# Modifying Ligand-Induced and Constitutive Signaling of the Human 5-HT4 Receptor

Wei Chun Chang, Jennifer K Ng, Trieu Nguyen, Lucie P. Pellissier, Sylvie

Claeysen, Edward C Hsiao, Bruce R Conklin

► To cite this version:

Wei Chun Chang, Jennifer K Ng, Trieu Nguyen, Lucie P. Pellissier, Sylvie Claeysen, et al. Modifying Ligand-Induced and Constitutive Signaling of the Human 5-HT4 Receptor. PLoS ONE, 2007, 2 (12), pp.e1317. 10.1371/journal.pone.0001317. [hal-02483770](https://hal.science/hal-02483770v1)

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# Modifying Ligand-Induced and Constitutive Signaling of the Human 5-HT₄ Receptor

Wei Chun Chang¹,⁴*, Jennifer K. Ng¹, Trieu Nguyen¹, Lucie Pellissier²,³, Sylvie Claeysen²,³, Edward C. Hsiao¹,⁵, Bruce R. Conklin¹,⁵,⁶*

1 Gladstone Institute of Cardiovascular Disease, University of California at San Francisco, San Francisco, California, United States of America, 2 Institut de Génomique Fonctionnelle, Universités de Montpellier, CNRS UMR 5203, Montpellier, France, 3 INSERM U661, Montpellier, France, 4 Graduate Program in Pharmaceutical Sciences and Pharmacogenomics, University of California at San Francisco, San Francisco, California, United States of America, 5 Department of Medicine, University of California at San Francisco, San Francisco, California, United States of America, 6 Department of Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, California, United States of America

G protein-coupled receptors (GPCRs) signal through a limited number of G-protein pathways and play crucial roles in many biological processes. Studies of their *in vivo* functions have been hampered by the molecular and functional diversity of GPCRs and the paucity of ligands with specific signaling effects. To better compare the effects of activating different G-protein signaling pathways through ligand-induced or constitutive signaling, we developed a new series of RASSLs (receptors activated solely by synthetic ligands) that activate different G-protein signaling pathways. These RASSLs are based on the human 5-HT₄b receptor, a GPCR with high constitutive G~s~ signaling and strong ligand-induced G-protein activation of the G~s~ and G~s/q~ pathways. The first receptor in this series, 5-HT₄-D¹⁰⁰A or Rs1 (RASSL serotonin 1), is not activated by its endogenous agonist, serotonin, but is selectively activated by the small synthetic molecules GR113808, GR125487, and RO110-0235. All agonists potently induced G~s~ signaling, but only a few (e.g., zacopride) also induced signaling via the G~q~ pathway. Zacopride-induced G~q~ signaling was enhanced by replacing the C-terminus of Rs1 with the C-terminus of the human 5-HT₂C receptor. Additional point mutations (D⁶⁶A and D⁶⁶N) blocked constitutive G~s~ signaling and lowered ligand-induced G~q~ signaling. Replacing the third intracellular loop of Rs1 with that of human 5-HT₁A conferred ligand-mediated G~i~ signaling. This G~i~-coupled RASSL, Rs1.3, exhibited no measurable signaling to the G~s~ or G~q~ pathway. These findings show that the signaling repertoire of Rs1 can be expanded and controlled by receptor engineering and drug selection.

Citation: Chang WC, Ng JK, Nguyen T, Pellissier L, Claeysen S, et al (2007) Modifying Ligand-Induced and Constitutive Signaling of the Human 5-HT₄ Receptor. PLoS ONE 2(12): e1317. doi:10.1371/journal.pone.0001317

## INTRODUCTION

Heptahelical G protein-coupled receptors (GPCRs) are the largest family of human cell-surface receptors, encompassing more than 340 hormone receptors and 350-460 olfactory receptors [1,2]. They are activated by peptide hormones, odorants, photons, biogenic amines, phospholipids, and many other extracellular signals. Upon activation, GPCRs undergo conformational changes that allow active and reversible signaling through a limited number of G-protein pathways (G~s~, G~i~, G~q~, G~i2/13~). These signals mediate a wide variety of physiological responses, including heart rate, chemotaxis, cell proliferation, neurotransmission, and hormonal responses. Owing to their physiological importance, GPCRs are of great medical interest. Indeed, they are targets for at least 40% of modern pharmaceuticals [3].

Although many drugs target GPCRs, studies of GPCR signaling *in vivo* have been hampered by the lack of specific agonists and antagonists for many of the receptors. GPCRs display molecular and functional diversity, such as the type of G-protein signaling pathway associations, different levels of constitutive activity, and different signaling responses due to different ligand-selective conformations [4,5]. This diversity enables the receptors to transmit unique extracellular signals but hampers efforts to sort out the relative contributions of each signaling pathway or the roles of constitutive signaling for each receptor.

To better study the diversity of GPCRs, we developed receptors activated solely by synthetic ligands (RASSLs) by modifying their structures to render them unresponsive to endogenous hormones. Instead, RASSLs are activated by small-molecule drugs [6], allowing them to be used to activate specific G-protein pathways rapidly and reversibly and to mimic the speed, localization, regulation, and amplification of endogenous GPCR signals [7].

Since it is impractical to convert all GPCRs into RASSLs, we and others have focused on representative G~s~, G~i~, and G~q~-coupled GPCRs, which stimulate adenylyl cyclase, inhibit adenylyl cyclase, and stimulate phospholipase-C, respectively. Ro1 (RASSL opioid 1), the prototype RASSL based on a G~i~-coupled κ-opioid receptor [8], provided a proof of concept for this strategy. Its G~i~ response to natural ligands is 0.001% of that of the wildtype receptor, but it is potently activated by the synthetic agonist spiradoline [8-10]. Ro1 decreases heart rate in mice [11] and affects taste sensation in the tongue [9]. In addition to serving as powerful tools to dissect the G-protein signaling *in vivo*, RASSLs can yield insights into fundamental aspects of receptor diversity [12]. For instance, constitutive signaling of Ro1 led to cardiomy-

Academic Editor: Hany El-Shemy, Cairo University, Egypt

Received July 27, 2007; Accepted November 18, 2007; Published December 19, 2007

Copyright: © 2007 Chang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This study was supported by NIH grant HL-60664 and American Heart Association Predoctoral Fellowship 0415005Y (to WCC), NIH Fellowship Training Grant 2T32DK07418-26 (to ECH), J David Gladstone CIRM Fellowship (to ECH), and Kirschstein NRSA Fellowship F32A624044-02 (to JKN).

Competing Interests: The authors have declared that no competing interests exist.

* To whom correspondence should be addressed. E-mail: bconklin@gladstone.ucsf.edu

a Current address: Lexicon Pharmaceuticals, The Woodlands, Texas, United States of America

<figure><img src="image_2.png" /></figure>
opathy [11], diminished bone formation [13], and induced
hydrocephalus [10]. These constitutive signaling phenotypes
would have been difficult or impossible to identify by studying
endogenous receptors.

Multiple RASSLs have since been made, including a G<sub>s</sub>-coupled RASSL based on the melanocortin-4 receptor [14], a G<sub>q</sub>-coupled RASSL based on the histamine 1 receptor [15], and a series of RASSLS based on muscarinic receptors [16]. These RASSLs are useful tools; however, it is still advantageous to derive a series of RASSLs with distinct G-protein signaling from the same parental GPCR. It can be difficult to compare the effects of RASSLs based on different parental GPCRs since these RASSLs could have different pharmacokinetics, constitutive activity, desensitization kinetics, and cellular localization.

To better study GPCRs, we built a series of RASSLs based on
the human 5-HT₄ receptor (Figure 1), which has several
advantages over other serotonin receptors. First, its pharmacolog-
ical properties are well established [17]. Second, its agonists have
milder effects (increased gastrokinesis [18], augmented memory
acquisition and retention [19], increased chronotropic and
inotropic cardiostimulation [20], and enhanced cortisol release
[21]) than other serotonergic drugs. Third, it has a large number

of synthetic ligands, which allows us to identify differences in their
effects on that receptor. Fourth, a single mutation (D<sup>100</sup>A) in the
mouse 5-HT<sub>4</sub> receptor dramatically reduced its affinity for
serotonin, its endogenous ligand. This mutation also allows
synthetic agonists and antagonists for the wildtype receptor to
activate the mutant, turning 5-HT<sub>4</sub>-D<sup>100</sup>A into a RASSL [22].

Finally, we reasoned that novel receptors coupling to other
signaling pathways could be created by making chimeras of the
5-HT₄ receptor with other family members. Altering the G-
protein selectivity of GPCRs is often difficult because it is based
on receptor conformation determined by multiple regions of the
receptor [23]. Changing multiple regions involves large internal
mutations that often lead to receptor instability. A better strategy
for altering G-protein signaling characteristics is to swap domains
between structurally similar receptors within the same family.
The 5-HT₄ receptor belongs to a family of at least 15 receptors,
each with different subfamilies that engage different G-protein
signaling pathways. The 5-HT₄, 5-HT₆, and 5-HT₇ subfamilies
are G<sub>s</sub> coupled, the 5-HT₁ subfamily is G<sub>i</sub> coupled, and the 5-
HT₂ subfamily is G<sub>q</sub> coupled [24]. These characteristics could
expedite our efforts to make purely G<sub>s</sub>-, G<sub>i</sub>-, and G<sub>q</sub>-coupled
RASSLs.

<figure><img src="image_3.png" /></figure>

**Figure 1. Human 5-HT₄-based RASSLs.** A signal peptide and the FLAG epitope were added to the N-terminus of the human 5-HT₄ receptor. A D<sup>100</sup>A mutation was introduced by site-directed mutagenesis to create the G<sub>s</sub>-coupled RASSL (Rs1). An additional point mutation (D<sup>66</sup>A, D<sup>66</sup>N, or W<sup>272</sup>A) was added to Rs1 to modulate constitutive signaling. Rs1.1 is the Rs1-C-5-HT<sub>2C</sub> chimera with enhanced G<sub>q</sub> signaling. Rs1.2 is Rs1 with an extra D<sup>66</sup>A mutation that decreased constitutive G<sub>s</sub> signaling. Rs1.3 is the Rs1-i3-5-HT<sub>1A</sub> chimera with G<sub>i</sub> signaling. The junctions in Rs1.1 and Rs1.3 indicate ends of domain swapping where the 5-HT<sub>4</sub> has been replaced by 5-HT<sub>2C</sub> or 5-HT<sub>1A</sub>, respectively.
doi:10.1371/journal.pone.0001317.g001

<figure><img src="image_4.png" /></figure>
Here, we describe a new series of RASSLs developed to modify the ligand-induced and constitutive signaling of the human 5-HT₄ receptor. These modified GPCRs will help us better study the effect of constitutive Gs signaling and ligand-induced Gs, Gs/Gq, and Gi signaling *in vivo*.

## RESULTS

### Human 5-HT₄ D¹⁰⁰A is a Gs-coupled RASSL

The D¹⁰⁰A mutation in the mouse 5-HT₄ receptor converts it into a RASSL [22], but its effects on the human 5-HT₄ receptor have not been tested. We now extend these findings to the human 5-HT₄ receptor (Figure 1). To determine if antagonists for the wildtype 5-HT₄ receptor also activate the human 5-HT₄-D¹⁰⁰A mutant, we tested a variety of compounds. The mutant receptor was not activated by serotonin (Figures 2, 3A), but it was activated by agonists (cisapride, zacopride), partial agonists (RS23597, RS39604, RS67333), antagonists (GR113808, RO110-0235), and an inverse agonist (GR125487) for the wildtype 5-HT₄ receptor (Figure 2). Interestingly, GR125487 showed a specific response, as demonstrated by the steep concentration-response curve (Figure 3D). In addition, GR113808, GR125487, and RO110-0235 potently activated Gs signaling of 5-HT₄-D¹⁰⁰A without stimulating the wildtype receptor (Figures 3B-E). The mutant receptor was selectively activated by multiple synthetic ligands (GR113808, GR125487, and RO110-0235) but not serotonin. We named it Rs1 (RASSL serotonin 1) (Figure 1).

### Rs1 has a high level of constitutive signaling

We next examined the constitutive signaling of Rs1 in more detail. Rs1 showed greater constitutive signaling than the wildtype receptor at all levels of transfection (Figure 4A). Constitutive activity was observable when only 25 ng of receptor cDNA per 5×10⁶ HEK293 cells was transfected (Figure 2). The highest level of constitutive activity, achieved with 5.4 µg of receptor cDNA per 5×10⁶ HEK293 cells, was 1.5 times greater than that of the wildtype 5-HT₄ receptor (49.6±1.25 nM vs. 32.5±4.04 nM, p<0.005) and >10-fold higher than that of the control receptors (the β₂-adrenergic and parathyroid hormone receptors), which have low levels of constitutive signaling (Figure 4A). Despite the high level of constitutive signaling, both the 5-HT₄ receptor and Rs1 could still be further activated by zacopride (Figure 4B).

### Ligand-specific Gq signaling in Rs1

Before constructing Rs1-5HT₂C chimeras to make a Gq-signaling RASSL, we assayed inositol phosphate 1 (IP1) accumulation by Rs1 via constitutive or ligand-induced signaling. Rs1 showed no measurable difference in constitutive Gq signaling (Figure 5) as compared to mock-transfected cells in the IP1 and calcium mobilization assays. Upon activation by cisapride, zacopride, RS23597, RS39604, or RS67333, Rs1 showed 2-3.5-fold higher Gq signaling than the wildtype 5-HT₄ receptor (p<0.005) (Figure 5). Surprisingly, GR113808, GR125487, and RO110-0235 activated predominately Gs (Figure 2) and little Gq signaling (Figure 5) by Rs1. These are the same ligands used to selectively

<figure><img src="image_5.png" /></figure>

**Figure 2.** Rs1 is a Gs-coupled RASSL. Rs1 was efficiently activated by small compounds known to be full agonists (cisapride, zacopride), partial agonists (RS39604, RS67333, and RS23597), antagonists (GR113808, RO110-0235), or inverse agonists (GR125487) for the wildtype 5-HT₄ receptor. It was not activated by its endogenous agonist (serotonin). Values are mean±SD of three independent experiments in which 25 ng of 5-HT₄ or Rs1 receptor cDNA was electroporated into 5×10⁶ cells. DMSO, dimethyl sulfoxide. doi:10.1371/journal.pone.0001317.g002

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<table><thead><tr><th>Receptor</th><th>Treatment</th><th>EC50 (nM)</th><th>S.E. (nM)</th></tr></thead><tbody><tr><td rowspan="5">5-HT<sub>4</sub></td><td>5HT</td><td>1.1</td><td>0.02</td></tr><tr><td>Zacopride</td><td>32.9</td><td>0.33</td></tr><tr><td>GR113808</td><td>*</td><td></td></tr><tr><td>GR125487</td><td>*</td><td></td></tr><tr><td>RO110-0235</td><td>*</td><td></td></tr><tr><td rowspan="4">Rs1</td><td>5HT</td><td>*</td><td></td></tr><tr><td>Zacopride</td><td>68.2</td><td>1.77</td></tr><tr><td>GR113808</td><td>13</td><td>0.62</td></tr><tr><td>GR125487</td><td>5.7</td><td>0.52</td></tr><tr><td></td><td>RO110-0235</td><td>15.1</td><td>0.22</td></tr></tbody></table>

* = Does not activate

Figure 3. Rs1 activation occurs in the nanomolar range. (A–E) Rs1 transfectants were stimulated with increasing amounts of drugs. The D<sup>100</sup>A mutation in Rs1 makes the receptor insensitive to serotonin. It was efficiently activated by GR113808, GR125487, and RO110-0235, which do not activate the wildtype 5-HT<sub>4</sub> receptor. Values are mean±standard deviation of three independent experiments in which 25 ng of 5-HT<sub>4</sub> or Rs1 receptor cDNA was electroporated into 5×10<sup>6</sup> HEK293 cells. (F) Best-fit estimate of the half-maximal effective concentration (EC50). Values are mean±SEM of three independent experiments.

doi:10.1371/journal.pone.0001317.g003

<figure><img src="image_12.png" /></figure>
A.

<figure><img src="image_13.png" /><figcaption>**Figure 4.** Rs1 has significant constitutive activity. (A) Rs1 and the wildtype 5-HT₄ receptor both had much higher constitutive activity than the β2-adrenergic receptor (β₂-ADR) and parathyroid hormone receptor (PTH). \***p<0.005, and \* p<0.05 vs. mock-transfected cells (t test). (B) Rs1 was still activated when 1.5 µg of receptor cDNA was electroporated into 5×10⁶ HEK293 cells. Rs1 only showed a 197% increase over constitutive signaling. 5-HT₄ and Rs1 were stimulated with 1 µM of zacopride. β₂-ADR and PTH were stimulated with 1 µM of isoproterenol and human PTH (1-34) peptide, respectively. Activation of any of the four receptors resulted in significantly higher cAMP over the PBS mock treated samples. \***p<0.005 (t test). All experiments were repeated three times. Values are mean±SD.
doi:10.1371/journal.pone.0001317.g004</figcaption></figure>

activate the Gs signaling of Rs1 without activating the Gs signaling of the wildtype 5-HT₄ receptor (Figure 2). Therefore, we could use drugs with distinct chemical structures (Figure S1) to activate Gs or Gs/Gq signaling of Rs1. The once controversial use of conformation-specific ligands to alter G-protein coupling and other receptor functions has now been demonstrated in several other GPCRs [25–29]. This is the first time that agonist-dependent functional selectivity has been shown in a RASSL.

## Replacing the C-terminus of Rs1 with 5-HT₂C increases Gq signaling

To make a purely Gq signaling RASSL from Rs1, we exchanged the intracellular loops of Rs1 with those of the Gq-coupled human 5-HT₂C receptor. By transferring domains at different junctions of intracellular loops, we made 12 different Rs1-5-HT₂C chimeras

(Figures S2, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17). To characterize them, we used RS23597 because it activated Gq signaling of Rs1 but not the wildtype 5-HT₄ receptor, as measured by calcium mobilization assays (Figure S3C).

Replacing the second (i2) or third intracellular loop (i3) of Rs1 eliminated both Gs and Gq signaling (Figure S3). Only the carboxyl chimera (Rs1-C-5-HT₂C) showed enhanced Gq signaling in response to cisapride, zacopride, RS23597, RS39604, and RS67333 (Figure 5), showing that the ligand-induced specificity of signaling was preserved. Constitutive and ligand-induced Gs signaling were also largely preserved (Figures S3A, S3B).

These data indicated that i2 and i3 are both necessary for Gs-coupling of the 5-HT₄ receptor. They also suggested that the C-terminus of 5-HT₂C contains a G-protein coupling domain for Gq signaling or that the C-terminus of 5-HT₄ receptor promotes Gq signaling. However, we were unable to completely alter the G-protein preference of this receptor for Gq signaling. We amplified the Gq signaling of Rs1 by domain swapping the C-terminus with 5-HT₂C. None of the other 12 chimeras showed enhanced Gq signaling, even when multiple internal segments were combined. We did not proceed further with these experiments because substitutions of multiple internal domains also decreased cell-surface expression of the receptors (data not shown). Since Gq signaling of Rs1-C-5-HT₂C was activated by cisapride, zacopride, and RS23597 but not by serotonin, we named it Rs1.1 (Table 1).

Moreover, when Rs1-C-5-HT₂C was activated by cisapride, zacopride, RS23597, RS39604, or RS67333, signaling was 4.5–6.8-fold higher than constitutive signaling (p<0.005). Signaling increased 1.3-fold in response to GR125487 (p<0.05), 1.7-fold in response to GR113808 (p<0.05), and 2.2-fold in response to RO110-0235 (p<0.005). This suggests that the carboxylic tail may play a role in functional selectivity in 5-HT₄ or 5-HT₂C receptors (Figure S3).

## A purely Gs signaling RASSL with low levels of constitutive signaling

Since the high constitutive activity of the 5-HT₄-D¹⁰⁰A mutant causes significant phenotypes in transgenic mice [30] and could not be controlled by inverse agonists [31], we attempted to lower the Rs1 constitutive activity by making additional point mutations. We focused on the D⁶⁶N and W²⁷²A mutations, which reduce constitutive signaling of the mouse 5-HT₄ receptor [31,32]. Rs1-D⁶⁶A, Rs1-D⁶⁶N, and Rs1-W²⁷²A significantly reduced constitutive signaling (Figure 6A). The cell-surface expression of Rs1-D⁶⁶A and Rs1-D⁶⁶N was similar to that of Rs1 (Figure 6B), so the reduction in constitutive signaling was probably not linked to lower cell-surface expression. Surprisingly, the D⁶⁶A and D⁶⁶N mutations also abolished zacopride-induced Gq signaling (Figure 6C). Thus, we created two RASSLs exhibiting pure Gs signaling and low constitutive signaling. Unfortunately, the efficacy of the ligand-induced Gs response was also significantly compromised, diminishing the utility of these receptors.

## Engineering Rs1 for Gi Signaling

To engineer a Gi-signaling RASSL based on Rs1, we replaced its intracellular loops with those of 5-HT₁A, a Gi-signaling receptor [33]. Of four Rs1-5HT₁A chimeras (Figures 7, S4, S18, S19, S20, S21), only the two containing i2 and i3 from Rs1 were expressed at a level similar to that of Rs1 (Figure S5D). Replacing those loops abolished constitutive and ligand-induced Gs signaling at both low and high levels of receptor cDNA (25 ng and 4.8 µg per 5×10⁶ cells) (Figures S5A, S5B). Interestingly, this RASSL showed no evidence of constitutive signaling via the Gi or Gs pathway. These findings

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /><figcaption>**Figure 5.** The wildtype 5-HT<sub>4</sub> receptor, Rs1, and the Rs1-C-5-HT<sub>2C</sub> chimera exhibit different G<sub>q</sub> signaling properties. G<sub>q</sub> signaling was analyzed by measuring the accumulation of inositol 1 phosphate (IP1). The 5-HT<sub>4</sub> receptor showed increased G<sub>q</sub> signaling when activated by serotonin, cisapride, and zacopride but not by RS39604 or RS67333, relative to mock treatment with HBSS. Rs1 showed significantly higher G<sub>q</sub> signaling than the wildtype 5-HT<sub>4</sub> receptor when activated by cisapride, zacopride, RS39604, and RS67333 but not by serotonin. G<sub>q</sub> signaling of Rs1-C-5-HT<sub>2C</sub> chimera was activated by cisapride, zacopride, RS23597, RS39604, and RS67333 and was minimally activated by RO110-0235. GR113808 and GR125487 did not activate G<sub>q</sub> signaling by any of the three receptors. ***p<0.005, * p<0.05 vs. mock-transfected cells (t test). Values are ±SD of three experiments. doi:10.1371/journal.pone.0001317.g005</figcaption></figure>

strongly imply that both i2 and i3 are required for G<sub>s</sub> signaling of Rs1. In addition, activation of the Rs1-i3-5-HT<sub>1A</sub> chimera with zacopride significantly inhibited cAMP accumulation induced by 10 µM apomorphine (agonist for dopamine 1 receptors) in HEK293 cells co-transfected with 1.5 µg of Rs1 receptor and 0.5 µg of dopamine 1 receptor (per 5×10<sup>6</sup> cells; Figure S5C and Figure S8A). This inhibition was smaller than that of µ-opioid receptor stimulated by [D-Ala2, D-Leu5]-enkephalin (DADLE). Both responses were abolished by 50 nM pertussis toxin, indicating the involvement of G<sub>i</sub> signaling (Figure 7B). Unfortunately, the potency (amount of drug needed to reach an effect) of ligand-induced G<sub>i</sub> signaling was significantly reduced (Figure 7C). While these results are encouraging, future experiments are needed to determine if low potency may be due to nonspecific effects, or will reproduce in other cell types. Since Rs1-i3-5-HT<sub>1A</sub> exhibited G<sub>i</sub> but not G<sub>s</sub> signaling, we named it Rs1.3 (Figure 1, Table 1).

## DISCUSSION

### A new series of RASSLs

We report here a new series of RASSLs to study multiple G-protein signaling pathways. Many GPCRs activate multiple G-protein signaling pathways and exhibit a wide range of constitutive signaling activities. Our new RASSLs will help us better study the effect of stimulating canonical signaling pathways (G<sub>s</sub>/G<sub>q</sub>, G<sub>s</sub>, G<sub>i</sub>), with different constitutive signaling using a single receptor

backbone and different synthetic agonists. These new RASSLs will also allow a systematic examination of the different functional domains of the 5-HT<sub>4B</sub> receptor.

These new RASSLs could help us better dissect the physiological significance of constitutive signaling *in vivo*. Constitutive

<table><caption>**Table 1.** Controlling the G-protein signaling of Rs1</caption><thead><tr><th rowspan="2">Receptors</th><th rowspan="2">Description</th><th rowspan="2">Constitutive signalling</th><th colspan="3">G-protein signaling</th></tr><tr><th>G<sub>s</sub></th><th>G<sub>i</sub></th><th>G<sub>s</sub></th><th>G<sub>q</sub></th></tr></thead><tbody><tr><td>5-HT<sub>4</sub></td><td>5-HT<sub>4</sub></td><td>++</td><td>-</td><td>+++</td><td>+</td></tr><tr><td>Rs1</td><td>5-HT<sub>4</sub>-D<sup>100</sup>A</td><td>+++</td><td>-</td><td>+++</td><td>+</td></tr><tr><td>Rs1.1</td><td>Rs1-C-5-HT<sub>2C</sub></td><td>+++</td><td>N/A</td><td>+++</td><td>++</td></tr><tr><td>Rs1.2</td><td>Rs1-D<sup>66</sup>A</td><td>+</td><td>N/A</td><td>+</td><td>-</td></tr><tr><td>Rs1.3</td><td>Rs1-i3-5-HT<sub>1A</sub></td><td>-</td><td>+</td><td>-</td><td>N/A</td></tr></tbody></table>

Constitutive signaling and ligand-induced signaling of Rs1 were successfully controlled by point mutations, drug choice, and domain swapping. G<sub>q</sub> signaling of Rs1 could be activated by zacopride or RS23597 but not GR113808, GR125487 or RO110-0235. The signaling was significantly increased by switching the carboxyl tail (Rs1.1). Attempts to decrease constitutive activity also decreased ligand-induced G<sub>s</sub> signaling and abolished G<sub>q</sub> signaling (Rs1.2). Replacing i3 of Rs1 with that of 5-HT<sub>1A</sub> resulted in a G<sub>i</sub> coupled RASSL with no G<sub>s/q</sub> signaling and G<sub>i</sub> signaling (Rs1.3). doi:10.1371/journal.pone.0001317.t001

<figure><img src="image_16.png" /></figure>
<figure><img src="image_17.png" /><figcaption>**Figure 6.** Abolishing the constitutive activity of Rs1 eliminates ligand-induced G~s~ and G~q~ signaling. (A) The D<sup>66</sup>A, D<sup>66</sup>N, and W<sup>272</sup>A mutations each decreased the constitutive signaling of Rs1, as shown by cAMP HTRF HiRange assay. (B) Cell-surface expression levels of Rs1-D<sup>66</sup>A, Rs1-D<sup>66</sup>N, and Rs1 were similar and higher than that of Rs1-W<sup>272</sup>A. \*\*\* p<0.05 vs. Rs1 (t test). (C) The D<sup>66</sup>A or D<sup>66</sup>N mutations also abolished G~q~ signaling of Rs1, as shown by HTRF IP1 assay.
doi:10.1371/journal.pone.0001317.g006</figcaption></figure>

signaling is crucial for many physiological processes and many
diseases. Up to 40% of all GPCRs [34], including the 5-HT₄
receptor [35], show significant constitutive activity, and Rs1 could
be a good model for these receptors. Like the mouse 5-HT₄-D¹⁰⁰A
receptor [22], Rs1 had a higher level of constitutive signaling than
the wildtype 5-HT₄ receptor. Using the tetracycline transactivator
(Tet) system, we have already made an Rs1 transgenic mouse in
which Rs1 expression is driven by the osteoblast-specific Col1α-1
2.3-kb promoter fragment. These mice exhibited dramatically
increased bone formation [30]. These and other findings strongly
suggest that constitutive signaling can drive potent phenotypic
changes *in vivo*. We found only constitutive G~s~ signaling in Rs1 but
did not observe any constitutive G~q~ signaling. It would be
interesting to generate new or modify existing RASSLs with
increased G~i~ or G~q~ constitutive signaling in the future to examine

the effect of both agonist-mediated and constitutive signaling in
various physiological processes. Since signaling would only be
dependent on expression rather than on circulating hormones,
these RASSLs have an intrinsic advantage for studying constitutive
signaling when combined with a Tet system.

In addition, the 5-HT₄ RASSLs have a large set of agonists with
different activities that could be useful in future studies. Many of the
Rs1 agonists used in this study activated Rs1 with an EC₅₀ in the
nanomolar range, allowing to us activate Rs1 effectively. Having an
abundant selection of ligands for Rs1 helped us find strong evidence
of ligand-induced functional selectivity on Rs1. Having a greater
variety of drugs to choose from could be valuable for *in vivo* studies.
For instance, we could use GR125487 to activate Rs1 while
suppressing the basal activity of wildtype 5-HT₄. Alternatively, we
could potentially study Rs1 constitutive signaling by using drugs such

<figure><img src="image_18.png" /></figure>
<figure><img src="image_19.png" /><figcaption>**Figure 7.** Replacing i3 of Rs1 with that of 5-HT<sub>1A</sub> results in a receptor, Rs1-i3-5-HT<sub>1A</sub>, with weak G<sub>i</sub> signaling. (A) Rs1-i3-5-HT<sub>1A</sub> chimera decreased cAMP accumulation. It also showed little constitutive G<sub>s</sub> signaling, in contrast to Rs1. All HEK293 transfectants were electroporated with 0.6 µg of human dopamine 1 receptor and 1.5 µg of Rs1, Rs1-i3-5-HT<sub>1A</sub> chimera, or human mu-opioid receptor. The transfectants were stimulated with 10 µM apomorphine (agonist for the dopamine 1 receptor) to increase basal cAMP level in order to observe G<sub>i</sub> signaling. Rs1 and Rs1-i3-5-HT<sub>1A</sub> were then stimulated with 10 µM zacopride. The mu-opioid receptor was stimulated with 10 µM DADLE. ***p<0.005, *p<0.05 vs. apomorphine (t test). (B) Treatment with pertussis toxin (PTX) abolished the decreased cAMP accumulation of Rs1-i3-5-HT<sub>1A</sub> and mu-opioid receptor, indicating that the decreased cAMP accumulation seen in panel (A) was due to G<sub>i</sub> signaling. The results are representative of three independent experiments. Values are mean±SD. (C) Rs1-i3-5-HT<sub>1A</sub> required a large amount of zacopride for maximal G<sub>i</sub> response. The data are representative of two independent experiments. ***p<0.005, *p<0.05 (t test), zacopride vs. no treatment.

doi:10.1371/journal.pone.0001317.g007</figcaption></figure>

as RO116-0086 or RO116-1148 [31] to decrease constitutive signaling of the wildtype 5-HT<sub>4</sub> receptor but not of Rs1.

Finally, this new series of RASSLs may make it possible to perform *in vivo* studies in which Rs1 is activated with minimal side effects. Since knockout of the 5-HT<sub>4</sub> receptor does not cause overt side effects [36], treatment with GR113808, GR125487, and R0110-0235 (antagonists and inverse agonists for the wildtype receptor) may have minimal side effects as well. This eliminates the need to knock out the endogenous 5-HT<sub>1A</sub> receptors for most *in vivo* experiments [10]. Since the same agonists can be used to activate all of the RASSLs within this series and therefore+ engage

different G-protein signaling pathways (Table 1), we can more easily compare the effect of activating the G<sub>s</sub>, G<sub>s/q</sub>, or G<sub>i</sub> pathway.

While these new RASSLs may be useful for studying G protein signaling *in vivo*, they have several limitations. Despite significant efforts to screen all available antagonists, we could not identify inverse agonists that would lower constitutive signaling by Rs1. Fortunately, we can use conditional expression systems such as the tetracycline transactivator system to control Rs1 expression and constitutive activation. Although we were successful in finding mutations that reduced Rs1 constitutive signaling these same mutations adversely affected the agonist mediated signaling (Rs1.2), suggesting that the

<figure><img src="image_20.png" /></figure>
D<sup>66</sup>N and W<sup>272</sup>A mutations are critical for all aspects of G<sub>s</sub> signaling. Other mutations could be explored that may provide a more optimal reduction in G<sub>s</sub> basal activity without affecting ligand-induced receptor activation. In addition, the relatively small G<sub>i</sub> signaling of Rs1.3 can only be activated by a relatively high concentration of zacopride, thus limiting its usefulness *in vivo*.

## Insights into G-protein signaling by Rs1

Our study yielded insights into the G-protein selectivity and functional selectivity (differential effects of ligands on the same receptor) of Rs1. Of the 12 Rs1-5-HT<sub>2C</sub> chimeras that are expressed on the cell surface, none of the i2 or i3 chimeras showed any G<sub>s</sub> or G<sub>q</sub> signaling. Evidently, these intracellular loops of Rs1 are crucial for signaling via those pathways. The importance of i2 and i3 for G<sub>s</sub> signaling of Rs1 is further supported by the lack of G<sub>s</sub> signaling by the Rs1-i2-5-HT<sub>1A</sub> and Rs1-i3-5-HT<sub>1A</sub> chimeras. This is the first study showing the importance of i2 and i3 in both G<sub>s</sub> and G<sub>q</sub> signaling of the human 5-HT<sub>4</sub>-D<sup>100</sup>A receptor.

We also found that i3 domain swapping abolished all G<sub>s</sub> signaling and enabled Rs1 to stimulate G<sub>i</sub> signaling of 5-HT<sub>1A</sub>. The role of i2 and i3 in the G<sub>i</sub> signaling of 5-HT<sub>1A</sub> receptor has been extensively reported. The entire N-terminus [37] and C-terminus of i2 [38] of 5-HT<sub>1A</sub> are thought to be sufficient to support G-protein coupling, but not signaling. On the other hand, the N-terminus [39] and C-terminus of i3 of 5-HT<sub>1A</sub> [40,41] seem to be essential for the G<sub>i</sub> signaling of 5-HT<sub>1A</sub>. In fact, replacing the N-terminus of the i3 of the α<sub>2</sub>-adrenergic receptor with that of 5-HT<sub>1A</sub> resulted in a chimera that signals like a 5-HT<sub>1A</sub> receptor when stimulated by a α<sub>2</sub>-adrenergic receptor agonist [42]. Since Rs1-5-HT<sub>1A</sub> chimeras with multiple internal domains replaced are not significantly expressed on the cell surface (data not shown), it may be difficult to further improve the potency of the Rs1.3 using our current approach. We hypothesize that replacing the N- and C-terminal portions of i2 and i3 instead of the whole i2 and i3 loops may increase the potency of Rs1.

We also showed that various drugs can differentially activate G-protein signaling of Rs1. Functional selectivity has been reported for many receptors. It led to divergent fates of internalization for the dopamine D<sub>1</sub> receptor [25], various binding specificities for gonadotropin-releasing hormone receptors, and different levels of activation of G proteins for the β<sub>2</sub>-adrenergic [26], mu-opioid [27], dopamine D<sub>2</sub> [28], and human 5-HT<sub>2A</sub> [29] receptors.

The indoleamine derivatives GR113808, GR125487, and RO110-0235 did not fully activate G<sub>q</sub> signaling of Rs1, Rs1.1, or the 5-HT<sub>4</sub> receptor. On the other hand, the benzamide derivatives cisapride, zacopride, RS23597, RS39604, and RS67333 activated the G<sub>q</sub> signaling of Rs1. These findings may reflect distinct conformational changes caused by indoleamine and benzamide derivatives.

The possibility of functional selectivity is further supported by the results obtained with Rs1-C-5-HT<sub>2C</sub> and Rs1 point mutants (Rs1-D<sup>66</sup>A and Rs1-D<sup>66</sup>N). The D<sup>100</sup>A mutation and replacement of the C-terminus amplified G<sub>q</sub> signaling by the 5-HT<sub>4</sub> receptor. The addition of D<sup>66</sup>A and D<sup>66</sup>N abolished G<sub>q</sub> signaling. Since D<sup>100</sup>A is located in the binding pocket of the 5-HT<sub>4</sub> receptor, this mutation in Rs1 may have changed the configuration of the binding pocket, making the receptor more susceptible to G<sub>q</sub> activation by zacopride and RS23597. This response was even more pronounced when the D<sup>100</sup>A mutation was combined with domain swapping of the C-terminus with that of 5-HT<sub>2C</sub>. Thus, it is reasonable to hypothesize that these changes modified the ligand-selective receptor conformation [5], changing the receptor susceptibility to functional selectivity.

## Conclusions

Our studies with Rs1 provide a proof-of-concept for making a series of RASSLs with different signaling properties. Recently, Armbruster *et al.* made a series of RASSLs based on the muscarinic M3 and M4 receptors, which have low constitutive activity. These RASSLs each couple different G-protein signaling pathways and can be activated by clozapine-N-oxide, an inert ligand with high bioavailability [16]. These RASSLs nicely complement our Rs1 RASSLs with varying constitutive activity. In addition, we predict that some RASSLs with the same canonical G-protein signaling (G<sub>s</sub>, G<sub>i</sub>, or G<sub>q</sub>) will have different *in vivo* phenotypes due to noncanonical signaling. This growing collection of RASSLs will greatly facilitate our efforts to understand the physiological significance of the inherent signaling diversity of GPCRs.

An ideal series of RASSLs would have receptors with different combinations of low and high basal signaling, with robust ligand-induced effects for each major pathway, and potent inverse agonists. Although we have not achieved this goal with the Rs1 series, we are hopeful that it can be achieved with other receptors in the future. Indeed, RASSLs based on the muscarinic receptors [16] show great promise, as there are naturally occurring, or published mutants of the muscarinic reports that activate each of the major G protein signaling pathways.

## MATERIALS AND METHODS

### Constructing human 5-HT<sub>4</sub> mutant cDNA and Rs1-5-HT<sub>1A</sub>, and Rs1-5-HT<sub>2C</sub> chimeras

The human 5-HT<sub>4</sub> receptor cDNA (a gift from Dr. Bryan Roth, University of North Carolina) was used in all experiments. To improve expression and allow detection of the receptor, we added a signal peptide from influenza hemagglutinin [43] and a FLAG epitope (DYKDDDDA) at the N-terminus. 5-HT<sub>4</sub> was then subcloned by PCR; the primers, ATCGATCGgcggccgcGTGAG-CAAGGGCGAGGAGCTGTTC and ATCGATCG gcggccgc-CTAAGTGTC ACTGGGCTGAGCAGCC, were inserted into the Not1 restriction site (gcggccgc) of the pUNIV-5-HT<sub>2C</sub>-INI plasmid to replace the 5-HT<sub>2C</sub>-INI (a gift from Dr. Bryan Roth) in frame with the signal peptide and the FLAG epitope. The receptor was then mutated (D<sup>100</sup>A) with a Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA) with primer GTCTT-GTTTCGGACATCTCTG**gcc**GT CCTGCTCACAACGGCAT-CG (Figures 1, S2). The following mutant sense primers were used: 5-HT<sub>4</sub>-D<sup>66</sup>A, TTCATTGTATCTCTTGCTTTTGCG**gca**CT-GCT GGTTCGGTGCTGGT-GATG; 5-HT<sub>4</sub>-D<sup>66</sup>N, TTCA-TTGTATCTCTTGCTTTTGCG**aac**C TGCTGGTTTCGGT-GCT-GGTGATG; and 5-HT<sub>4</sub>-W<sup>272</sup>A, GTTGCTTCTGCC-TCTGCT GGGCGCCA**gcc**TTTGTC-CCAATATTGTGG. The sense primer used to replace the carboxyl chimera for Rs1.1 (Table 1) was AGTTACTCTTCCgcgGCCGCGAATT-CAG TGGATCCACTAGTAAC. The Rs1-5-HT<sub>1A</sub> and Rs1-5-HT<sub>2C</sub> chimeras were made by PCR fusion. The mutations are indicated by boldface, lower-case letters.

### HEK293 maintenance and electroporation

Early-passage (≤20) HEK293 cells were maintained in high-glucose DMEM (Invitrogen, Carlsbad, CA) supplemented with sodium pyruvate (Invitrogen) and 10% Fetalplex (Gemini Bio-Products, West Sacramento, CA). Receptors were electroporated into HEK293 cells as described [44]. The electroporated cells were reconstituted into a suspension using DMEM with 10% heat-inactivated, dialyzed fetal bovine serum (Thermo-Fisher Scientific,

<figure><img src="image_21.png" /></figure>
Logan, UT). The transfection efficiency was monitored by flow cytometry, and the cell-surface expression of the receptor was determined by FLAG ELISA (enzyme-linked immunosorbent assay) the next day.

## Drugs

5-HT, isoproterenol, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma-Aldrich (St. Louis, MO). Cisapride, zacopride, GR113808, GR1254875, RS23597-190 HCl, RS39604 HCl, and RS67333 HCl were from Tocris (Bristol, UK). Human parathyroid hormone peptide (amino acids 1–34) was from Bachem Biosciences (King of Prussia, PA). RO110-0235 was generously donated by Renee Martin (Roche, Palo Alto, CA).

## Measuring cell-surface expression by FLAG ELISA

Cell-surface receptor expression was measured with a FLAG ELISA as described [45]. Cells seeded in poly-D-lysine-coated 96-well plates were fixed with 100 µl of 4% paraformaldehyde for 10 min at room temperature, washed, and stained with 100 µl of staining buffer (DMEM, 10% FBS, and 1 mM CaCl₂) containing anti-FLAG M1 antibody (1:1000; Sigma-Aldrich) for 1 h at 25°C. The samples were washed three times with wash buffer (PBS and 1 mM CaCl₂) and stained with 100 µl of staining buffer with rat anti-mouse IgG antibody conjugated with horseradish peroxidase (1:1000; Bio-Rad Laboratories, Hercules, CA). After 30 min, the samples were washed with wash buffer, placed on a rocker for 10 min, and washed again. This process was repeated two more times. Then, 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) liquid substrate (200 µl; Sigma-Aldrich) was added to the samples. After rocking for 30 min, 200 µl of the substrate was transferred to new 96-well plates, and optical density was measured with Victor 3 (PerkinElmer, Waltham, MA) at 405 nm. All samples contain three replicates, and all experiments were repeated at least three times.

## Measurement of cAMP production in intact cells

To improve assay consistency and minimize pipetting error in the 384-well plates, we modified the high-range HTRF assay (CisBio International, Bagnols-sur-Cèze, France) by seeding, stimulating, and lysing the cells in 96-well plates and using the lysate instead of live cells to determine cAMP production. The remainder of the analysis was performed according to the manufacturer's instructions

### Gᵢ Assay

Gᵢ signaling was examined in cells transfected with 1.5 µg of receptor cDNA, 0.6 µg of human dopamine 1 receptor cDNA, and pcDNA3 (up to 6 µg). The co-transfectants were stimulated first with 100 µl of KRBG buffer containing IBMX for 10 min at room temperature and then with 50 µl of PBS containing 10 µM apomorphine (agonist for the dopamine 1 receptor) and 10 µM zacopride for 10 min at 37°C. The cells were lysed in 50 µl of lysis buffer, and 5 µl of lysate was used in the HiRange HTRF assay.

## Fluorometric imaging plate reader assay to measure calcium mobilization

To measure calcium mobilization, 4.8 µg of receptor cDNA, 0.6 µg of DsRed plasmid, and 0.6 µg of human bombesin receptor cDNA were electroporated into $5×10^6$ HEK293 cells as above [44]. Hank's balanced salt solution (10 ml) with 20 mM HEPES, 0.25 mM probenecid acid (Sigma-Aldrich), and 2% pluronic acid (Sigma-

Aldrich) was added to each bottle of Calcium 4 (Molecular Devices, Sunnyvale, CA), and 100 µl of the resulting solution was added to each well for 1 h at 37°C before measurement. Assays were performed with a FLEX Station (Molecular Devices), with excitation of 485 nm, emission of 525 nm, and cut-off of 515 nm, as recommended by the manufacturer.

## Determination of IP1 production in intact cells

A modified version of the IP1 protocol was used (CisBio International). HEK293 cells were washed once with calcium-free PBS and dissociated from flasks with cell dissociation buffer (Invitrogen). Cells ($5×10^6$) were electroporated as described above. Then, $10^5$ cells were placed in DMEM supplemented with 10% decomplemented, dialyzed against FBS, and seeded onto 96-well plates coated with poly-D-lysine. The next day, the cells were stimulated with agonists in 50 µl of 1× stimulation buffer for 30 min at 37°C and lysed for 10 min with 9 µl of lysis/detection buffer. Then, 14 µl of lysate was added to 384-well plates and subjected to High-Range HTRF assay as described above, except that 3 µl of cAMP-d2 and anti-cAMP-cryptate solution were added to each well.

## Data analysis

cAMP and IP1 values were analyzed with GraphPad Prism 4 (GraphPad Software, San Diego, CA). Calcium mobilization results were analyzed with SoftMax Pro v5 (Molecular Devices). Statistical significance was determined with paired Student's *t* tests.

## SUPPORTING INFORMATION

**Figure S1** Chemical structures of the compounds used in the study. All the chemicals used in the experimental are shown. Found at: doi:10.1371/journal.pone.0001317.s001 (1.92 MB EPS)

**Figure S2** Rs1-5-HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment. Found at: doi:10.1371/journal.pone.0001317.s002 (12.17 MB EPS)

**Figure S3** The second and third intracellular loops (i2 and i3) of Rs1 are crucial for Gs and Gq signaling. (A, B) Rs1-5-HT2C chimeras with swaps of i2 and i3 could no longer process Gs signals, at either 25 ng or 4.8 µg of receptor cDNA per $5×10^6$ HEK293 cells. (C) Gq signaling of Rs1 was abolished when i2 and i3 of Rs1 were replaced with those of 5-HT2C. The Gq signaling was measured by calcium mobilization assay. (D) Only chimeras with a single domain swap were expressed on the cell surface. The results represent three independent experiments. All figures were representative of three independent experiments. Found at: doi:10.1371/journal.pone.0001317.s003 (2.03 MB EPS)

**Figure S4** Rs1-5-HT1A chimeras. All modifications were made on Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment. Found at: doi:10.1371/journal.pone.0001317.s004 (3.82 MB EPS)

**Figure S5** Replacing the second or third intracellular loop (i2 or i3) of Rs1 with 5-HT1A alters G protein signaling. (A, B) Rs1-5-HT1A chimeras with swaps of the second (i2) and third intracellular (i3) loops no longer signal via the Gs pathway, regardless of whether cells were transfected with 25 ng or 4.8 µg of receptor cDNA per $5×10^6$ HEK293 cells. This suggests that the second and third intracellular loops are crucial for acute Gs

<figure><img src="image_22.png" /></figure>
signaling of Rs1. ***p<0.001 vs. mock transfected (t test). (C)
Replacing i3 of Rs1 resulted in a Gi signaling receptor. All
HEK293 transfectants were electroporated with 0.6 µg of the
human dopamine 1 receptor and 1.5 µg of Rs1, Rs1-5HT1A
chimeras, or the mu-opioid receptor. Rs1 and Rs1-5HT1a
chimeras were treated with 10 µM apomorphine (an agonist for
dopamine 1 receptor) or with 10 µM apomorphine and 10 µM
zacopride. Transfectants with 0.6 µg of the human dopamine 1
receptor and 1.5 µg of the mu-opioid receptor served as positive
controls. 10 µM DADLE was used in place of zacopride to
stimulate mu-opioid receptors. ***p<0.005, *p<0.05 vs. apomor-
phine (t test). (D) Rs1, (i1, i2 and i30 were expressed on the cell
surface. Cell-surface expression and calcium mobilization of the
chimeras were examined at 4.8 µg of receptor DNA per 5×106
HEK293 cells. The results are representative of three independent
experiments. Values are mean±SD. ***p<0.001 vs. mock
transfected (t test).

Found at: doi:10.1371/journal.pone.0001317.s005 (1.67 MB EPS)

**Figure S6** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s006 (2.98 MB EPS)

**Figure S7** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s007 (2.96 MB EPS)

**Figure S8** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s008 (3.00 MB EPS)

**Figure S9** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s009 (2.99 MB EPS)

**Figure S10** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s010 (2.99 MB EPS)

**Figure S11** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s011 (2.99 MB EPS)

**Figure S12** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s012 (3.00 MB EPS)

**Figure S13** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s013 (2.99 MB EPS)

**Figure S14** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s014 (3.00 MB DOC)

**Figure S15** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s015 (2.99 MB EPS)

**Figure S16** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s016 (3.00 MB EPS)

**Figure S17** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s017 (2.96 MB EPS)

**Figure S18** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s018 (3.00 MB EPS)

**Figure S19** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s019 (2.96 MB DOC)

**Figure S20** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s020 (3.40 MB EPS)

**Figure S21** High-resolution representation of Rs1-5HT1A and Rs1-5HT2C chimeras. All modifications made to Rs1 (Figure 1). Red lines indicate the junctions of chimeras. The amino acids exchanged are shown by the amino acid alignment.

Found at: doi:10.1371/journal.pone.0001317.s021 (3.03 MB EPS)

## ACKNOWLEDGMENTS

We thank Kimberly Scearce-Levie and Renee Martin for insightful review of the manuscript, Bryan Roth for the human 5-HT<sub>4B</sub> and 5-HT<sub>2C</sub> receptors cDNA, Robert J. Lefkowitz for the human 5-HT<sub>1A</sub> receptor cDNA, Mark von Zastrow for the human β<sub>2</sub>-adrenergic receptor cDNA, human μ-opioid receptor cDNA, and DADLE, Renee Martin for RO110-0235 (Roche Pharmaceuticals Division, Palo Alto, CA), Mary Weglarz for manuscript preparation and Gary Howard and Stephen Ordway for editorial review.

## Author Contributions

Conceived and designed the experiments: BC WC JN TN LP SC EH.
Performed the experiments: WC. Analyzed the data: BC WC JN TN LP
SC EH. Contributed reagents/materials/analysis tools: BC. Wrote the
paper: WC.

<figure><img src="image_23.png" /></figure>
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