Agonist-directed signaling of the serotonin 2Areceptor depends on �-arrestin-2 interactions in vivoCullen 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-chiatric disorders, such as schizophrenia, and they also can beinduced by the use or abuse of certain drugs. The heptahelicalserotonin 2A receptors (5-HT2ARs) are molecular targets for drug-induced hallucinations. However, the cellular mechanisms bywhich the 5-HT2AR mediates these effects are not well understood.Drugs acting at the 5-HT2AR can trigger diverse signaling path-ways that may be directed by the chemical properties of the drug.�-arrestins are intracellular proteins that bind to heptahelicalreceptors and represent a point where such divergences in ligand-directed functional signaling could occur. Here we compare theendogenous agonist, serotonin, to a synthetic 5-HT2AR hallucino-genic agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI), in micelacking �-arrestin-2, as well as in cells lacking �-arrestins. In mice,we find that serotonin induces a head twitch response by a�-arrestin-2-dependent mechanism. However, DOI invokes thebehavior independent of �-arrestin-2. The two structurally distinctagonists elicit different signal transduction and trafficking patternsupon activation of 5-HT2AR, which hinge on the presence of�-arrestins. Our study suggests that the 5-HT2AR–�-arrestin inter-action may be particularly important in receptor function in re-sponse to endogenous serotonin levels, which could have majorimplications in drug development for treating neuropsychiatricdisorders such as depression and schizophrenia.

5-HT2A receptor � G protein-coupled receptor � internalization �MAP kinase � schizophrenia

G protein-coupled receptors (GPCRs) are major drug targets,yet different compounds acting at a given receptor can elicit

substantially different biological responses. The growing body ofevidence supports a model wherein GPCR regulation and subse-quent signaling are determined by proteins that interact with thereceptor within distinct cellular environments (1, 2). Moreover, thechemical nature of the ligand can dictate the receptor’s ability torecruit and interact with such proteins and can thereby determinethe extent of overall drug responsiveness. Proteins that regulateGPCR signaling, such as GPCR kinases and �-arrestins, have beenshown to define receptor responsiveness to drugs and endogenousneurotransmitters in vivo (3–9). Because �-arrestins can bothdesensitize and promote GPCR signaling, they are particularly wellpositioned to play a significant role in ligand-directed functionalsignaling (2).

The heptahelical serotonin 2A receptor (5-HT2AR) is a GPCRthat couples primarily with Gq proteins, yet several cellular studieshave shown that this receptor can have different signaling andtrafficking profiles depending on the nature of the ligand bound (1,10–15). However, such divergences in ligand-directed 5-HT2ARsignaling have yet to be correlated with drug-induced behaviors.Serotonergic drugs that induce hallucinations in humans alsoproduce a head twitch response in mice. Extensive pharmacologicalstudies strongly implicate the 5-HT2AR in mediating this effect(16). Furthermore, 5-HT2AR knockout mice do not exhibit headtwitches in response to a wide panel of hallucinogenic drugs, furthersupporting the 5-HT2AR as a principle target in drug-inducedhallucinations (10).

Regulation of GPCRs can set the tone for receptor sensitivity tobasal levels of neurotransmitters (4, 17). Although �-arrestins areimportant for the regulation of many GPCRs, their role in5-HT2AR regulation and signaling remains unclear. Previous workhas shown that the 5-HT2AR colocalizes with �-arrestin-1 and �2in cortical neurons, and some colocalization is apparent in intra-cellular vesicles (18). Previous studies have shown that the role of�-arrestins in mediating 5-HT2AR internalization can vary be-tween cell lines (19), further emphasizing the importance of eval-uating �-arrestin’s impact on 5-HT2AR function and trafficking invivo. In the current study, we test whether 5-HT2AR regulation by�-arrestins can contribute to serotonergic responsiveness in vivo byassessing behavioral responses and examining 5-HT2AR traffickingand signaling in mice that lack �-arrestin-2. Understanding thefunctional significance of 5-HT2AR ligand-directed signaling andits impact on behavioral responsiveness in vivo may point to newavenues in serotonergic drug development.

Fig. 1A shows that treatment with the serotonin precursor,L-5-hydroxytryptophan (5-HTP), produces the expected display ofthe head twitch response in wild-type (WT) mice (16). However,this response is greatly attenuated in �-arrestin-2-KO mice (Fig.1A). A gene dosage effect was seen in the �-arrestin-2 heterozygousmice because they displayed significantly fewer head twitches,compared with WT mice (Fig. 1A). To determine whether �-ar-restin-2-KO mice respond to any biological effects resulting fromthe surge in endogenous serotonin produced by the systemic 5-HTPinjection, we simultaneously assessed additional behavioral re-sponses attributed to high serotonin levels and found that hypo-thermia (�3.5°C decrease in body temperature in 30 min) and theonset and severity of diarrhea did not differ between the twogenotypes (Fig. 1 B and C). These physiological responses aregenerally attributed to actions of other serotonin receptor subtypesand not the 5-HT2AR (20, 21). Surprisingly, treatment with thehallucinogenic drug, 2,5-dimethoxy-4-iodoamphetamine (DOI)(22), produced head twitch responses of equal magnitude in bothgenotypes (Fig. 1D). Taken together, these findings suggest that�-arrestin-2 mediates 5-HTP-induced head twitches, whereas DOIproduces this behavior in a �-arrestin-2-independent manner. The5-HT2AR-selective antagonist, R(�)-�-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907) (23),blocked the head twitch response in the WT mice after both drugtreatments, as well as in the �-arrestin-2-KO mice after DOItreatment, which further indicates that this behavior is due to5-HT2AR activation (Fig. 1 E and F).

Classically, �-arrestins are known for their role in promotingGPCR internalization (24). Previously, Gelber et al. (18) showed

Author contributions: C.L.S., K.M.R., and L.M.B. designed research, performed research,analyzed data, and wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

See Commentary on page 831.

*To whom correspondence should be addressed. E-mail: laura.bohn@osumc.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708862105/DC1.

© 2008 by The National Academy of Sciences of the USA

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that �-arrestin-2 and 5-HT2AR can be colocalized in intracellularvesicles of pyramidal neurons within cortical sections from un-treated rats. Here we examined the endocytic profile of the5-HT2AR in primary cortical neuronal cultures isolated from WTand �-arrestin-2-KO postnatal day 1 (P1) mice. The majority ofendogenous 5-HT2AR are found within the intracellular region ofWT neurons, which is consistent with previous studies (Fig. 2AUpper) (18, 25). However, neurons from the �-arrestin-2-KO micedisplay more prominent membrane staining of the endogenousreceptor (Fig. 2B Upper). To determine whether receptors aretrafficking from the cell surface to the intracellular vesicles, wetransfected neurons with an N-terminally HA-tagged 5-HT2ARand performed live cell antibody staining. We find that WT neuronsinternalized the cell surface-labeled receptors, whereas the �-arrestin-2-KO neurons retained more prominent antibody stainingon the cell surface (Fig. 2A Lower). The 5-HT2AR on the cellsurface of �-arrestin-2-KO neurons could be internalized uponexpression of �-arrestin-2-YFP (Fig. 2C). Therefore, our datasuggest that �-arrestin-2 plays an important role in determining5-HT2AR trafficking in cortical neuron cultures.

Serotonin is present in neuronal culturing conditions and likelycontributes to the 5-HT2AR trafficking seen in the WT neurons.Because manipulations of neuronal culturing conditions can bedetrimental to neuron survival, we further explored �-arrestin’scontributions to trafficking and signaling by using transfected

mouse embryonic fibroblasts (MEFs) derived from WT and�-arrestin-1- and �-arrestin-2-KO embryos (26). Studies by Rothand colleagues (19, 27–29) reported 5-HT2AR internalization inresponse to both agonists and antagonists and found that suchinternalization can occur in a �-arrestin-1-independent manner.Upon culturing WT MEFs transfected with a YFP C-terminallytagged 5-HT2AR in complete media containing 10% FBS, we findthat the majority of the 5-HT2AR-YFP is localized intracellularly.This pattern of expression can be reversed by removal of the serumfrom the media for 2 h of incubation, implicating the presence ofthe agonist (serotonin) within the complete media in contributingto receptor internalization. Adding serotonin directly to the serum-free media induces 5-HT2AR-YFP internalization in the WTMEFs (Fig. 3A). The �-arrestin-1- and �-arrestin-2-KO MEFsretain 5-HT2AR-YFP surface expression regardless of serum me-dia content and do not internalize the receptor upon addition ofserotonin (Fig. 3B). DOI, however, produces 5-HT2AR-YFP in-ternalization in both WT and �-arrestin-1- and �-arrestin-2-KOMEFs (Fig. 3 A and B). Longer agonist incubations (60 or 120 min)do not change receptor internalization profiles (data not shown).

To quantitatively assess the internalization profiles of the MEFs,we performed cell surface biotinylation studies. Serotonin and DOIboth induce an increase in the amount of protected biotinylated cellsurface receptor after glutathione stripping, suggesting an increasein receptor internalization in the WT cells. However, only DOI

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 inducea 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 headtwitches than �arr2-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 �arr2-HT, 8 �arr2-KO). (B) Change in body temperature 30 min after administration of 5-HTP. Both genotypes exhibiteda 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 twitchresponse after 5-HTP treatment in the same animals analyzed previously. Both genotypes experienced the effects of 5-HTP on gastrointestinal function to a similarextent (two-way ANOVA, F(1, 108) � 0.01, P � 0.9094; n � 12 WT, 8 �arr2-KO). The means � SEM are shown. (D) DOI (1 mg/kg, i.p.) induced equivalent head twitchresponses 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) �0.71, P � 0.4023; n � 8 WT, 5 �arr2-KO). The means � SEM are shown. (E and F) Sum number of head twitches produced over the testing period. (E) 5-HTP-inducedhead 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 withBonferroni 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 headtwitches 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.

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significantly induced receptor internalization in the �-arrestin-1-and �-arrestin-2-KO MEFs, which is consistent with the confocalmicroscopy studies (Fig. 3 A and B). Taken together, our findingsindicate that DOI-induced 5-HT2AR internalization is �-arrestin-independent, whereas serotonin-induced internalization requires�-arrestins.

We next examined whether �-arrestins play a role in 5-HT2ARsignaling in response to serotonin or DOI because �-arrestins canmediate MAP kinase activation by GPCR stimulation (30). Sero-tonin induces robust ERK1/2 phosphorylation in serum-fasted WTMEFs, which is significantly greater than that seen for DOI (Fig.4A). Both serotonin and DOI induce ERK1/2 phosphorylation inthe KO cells. However, the degree of 5-HT stimulation is muchlower than that observed in the WT cells (Fig. 4 A and B). Overall5-HT2AR expression levels were similar for both cell lines, asassessed by radioligand-binding assays [supporting information (SI)Fig. 6A]. Furthermore, mock-transfected cells do not express

5-HT2AR or respond to either agonist (SI Fig. 6B). Time coursestudies in the �-arrestin-1- and �-arrestin-2-KO MEFs do not revealhigher ERK1/2 activation at earlier or later time points (data notshown). These findings suggest that �-arrestins play a key role inserotonin-induced activation of ERK1/2 by the 5-HT2AR.

Signaling of the 5-HT2AR can transduce by multiple G protein-coupling pathways, including the Gq stimulation of phospholipaseC (PLC) (15). Therefore, we tested the contribution of PLCsignaling to ERK1/2 activation with the selective inhibitor, U73122(15). Inhibition of PLC blocked approximately one-third of theserotonin-mediated activation of ERK1/2 in the WT cells and alsoprevented DOI-induced activation. In the �-arrestin-null MEFs,U73122 pretreatment prevented all ERK1/2 phosphorylation in-duced by both serotonin and DOI (Fig. 4 B and C). These findingsdemonstrate that the two diverse agonists direct differential sig-naling by the 5-HT2AR. When the 5-HT2AR is expressed in MEFcells, DOI stimulates ERK1/2 primarily through a PLC-dependent

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-terminallytagged HA-5-HT2AR. Expression profiles were quantified by counting neurons based on robust, weak, or absent membrane staining. WT: 55 of 369 had weakstaining, 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 surfacestaining, and 21 of 411 had no discernable surface staining. (C) �-arrestin-2-KO neurons were transfected with �-arrestin-2-YFP (�arr2-YFP) and stained forendogenous 5-HT2AR [shown as �arr2-YFP (Left, green), 5-HT2AR (Center, red), and merged image (Right)]. Note the localization of the endogenous 5-HT2ARon the cell surface of the nontransfected neuron, compared with the internalized receptors in the neuron expressing �-arrestin-2-YFP as indicated.

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pathway, whereas serotonin activates ERK1/2 predominantlythrough a �-arrestin-dependent pathway.

Behavioral responses to serotonin are greatly attenuated in the�-arrestin-2-KO mice. Therefore, we tested the effects of serotoninon MAP kinase signaling in vivo. The degree of ERK1/2 phos-phorylation levels in frontal cortex lysates after 5-HTP and DOItreatment were assessed in WT and �-arrestin-2-KO mice. Micewere treated in the same manner as described in Fig. 1. However,frontal cortex was dissected 15 min after drug treatment, whenbehavioral effects peaked. Serotonin induced ERK1/2 phosphor-ylation in the WT mice but did not induce significant ERK1/2phosphorylation over vehicle treatments in the �-arrestin-2-KOmice (Fig. 5). Treatment with DOI promoted significant ERK1/2activation in both genotypes (Fig. 5). Total receptor levels did notdiffer between the genotypes as determined by radioligand-bindingstudies; phospho-ERK (P-ERK) and total ERK (T-ERK) levelsalso did not differ between saline-treated genotypes (SI Fig. 7).These findings indicate that �-arrestin-2 is critical for serotonin-induced ERK1/2 phosphorylation in the frontal cortex, whereasDOI can activate ERK1/2 in the absence of �-arrestins.

DiscussionOur findings indicate that �-arrestin-2 plays a major role in deter-mining 5-HT2AR responsiveness in vivo. In the absence of �-ar-restin-2, serotonin no longer induces the head twitch response inmice. In �-arrestin-2-KO cortical neurons, the 5-HT2AR becomespredominantly localized to the cell surface, as opposed to theintracellular distributions observed in normal mice and rats (18).Finally, serotonin no longer leads to ERK activation in the frontalcortex when �-arrestin-2 is genetically ablated. These observationssuggest that �-arrestin-2 mediates ERK activation and intracellulartrafficking of the 5-HT2AR and that both cellular events may playa role in the induction of head twitches in response to elevatedserotonin levels.

Alternatively, we found that DOI, a synthetic 5-HT2AR agonist,produces the head twitch response in mice that lack �-arrestin-2 tothe same extent as that seen in WT mice. �-arrestins are notrequired for DOI-induced receptor internalization in MEFs, nor is�-arrestin-2 required for DOI-induced ERK1/2 activation in thefrontal cortex. These findings suggest that DOI activates 5-HT2ARsignaling pathways that do not require �-arrestins and that DOI-

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 completemedia 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 drugor vehicle for 1 h. (Left) In the representative 5-HT2AR immunoblot, 100% represents surface biotinylation without glutathione stripping, and strip representscells 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, followedby Bonferroni post hoc analysis. WT: treated vs. vehicle, ***, P � 0.001; �arr1&2-KO: treated vs. vehicle, **, P � 0.01. WT plus DOI vs. �arr1&2-KO plus DOI didnot significantly differ (P � 0.05; n � 9–10 WT treatments in five separate experiments; n � 4–8 KO in three separate experiments).

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induced head twitches are mediated by �-arrestin-2-independentpathways.

�-arrestins have been shown to mediate ERK1/2 activation bysome GPCRs (2, 30). Our data suggest that the 5-HT2AR is amongthese receptors. Interestingly, �-arrestins mediate 5-HT2AR-induced ERK1/2 activation upon binding the endogenous agonist,serotonin, and not the synthetic hallucinogen, DOI. The normalaction of serotonin in vivo may depend on the interaction betweenthe 5-HT2AR and �-arrestin-2. An increase in the associationbetween these components could lead to a greater degree ofresponsiveness to the normal levels of serotonin. Current atypicalantipsychotics, such as clozapine, act as antagonists at 5-HT2AR;their function may be to offset hyperserotonergic responsiveness(31). Interestingly, clozapine induces 5-HT2AR internalization;however, it does not induce ERK activation in WT MEFs or in theprefrontal cortex of WT mice (refs. 29 and 32 and C.L.S., K.M.R.,and L.M.B., unpublished data). It is attractive to speculate thatclozapine may act by preventing downstream ERK 1/2 activation,although it does not prevent receptor internalization. However, therole of 5-HT2AR internalization versus ERK 1/2 activation in

determining hallucinogenic drug properties remains to be deter-mined.

Our data emphasize the contribution of the nature of the ligandto determining the receptor signaling pathway and, ultimately, thephysiological responses induced by the compound. Regulation ofthe 5-HT2AR in vivo may set the tone for neuronal sensitivity toendogenous levels of serotonin, as well as the responsiveness topharmacological agents. Moreover, drugs that disrupt the5-HT2AR–�-arrestin interaction might provide a means to alter thesensitivity of the receptor to the levels of serotonin present in brainand maintain a desired basal serotonergic tone while eliminatingexcessive receptor responsiveness to endogenous serotonin. Suchmanipulations may provide an approach in drug development fortreating neuropsychiatric disorders.

MethodsDrugs. DOI (Sigma–Aldrich) and 5-HTP (Sigma–Aldrich) were prepared in 0.9%saline. Serotonin hydrochloride [(5-HT) Sigma–Aldrich] was prepared in 20 mMascorbate in saline. M100907 (kindly provided by Kenner Rice, National Instituteon Drug Abuse/National Institutes of Health, Bethesda, MD) was prepared insaline plus 0.02% Tween 80.

Behavioral Experiments. Subjects used in behavioral experiments included male�-arrestin-2-KO, heterozygotes, and WT littermates between 3 and 6 months ofage and were derived by heterozygous breeding (3). Mice were treated withvehicle (0.9% saline) or drug (1 mg/kg DOI or 100 mg/kg 5-HTP) given i.p. at avolume of 10 �l per gram of body weight. Immediately after the injection, eachmouse was placed individually into a Plexiglas box. The number of head twitcheswas counted by two observers in 5- or 10-min increments. Body temperature wasassessed by using an electronic thermometer (TH5; Physitemp) connected to arectal temperature probe (RET-3; Physitemp) before and after the 30-min drugtreatment for the 5-HTP studies (3). Animals treated with 5-HTP also were scoredfor severity of diarrhea concurrently with head twitches. Severity of diarrhea wasscored as follows: 0, normal or no fecal boli (no diarrhea); 1, visibly wet fecal boli(moderate); 2, liquid fecal boli lacking form (severe) (33). In some studies, 0.05mg/kg M100907 was injected i.p. 10 min before 5-HTP or DOI. In all cases, micewere used only once for any drug treatment. All experiments were performedwith the approval of the Institutional Animal Care and Use Committee of TheOhio State University.

Fig. 4. Agonist-induced ERK1/2 phosphorylation in WT and �-arrestin-1- and�-arrestin-2-KO (�arr1&2-KO) MEFs expressing HA-5-HT2AR. ERK1/2 phos-phorylation was assessed by Western blot and densitometric analysis.P-ERK1/2 levels were normalized first to T-ERK1/2 levels, and then drugstimulation was normalized to the vehicle (1% ascorbate in saline) and ex-pressed as fold stimulation over control. The means � SEM are shown. (A)Addition of 1 �M serotonin (5-HT) and 1 �M DOI for 10 min stimulates ERK1/2to a greater extent in WT than in �arr1&2-KO MEFs. WT vehicle versus WT plusdrug, ***, 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 plusDOI, #, 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 �MU73122 (U7) 30-min pretreatment was used to inhibit PLC activation of ERK inWT and KO MEFs. (B) WT MEFs: vehicle versus 5-HT, ***, P � 0.001; 5HT versus5HT 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) �-arr-1&2-KO MEFs: vehicle versus 5-HT, ***, P � 0.001; 5HT versus5HT plus U73122, ***, P � 0.001; vehicle versus DOI, ***, P � 0.001; DOI versusDOI plus U73122, **, P � 0.01. Drug plus U73122 did not differ from vehicletreatment (P � 0.05). One-way ANOVA, followed by Bonferroni post hoccomparison of treatments (n � 5–6 for three independent transfections, witheach treatment performed in duplicate or triplicate). (D) Representative West-ern 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�-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 densitometryas described in Fig. 4. The serotonin precursor (5-HTP) significantly stimulatedERK1/2 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 � 0.01;

*, P � 0.05). One-way ANOVA performed within each genotype, followed byBonferroni post hoc analysis. Data are the mean � SEM (n � 9–13 mice pergenotype per treatment). A representative blot of P-ERK and T-ERK is shown.

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Neurons. Neuron cultures. Primary cortical neuronal cultures were obtained fromP1 mouse pups generated from both homo- and heterozygous breeding of�-arrestin-2mice. Frontal cortexneuroncultureswerepreparedasdescribed (34).Neurons were grown on poly-L-lysine-coated glass coverslip culture plates (Mat-Tek) at 37°C and in 5% CO2. One day after plating, 10 �l of cytosine B-Darabinofuranoside (Sigma–Aldrich) was added per 1 ml of neurobasal medium.Neurons were fixed, permeabilized, and blocked as described by Mu et al. (35).For endogenous receptor staining, neurons were fixed 4 days after culturing.Neurons were transfected with 500 ng of HA-5-HT2AR or 200 ng of �-arrestin-2-YFP cDNA by Lipofectamine 2000 (Invitrogen) according to the manufacturer’sdirections 3 or 4 days after culturing. Media was changed the next day, andneurons were fixed and stained 24 h after transfection. Neurons were incubatedwith 5-HT2AR polyclonal rabbit (1:100; Neuromics) and Map2 monoclonal mouse(1:20,000; Abcam) primary antibodies overnight (Map2) or for 36 h (5-HT2AR) at4°C in 5% BSA, 5% goat serum, and 0.02% sodium azide; washed with PBS; andincubated in Alexa Fluor goat anti-mouse 488 and goat anti-rabbit 568 (1:5,000;Molecular Probes/Invitrogen) secondary antibodies at room temperature for 1 h.An Olympus Fluoview 300 confocal microscope with green-helium neon andargon lasers was used to capture images. All experiments were performed on atleast seven separate neuronal preparations of each genotype. Several neuronswere imaged per preparation in each plate, and four to five plates of eachstaining/transfection condition were generated per preparation. Counting ofneurons was performed on three separate plates from three separate neuronalpreparations.At least100cellswerecountedperplatebytwoseparateobservers,one of whom was blinded to the experimental conditions.Neuronal receptor trafficking. For live cell antibody staining of transfected HA-5-HT2AR, 24 h after transfection, neurons were incubated for 45 min in neurobasalmedia containing anti-HA Alexa Fluor 594 conjugate (1:100; Molecular Probes/Invitrogen). Plates were then washed, fixed, permeabilized, stained for Map2,and imaged as described above.

MEFs. MEF trafficking. MEF cells were transiently transfected with 2–2.5 �g of5-HT2AR-YFP cDNA by using the Gene Pulser Xcell electroporation system asdescribed previously (Bio-Rad) (36). Images presented are representative of atleast four separate transfections and drug treatments.HA-5-HT2AR MEF stable cell lines. Stable and efficient HA-5-HT2AR expression inWT and �-arrestin-1- and �-arrestin-2-KO MEFs were obtained by using murinestem cell retroviral expression vectors. To avoid posttransfection cellular adapta-tions, cells were not maintained over two passages after viral transfection. Cellswere grown in complete media (DMEM plus 10% heat-inactivated FBS) at 37°Cunder 5% CO2. Transfection efficiency was determined by live cell confocal

imaging by using HA-488 Alexa Fluor antibody staining in live cells (1:100;Molecular Probes/Invitrogen) and Western blotting (SI Fig. 6).MAP kinase assays. To assess agonist-induced ERK1/2 phosphorylation, WT and�-arrestin-1- and �-arrestin-2-KO MEFs were serum starved for 2 h, followed by 1�M drug treatment for 10 min. Pretreatment with 1 �M U73122 (in 0.1% DMSOfinal concentration) occurred during the last 30 min of serum fasting. For studieswith U73122, all other treatments within the group received the same vehicle.After drug treatment, cell lysates were prepared, and Western blots were per-formed as previously described (37). Membranes were immunoblotted forT-ERK1/2 levels (p44/42 MAP kinase antibody; Cell Signaling Technology) andP-ERK1/2 levels (p-ERK E-4; Santa Cruz Biotechnology). Chemiluminescence wasdetected and quantified by using a Kodak 2000R imaging system (EastmanKodak).Cell surface biotinylation assay. MEF WT and �-arrestin-1- and �-arrestin-2-KO cellsexpressing HA-5-HT2AR were serum starved for 2 h before the assay as previouslydescribed (37). Anti-HA antibody-conjugated beads (Sigma–Aldrich) were usedto immunoprecipitate the receptor, and immunoblotting was performed withthe 5-HT2AR polyclonal rabbit (1:500; Neuromics).

Frontal Cortex Map Kinase Assays. Mice were treated with the drug as describedfor behavior experiments. Fifteen minutes after drug treatment, animals werekilled by cervical dislocation. Frontal cortex was isolated and frozen immediatelyin liquid nitrogen. Tissue was homogenized in solubilization lysis buffer (37) witha polytronic tissue grinder. To assess drug-induced effects, WT and �-arrestin-2-KO samples were run on independent gels. Vehicle-treated controls were runon the same gels with drug-treated samples. Fold stimulation over vehicle controlwas calculated by dividing the P-ERK intensity by the total-ERK intensity deter-mined by densitometric analysis and normalizing all values on the gel to theaverage of the vehicles on the same gel.

Statistical Analysis. Statistical analyses are indicated in the figure legends. Alltests were performed by using GraphPad Prism 3.0 software.

ACKNOWLEDGMENTS. We thank Dr. Robert J. Lefkowitz (Duke University,Durham, NC) for WT and �-arrestin-1- and �-arrestin-2-KO MEFs, as well as the�-arrestin-2 mice; Dr. Marc G. Caron (Duke University) for the �-arrestin-2-YFPcDNA construct; and Lori Hudson for mouse colony maintenance and technicalassistance. This work was supported by The National Institute on Drug Abuse viaTraining Fellowship F31DA219532 (to K.M.R.) and Grants K01 DA014600 and R01DA18860 to (L.M.B.).

1. Urban JD, Clarke WP, von Zastrow M, Nichols DE, Kobilka B, Weinstein H, Javitch JA,Roth BL, Christopoulos A, Sexton PM, et al. (2007) J Pharmacol Exp Ther 320:1–13.

2. Violin JD, Lefkowitz RJ (2007) Trends Pharmacol Sci 28:416–422.3. Bohn LM, Lefkowitz RJ, Gainetdinov RR, Peppel K, Caron MG, Lin FT (1999) Science

286:2495–2498.4. Bohn LM, Gainetdinov RR, Caron MG (2004) Neuromol Med 5:41–50.5. Raehal KM, Walker JK, Bohn LM (2005) J Pharmacol Exp Ther 314:1195–1201.6. Wang Q, Zhao J, Brady AE, Feng J, Allen PB, Lefkowitz RJ, Greengard P, Limbird LE

(2004) Science 304:1940–1944.7. Beaulieu JM, Sotnikova TD, Marion S, Lefkowitz RJ, Gainetdinov RR, Caron MG (2005)

Cell 122:261–273.8. Gainetdinov RR, Bohn LM, Sotnikova TD, Cyr M, Laakso A, Macrae AD, Torres GE, Kim

KM, Lefkowitz RJ, Caron MG, et al. (2003) Neuron 38:291–303.9. Bohn LM, Lefkowitz RJ, Caron MG (2002) J Neurosci 22:10494–10500.

10. Gonzalez-Maeso J, Weisstaub NV, Zhou M, Chan P, Ivic L, Ang R, Lira A, Bradley-MooreM, Ge Y, Zhou Q, et al. (2007) Neuron 53:439–452.

11. Nichols DE (2004) Pharmacol Ther 101:131–181.12. McLean TH, Parrish JC, Braden MR, Marona-Lewicka D, Gallardo-Godoy A, Nichols DE

(2006) J Med Chem 49:5794–5803.13. Bhatnagar A, Sheffler DJ, Kroeze WK, Compton-Toth B, Roth BL (2004) J Biol Chem

279:34614–34623.14. Gray JA, Roth BL (2001) Brain Res Bull 56:441–451.15. Kurrasch-Orbaugh DM, Parrish JC, Watts VJ, Nichols DE (2003) J Neurochem 86:980–

991.16. Corne SJ, Pickering RW (1967) Psychopharmacologia 11:65–78.17. Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, Caron MG (2004) Annu Rev

Neurosci 27:107–144.18. Gelber EI, Kroeze WK, Willins DL, Gray JA, Sinar CA, Hyde EG, Gurevich V, Benovic J,

Roth BL (1999) J Neurochem 72:2206–2214.19. Gray JA, Sheffler DJ, Bhatnagar A, Woods JA, Hufeisen SJ, Benovic JL, Roth BL (2001)

Mol Pharmacol 60:1020–1030.

20. Fiorica-Howells E, Hen R, Gingrich J, Li Z, Gershon MD (2002) Am J Physiol GastrointestLiver Physiol 282:G877–G893.

21. Hedlund PB, Danielson PE, Thomas EA, Slanina K, Carson MJ, Sutcliffe JG (2003) ProcNatl Acad Sci USA 100:1375–1380.

22. Glennon RA (1986) Life Sci 39:825–830.23. Sorensen SM, Kehne JH, Fadayel GM, Humphreys TM, Ketteler HJ, Sullivan CK, Taylor

VL, Schmidt CJ (1993) J Pharmacol Exp Ther 266:684–691.24. Pierce KL, Lefkowitz RJ (2001) Nat Rev Neurosci 2:727–733.25. Xia Z, Hufeisen SJ, Gray JA, Roth BL (2003) Neuroscience 122:907–920.26. Kohout TA, Lin FS, Perry SJ, Conner DA, Lefkowitz RJ (2001) Proc Natl Acad Sci USA

98:1601–1606.27. Gray JA, Bhatnagar A, Gurevich VV, Roth BL (2003) Mol Pharmacol 63:961–972.28. Berry SA, Shah MC, Khan N, Roth BL (1996) Mol Pharmacol 50:306–313.29. Willins DL, Berry SA, Alsayegh L, Backstrom JR, Sanders-Bush E, Friedman L, Roth BL

(1999) Neuroscience 91:599–606.30. Luttrell LM, Roudabush FL, Choy EW, Miller WE, Field ME, Pierce KL, Lefkowitz RJ (2001)

Proc Natl Acad Sci USA 98:2449–2454.31. Meltzer HY (2002) in Neuropsychopharmacology: The Fifth Generation of Progress,

eds Davis KL, Charney D, Coyle JT, Nemeroff C (Raven, New York), pp 819–832.32. Willins DL, Alsayegh L, Berry SA, Backstrom JR, Sanders-Bush E, Friedman L, Khan N,

Roth BL (1998) Ann NY Acad Sci 861:121–127.33. Gainetdinov RR, Bohn LM, Walker JK, Laporte SA, Macrae AD, Caron MG, Lefkowitz RJ,

Premont RT (1999) Neuron 24:1029–1036.34. Askwith CC, Wemmie JA, Price MP, Rokhlina T, Welsh MJ (2004) J Biol Chem 279:18296–

18305.35. Mu Y, Otsuka T, Horton AC, Scott DB, Ehlers MD (2003) Neuron 40:581–594.36. Bohn LM, Dykstra LA, Lefkowitz RJ, Caron MG, Barak LS (2004) Mol Pharmacol

66:106–112.37. Groer CE, Tidgewell K, Moyer RA, Harding WW, Rothman RB, Prisinzano TE, Bohn LM

(2007) Mol Pharmacol 71:549–557.

1084 � www.pnas.org�cgi�doi�10.1073�pnas.0708862105 Schmid et al.