 39:825–830. 4. Bohn LM, Gainetdinov RR, Caron MG (2004) Neuromol Med 5:41–50. 23. Sorensen SM, Kehne JH, Fadayel GM, Humphreys TM, Ketteler HJ, Sullivan CK, Taylor 5. Raehal KM, Walker JK, Bohn LM (2005) J Pharmacol Exp Ther 314:1195–1201. VL, Schmidt CJ (1993) J Pharmacol Exp Ther 266:684–691. 6. Wang Q, Zhao J, Brady AE, Feng J, Allen PB, Lefkowitz RJ, Greengard P, Limbird LE 24. Pierce KL, Lefkowitz RJ (2001) Nat Rev Neurosci 2:727–733. (2004) Science 304:1940–1944. 25. Xia Z, Hufeisen SJ, Gray JA, Roth BL (2003) 122:907–920. 7. Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG (2005) 26. Kohout TA, Lin FS, Perry SJ, Conner DA, Lefkowitz RJ (2001) Proc Natl Acad Sci USA Cell 122:261–273. 98:1601–1606. 8. Gainetdinov RR, Bohn LM, Sotnikova TD, Cyr M, Laakso A, Macrae AD, Torres GE, Kim 27. Gray JA, Bhatnagar A, Gurevich VV, Roth BL (2003) Mol Pharmacol 63:961–972. KM, Lefkowitz RJ, Caron MG, et al. (2003) Neuron 38:291–303. 28. Berry SA, Shah MC, Khan N, Roth BL (1996) Mol Pharmacol 50:306–313. 9. Bohn LM, Lefkowitz RJ, Caron MG (2002) J Neurosci 22:10494–10500. 29. Willins DL, Berry SA, Alsayegh L, Backstrom JR, Sanders-Bush E, Friedman L, Roth BL 10. Gonzalez-Maeso J, Weisstaub NV, Zhou M, Chan P, Ivic L, Ang R, Lira A, Bradley-Moore (1999) Neuroscience 91:599–606. M, Ge Y, Zhou Q, et al. (2007) Neuron 53:439–452. 30. Luttrell LM, Roudabush FL, Choy EW, Miller WE, Field ME, Pierce KL, Lefkowitz RJ (2001) 11. Nichols DE (2004) Pharmacol Ther 101:131–181. Proc Natl Acad Sci USA 98:2449–2454. 12. McLean TH, Parrish JC, Braden MR, Marona-Lewicka D, Gallardo-Godoy A, Nichols DE 31. Meltzer HY (2002) in : The Fifth Generation of Progress, (2006) J Med Chem 49:5794–5803. eds Davis KL, Charney D, Coyle JT, Nemeroff C (Raven, New York), pp 819–832. 13. Bhatnagar A, Sheffler DJ, Kroeze WK, Compton-Toth B, Roth BL (2004) J Biol Chem 279:34614–34623. 32. Willins DL, Alsayegh L, Berry SA, Backstrom JR, Sanders-Bush E, Friedman L, Khan N, 14. Gray JA, Roth BL (2001) Brain Res Bull 56:441–451. Roth BL (1998) Ann NY Acad Sci 861:121–127. 15. Kurrasch-Orbaugh DM, Parrish JC, Watts VJ, Nichols DE (2003) J Neurochem 86:980– 33. Gainetdinov RR, Bohn LM, Walker JK, Laporte SA, Macrae AD, Caron MG, Lefkowitz RJ, 991. Premont RT (1999) Neuron 24:1029–1036. 16. Corne SJ, Pickering RW (1967) Psychopharmacologia 11:65–78. 34. Askwith CC, Wemmie JA, Price MP, Rokhlina T, Welsh MJ (2004) J Biol Chem 279:18296– 17. Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, Caron MG (2004) Annu Rev 18305. Neurosci 27:107–144. 35. Mu Y, Otsuka T, Horton AC, Scott DB, Ehlers MD (2003) Neuron 40:581–594. 18. Gelber EI, Kroeze WK, Willins DL, Gray JA, Sinar CA, Hyde EG, Gurevich V, Benovic J, 36. Bohn LM, Dykstra LA, Lefkowitz RJ, Caron MG, Barak LS (2004) Mol Pharmacol Roth BL (1999) J Neurochem 72:2206–2214. 66:106–112. 19. Gray JA, Sheffler DJ, Bhatnagar A, Woods JA, Hufeisen SJ, Benovic JL, Roth BL (2001) 37. Groer CE, Tidgewell K, Moyer RA, Harding WW, Rothman RB, Prisinzano TE, Bohn LM Mol Pharmacol 60:1020–1030. (2007) Mol Pharmacol 71:549–557.

1084 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0708862105 Schmid et al. Downloaded by guest on September 26, 2021