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Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

  • Yasuyuki Nakamura,

    Affiliation Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan

  • Jun Ishii,

    Affiliation Organization of Advanced Science and Technology, Kobe University, Kobe, Japan

  • Akihiko Kondo

    akondo@kobe-u.ac.jp

    Affiliation Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan

Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

  • Yasuyuki Nakamura, 
  • Jun Ishii, 
  • Akihiko Kondo
PLOS
x

Abstract

Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.

Introduction

The potentially large functional and physiological diversity of dimerization among G-protein-coupled receptors (GPCRs) has generated a great deal of excitement due to the opportunity for novel drug discovery [1], [2]. The findings of physiologically relevant GPCR dimers raise the prospect of developing new drugs against a wide range of diseases by focusing on the machinery of targeted dimers because ligand-induced conformational changes in GPCR dimers could affect ligand affinity and signaling function [3], [4]. Since the human genome encodes more than 800 GPCR genes [5], the possible combinations of physiologically significant GPCR heterodimers would be immeasurable. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood.

Techniques to observe the dimerization of GPCRs include atomic force microscopy, electrophoresis, co-immunoprecipitation, cross-linkage, and fluorescence and bioluminescence resonance energy transfer (FRET and BRET) [3], [4], [6]. The FRET and BRET approaches are especially helpful for in vivo analysis and therefore are widely used for the studies of dimerized GPCRs. However, although the FRET and BRET techniques permit the direct monitoring of GPCR dimerization, it might be difficult to use these techniques to achieve rapid and facile identification of dimerizable candidates among numerous GPCR combinations.

To overcome this limitation, here we established a specialized method to screen candidate heterodimer partners for target GPCRs based on the split-ubiquitin membrane yeast two-hybrid method. In addition, since our system is independent from the activation of mitogen-activated protein kinase (MAPK) signal, it permits not only the identification of heterodimer partners, but also the monitoring of ligand-induced conformational changes.

Results and Discussion

While the original split-ubiquitin system enables comprehensive screening of protein-protein interactions [7], it is intrinsically possible that the addition of ligand triggers activation of pheromone signaling via endogenous yeast heterotrimeric G-protein [8]. The activation of pheromone signaling in yeast induces cell cycle arrest in G1 phase and triggers global changes in transcription of mating-related genes [9]. The ligand-induced G1 arrest that is exposed as robust growth inhibition in yeast cells [10] might lead to an inadequate assessment of reporter gene activity. Therefore, we constructed a yeast deletion mutant lacking the STE20 or STE11 gene involved in the activation of the MAPK cascade by using NMY51 as the parental strain with an aim to enable screening of GPCR dimers with and without ligand (Fig. 1 and Table 1). Halo bioassays responding to α-factor pheromone showed the formations of a thin halo and no halos with 100 µg of α-factor in NMY61 (ste20Δ) and NMY62 (ste11Δ) yeast strains, respectively, revealing that ste11Δ allele provides more strict avoidance of signal-promoted growth arrest in the presence of ligand (Fig. 2A). For checking the expressions of reporter genes, the growth assays in the presence of ligand were carried out (Fig. 2B). While the strains harboring mock vectors pBT3-C and pPR3-C were used as negative controls, those harboring pCCW-Alg5 and pAI-Alg5 to express Alg5-NubI and Alg5-Cub-LexA-VP16 were used as positive controls. Alg5-NubI is a yeast membrane protein fused with a WT Nub tag. The NubI tag interacts spontaneously with any Cub tag-containing constructs [11], [12]. The deletion mutants (ste20Δ and ste11Δ) avoided the robust growth inhibition and therefore could allow the growth assays with ADE2 and HIS3 reporter genes even in the presence of ligand (Fig. 2B). We used the MAPK-defective NMY62 yeast strain for the following experiments.

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Figure 1. Schematic illustration of yeast pheromone signaling pathway and principle for GPCR dimerization assay based on split-ubiquitin system in yeast.

Agonistic ligand binding to the GPCR leads to the activation of heterotrimeric G-proteins, the mitogen-activated protein kinase (MAPK) cascade and a cyclin-dependent kinase inhibitor Far1p. Phosphorylated Far1p induces G1 cell-cycle arrest. The STE20 or STE11 gene located upstream of the MAPK cascade was disrupted in the NMY51 strain. In the split-ubiquitin yeast two-hybrid system, NubG will only efficiently interact with Cub when the proteins to which the two split tags are attached interact with each other, resulting in the formation of a NubG/Cub complex. This complex is recognized by ubiquitin-specific proteases (UBPs), which release the artificial transcription factor (LexA-VP16) from the Cub-containing construct. LexA-VP16 then enters the nucleus via diffusion and binds to the LexA-binding sites upstream of the reporter genes. In this study, the GPCRs are fused to the split-ubiquitin and are expressed in MAPK-defective mutant yeast strain of NMY51 to allow the monitoring of GPCR dimerizations and conformational changes responding to binding of ligand.

https://doi.org/10.1371/journal.pone.0066793.g001

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Figure 2. ste11Δ allele allowed more strict avoidance of signal-promoted growth arrest in the presence of ligand.

(A) Halo-bioassay (agonist-induced growth arrest assay) for STE20-, STE11- and STE2-gene-disrupted strains: NMY51 (WT); NMY61 (ste20Δ); NMY62 (ste11Δ); and NMY63 (ste11Δ ste2Δ). Each paper filter disk was spotted with the indicated amount of α-factor. (B) Growth assay of NMY51 (WT; a,b), NMY61 (ste20Δ; c,d) and NMY62 (ste11Δ; e,f) strains on SD –Leu, Trp, Ade and His dropout plates. Yeast strains harboring pBT3-C/pPR3-C or pCCW-Alg5/pAI-Alg5 respectively expressed Cub/NubG (negative control; a,c,e) or Alg5-Cub/Alg5-NubI (positive control; b,d,f). Each cell was spotted in serial 10-fold dilutions on selective agar plates with or without 5 µM of α-factor. NubI is a WT Nub tag and interacts spontaneously with Cub.

https://doi.org/10.1371/journal.pone.0066793.g002

To test the viability of split-ubiquitin–based reporter gene assays for detecting GPCR dimers, we first analyzed the homodimerization of endogenous yeast pheromone receptor (Ste2p) in the NMY62 yeast strain. The N-terminal moiety of split-ubiquitin with an I13G mutation (NubG) and the C-terminal ubiquitin moiety linked to an artificial transcription factor (Cub-LexA-VP16) [7] were respectively designed to genetically fuse to the C-termini of Ste2p receptors by using original pPR3-C (prey) and pBT3-C (bait) split-ubiquitin vectors (Table S2). Upon in vivo protein-protein interaction, the reconstituted ubiquitin leads to cleavage and release of LexA-VP16 by ubiquitin-specific proteases (UBPs) [7]; therefore, the dimerization of Ste2p should be detected via the transcription activation of the reporter genes (ADE2, HIS3, and lacZ) (Fig. 1 and Table 1). However, the cells coexpressing Ste2p-NubG and Ste2p-Cub-LexA-VP16 never grew on the adenine/histidine-deficient selectable media (Fig. S1A). Therefore, we replaced the weak CYC1 promoter of the original pBT3-C bait vector by comparatively strong PHO5, TPI1 and TDH3 promoters (PCYC1<PPHO5<PTPI1<PTDH3). As a result, the expression of Ste2p-Cub-LexA-VP16 by the TPI1 and TDH3 promoters prompted cell growth on the selection media when combined with the expression of Ste2p-NubG (Fig. S1B and C). Even though previous report expressed the Ste2p in relatively low expression manner [13], our result did not show evidence of dimerization for the Ste2p expressed under the control of the CYC1 promoter in the split-ubiquitin system. This is assumed that attached transcription factor might be not present at levels adequate to activate the response.

Then, we replaced the full-length Ste2p receptors with a truncated version that lacks the C-terminal tails (Ste2ΔC; amino acids 1–304) to adjust the distance between the C-termini of the receptors [13], providing an increased signal-to-noise (S/N) ratio (under the control of the TPI1 promoter; Fig. S2A and B). Furthermore, we replaced the TPI1 promoter with the CYC1 promoter of the bait vector again, which resulted in much lower background cell growth and a drastically improved S/N ratio (Fig. 3A and B). Since the truncation of the C-termini of Ste2p receptors had been reported to increase in the number of receptor sites [14], the greatly enriched receptors at the plasma membrane might have provided the drastic improvement of S/N ratio. These results indicate that the bait receptor is predominant for successful detection of the dimerized receptors. Thus, the consideration of the receptor’s expression manner is important to screen the GPCR dimer partners, because the leaky background cell growth often brings unanticipated candidates.

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Figure 3. Detection for dimerization of yeast truncated Ste2p lacking the carboxy-terminal tail (Ste2ΔC) receptor in NMY62 strain.

Growth and quantitative β-galactosidase activity of yeast cells expressing various combinations of Cub and NubG fusions. The control bait plasmid was pBT3-C mock vector (empty vector). The control prey plasmids were pPR3-C mock vector (empty vector) and pPR3-HXT1. (A) Growth assay without α-factor (left panels) and with 5 µM α-factor (right panels). Each cell was spotted in serial 10-fold dilutions on SD –Leu, Trp, Ade and His dropout plate. (B) Quantitative β-galactosidase assay. Error bars represent the standard deviations (n = 3).

https://doi.org/10.1371/journal.pone.0066793.g003

In the optimized system, Hxt1p never presented background cell growth in the dimerization assay with the Ste2ΔC receptor (Fig. 3A). Previously, Overton et al. had used the Hxt1p as the negative control for FRET analysis of Ste2p dimerization and confirmed the subcellular localization of it at the plasma membrane [15]. The β-galactosidase assay that reflects lacZ reporter enzyme activity also displayed similar trends (Fig. 3B). The addition of ligand had no effect on the dimerization events of Ste2ΔC, since the MAPK-defective NMY62 yeast strain displayed unchanged growth in the presence of α-factor (Fig. 3A). We additionally constructed two types of deletion mutants in which the TM6–7 domains and the TM1–5 domains were removed, respectively (TM1–5 (amino acids 1–236) and TM6–7 (amino acids 237–304)) (Fig. 4A). Overton et al. also indicated that self-association of TM6–7 had not been detected, and plasma membrane localization of the YFP-tagged TM1–5 and TM6–7 was observed [16]. As previously indicated [16], only TM1–5 formed the homodimer (Fig. 4B and C), showing that our system is applicable to examine critical domains involved in the dimerization of 7TM receptors.

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Figure 4. Detection for dimerization of yeast Ste2p deletion mutants (TM1–5 and TM6–7) in NMY62 strain.

(A) Schematic of Ste2ΔC and the deletion mutants. Transmembrane (TM) domains are indicated with pillar-type boxes, and the Cub (Cub-LexA-VP16) or NubG (Nub with I13G mutation) is depicted as a rounded rectangle. (B) Growth assay without α-factor (left panels) and with 5 µM α-factor (right panels). Each cell was spotted in serial 10-fold dilutions on SD –Leu, Trp, Ade and His dropout plate. (C) Quantitative β-galactosidase activity in yeast cells containing various combinations of plasmids. Error bars represent the standard deviations (n = 3). The control prey plasmids were pPR3-C mock vector (empty vector) and pPR3-HXT1.

https://doi.org/10.1371/journal.pone.0066793.g004

Subsequently, we validated the capability of our system to detect human GPCR heterodimer pairs. To avoid competitive dimerization with the endogenous yeast Ste2p receptor, we constructed an NMY63 mutant strain in which the STE2 gene was additionally deleted (Table 1 and Fig. S3). Since clear evidence for the functional role of GPCR homodimer and heterodimer pairs was first obtained for class C receptors, such as GABAB receptors [17], we tested the dimerization of GABAB2 receptor (GABBR2) with GABAB1a receptor (GABBR1a). The β-galactosidase assay clearly showed the specific activities both for GABAB2/GABAB2 and GABAB2/GABAB1a couples (Fig. 5A and Fig. S4A). This result was coincident with the fact that GABAB2 receptor could form not only heterodimer with GABAB1a receptor but also homodimer [18], [19], indicating that the split-ubiquitin-based approach could detect the homodimerization and heterodimerization of GABAB2 receptors. Additionally, the β-galactosidase assay for dimerization of GABAB1a receptor also showed similar results (Fig. S5).

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Figure 5. Dimerization assays of human GPCRs.

Quantitative β-galactosidase assay. (A) Detection of GABAB1a/GABAB2 (GABBR1a/GABBR2) heterodimers. (B) Detection of AT1/AT2 (AGTR1/AGTR2) heterodimers. Error bars represent the standard deviations (n = 3). The control prey plasmid was pPR3-C mock vector.

https://doi.org/10.1371/journal.pone.0066793.g005

In contrast to the widely accepted concept of class C GPCR dimerization, the significance of in vivo dimerization of class A GPCRs remains controversial [20]. Since a growing amount of evidence indicates that class A GPCRs are able to form dimers or higher-ordered oligomers in vivo [21], [22], we next evaluated class A GPCR heterodimer pairs. As class A GPCRs, AT1 and AT2 angiotensin receptors (AGTR1 and AGTR2) were selected. Consistent with previous reports [23], [24], the β-galactosidase assay also illustrated the formation of heterodimers between AGTR1 and AGTR2 (Fig. 5B and Fig. S4B).

Next, we aimed to apply our system to screen new candidate heterodimer partners of AGTR1 receptor within the class A GPCRs, except for AGTR2. The CYC1 promoter was selected for expressing AGTR1 as a bait protein (Fig. S4B). As a model candidate library, we constructed the prey vectors to express AGTR1 (as a positive control), β2-adrenergic receptor (ADRB2), 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A), somatostatin receptor 2 (SSTR2), somatostatin receptor 5 (SSTR5), endothelin receptor type B (EDNRB), neurotensin receptor 1 (NTSR1) and neurotensin receptor 2 (NTSR2) (Table S2 and S3) and then mixed equal amounts of these 9 prey vectors (containing pPR3-C mock vector). After introduction of the constructed library into the NMY63 yeast strains harboring AGTR1 bait vector, the selection with ADE2/HIS3 growth reporter genes was performed (Fig. 6A). A total of 30 colonies was generated on the adenine/histidine-deficient selection media (Fig. 6A). Following isolation of prey plasmids from each colony, the obtained GPCR clones were determined by sequencing analysis. Ten clones of AGTR1 were dominantly identified as the homodimer (33.3%), whereas 5 clones of SSTR2 (16.7%), 3 clones of ADRB2 (10.0%) and 3 clones of HTR1A (10.0%) were successfully screened as the candidate heterodimer partners for AGTR1.

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Figure 6. Screening of candidate heterodimer partners of AT1 angiotensin receptor (AGTR1).

(A) Workflow of a yeast two-hybrid screen. Prey library was transformed into the NMY63 yeast strains harboring AGTR1 bait vector, and the selection with growth reporter genes was performed. Following isolation of prey plasmids from each colony, the obtained GPCR clones were determined by sequencing analysis. (B) Quantitative β-galactosidase assays for homo- and hetero-dimerization of AGTR1 in NMY63 strain. NMY63 yeast strain was transformed with GPCR-NubG indicated at the left and AGTR1-Cub-LexA-VP16. The control prey plasmid was pPR3-C mock vector. Error bars represent the standard deviations (n = 3).

https://doi.org/10.1371/journal.pone.0066793.g006

To validate the success or failure of the screening, we measured the β-galactosidase activities of the yeast cells separately co-transformed with the AGTR1 bait vector and 9 other prey vectors including the previously reported AGTR1/ADRB2 heterodimer pairs [25], yeast Ste2p control receptor and mock control. The results likely reflected the occupancies of identified clones, indicating that our system succeeded in screening heterodimer candidates (Fig. 6B). Additionally, β-galactosidase activities measured with other GPCRs as bait proteins were fairly consistent with the results of the screening and also revealed new candidates for heterodimer pairs including SSTR2/HTR1A, SSTR2/ADRB2, and HTR1A/EDNRB (Fig. 7A–C and Fig. S4C–E). Our experiments indicated that Ste2p could not co-oligomerize with the human GPCRs (Fig. 6B and Fig. 7A–C).

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Figure 7. Quantitative β-galactosidase assays for homo- and hetero-dimerization between human-GPCRs in NMY63 strain.

NMY63 yeast strain was transformed with GPCR-NubG indicated at the left and SSTR2-Cub-LexA-VP16 (A), ADRB2-Cub-LexA-VP16 (B), or HTR1A-Cub-LexA-VP16 (C). The control prey plasmid was pPR3-C mock vector. Error bars represent the standard deviations (n = 3).

https://doi.org/10.1371/journal.pone.0066793.g007

Additionally, we measured the β-galactosidase activities of the yeast cells separately co-transformed with the AGTR1 bait vector and GABBR1a, GABBR2, MT1 and MT2 melatonin receptor (MTNR1A and MTNR1B) prey vectors. The results indicated new candidates for heterodimer pairs including AGTR1/GABBR1a and AGTR1/MTNR1B (Fig. 8A). Thus, the obtained results from all heterodimerization assays with the split-ubiquitin system might have implicated a general statement about the ability of various human GPCRs to heterooligomerize with each other.

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Figure 8. Detection for dimerization and ligand-induced conformational changes of human GPCRs.

(A) Quantitative β-galactosidase assays for heterodimerization of AGTR1 in NMY63 strain. NMY63 yeast strain was transformed with GPCR-NubG indicated at the bottom and AGTR1-Cub-LexA-VP16. (B–D) Ligand assays for detection of conformational changes in GPCR dimerizations. (B) AT1/AT2 (AGTR1/AGTR2) heterodimers. Incubation time, 18 h. Angiotensin II conc., 0 or 10 µM. (C) MT1/MT2 (MTNR1A/MTNR1B) heterodimers. Incubation time, 18 h. Melatonin conc., 0 or 10 µM. (D) GABAB1a/GABAB2 (GABBR1a/GABBR2) heterodimers. Incubation time, 18 h. GABA conc., 0 or 100 µM. The control prey plasmid was pPR3-C mock vector. Error bars represent the standard deviations (n = 3). (*P<0.05).

https://doi.org/10.1371/journal.pone.0066793.g008

Finally, we performed detection of not only the dimer formation of target human GPCRs but also the ligand-mediated conformational changes in living yeast cells. In the case of AGTR1 the addition of 10 µM of native ligand, angiotensin II, did not affect the states of the homodimerized and heterodimerized receptors with AGTR2 (Fig. 8B). MT1 and MT2 melatonin receptors (MTNR1A and MTNR1B) not only form heterodimers, but also induce a conformational change within the heterodimers [4]. In addition, it has been reported that expressions of MTNR1A and MTNR1B in yeast activated the pheromone signaling pathway via the endogenous yeast G-proteins in response to the native ligand melatonin [26], [27]. β-galactosidase assays based on the split-ubiquitin technique in the MAPK-defective NMY63 yeast strain allowed successful detection of the conformational change of MTNR1A/MTNR1B heterodimers in the presence of melatonin (Fig. 8C and Fig. S4F), suggesting that our system can detect ligand-mediated conformational changes as well as the heterodimer formations. Moreover, we also tested the detection of the conformational change of GABAB2/GABAB1a heterodimers in the presence of GABA (Fig. 8D). While the addition of 100 µM of GABA did not affect the states of the GABAB2 homodimers, the β-galactosidase assay exposed the conformational change of GABAB2/GABAB1a heterodimers in consistency with previous reports [28] (Fig. 8D).

Because a positive result in the assay potentially could come about through indirect association of receptors via a third protein or close co-localization, it is important to re-evaluate the irrefragability with other methods or in human cells. Additionally, there is no guarantee that association is actually physiologically relevant. However, it is also true that this system could narrow down the new candidates of GPCR heterodimers with a bit of effort. If one accepts these uncertainties, the assay provides a nice way of monitoring changes that does not depend on effective downstream signaling through the GPCR pathway.

In summary, we have developed a specialized method to screen candidate heterodimer partners for target GPCRs based on the split-ubiquitin membrane yeast two-hybrid method. This modified system permitted the rapid and facile detection of not only the heterodimer formation of target human GPCRs, but also the ligand-mediated conformational changes in living yeast cells. Since budding yeast Saccharomyces cerevisiae can functionally express human GPCRs [29], [30], construction of a large prey library would be beneficial for the identification of heterodimer candidates as the partners of target human GPCRs. Our system will be a useful tool to assist in the intermolecular mapping of interactions among GPCRs and uncover potential targets for the development of new therapeutic agents.

Materials and Methods

Media

Synthetic dextrose (SD) media contained 6.7 g/l yeast nitrogen base without amino acids (YNB) (BD Diagnostic Systems, Sparks, MD, USA) and 20 g/l glucose. YPDA media contained 10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose and 55 mg/l adenine. Amino acids and nucleotides (60 mg/l leucine, 40 mg/l tryptophan, 40 mg/l adenine, 20 mg/l histidine or 20 mg/l uracil) were supplemented into SD media to provide the relevant auxotrophic components. For solid plates, 2% agar was added to the media.

Yeast Strains

All yeast strains were generated from NMY51 (Dualsystems Biotech AG, Schlieren, Switzerland) as a parental backbone strain and are listed in Table 1. Transformation with linear DNA fragments was performed by using the lithium acetate method [31]. To eliminate the URA3 selectable marker in each transformation step, we basically followed previous procedures [32], [33] with the marker recycling method [34]. All oligonucleotides used for the strain constructions are listed in Table S1. To disrupt the target genes (STE20, STE11 and STE2), the first half of DNA fragments containing upstream regions of target genes and URA3 selectable marker were PCR-amplified from pGK406 [35] by using gene-specific oligonucleotides. The last half of DNA fragments containing downstream regions of target genes and homologous sequences to eliminate URA3 marker were PCR-amplified from NMY51 genomic DNA by using gene-specific oligonucleotides. These amplified fragments were then used as the templates for overlap PCR. The combined linear fragments were introduced into appropriate parental yeast strains, and the transformants were selected on SD solid media lacking uracil. After confirming integration of the fragments at the correct positions, the cells were maintained on SC media containing 1 mg/ml 5-fluoroorotic acid (5-FOA, Fluorochem, Derbyshire, UK) to eliminate URA3 marker.

Plasmids

Plasmid construction is described in Document S1. All plasmids used for the assays are listed in Table S2. The transformation procedure followed the lithium acetate method [31].

Agar Diffusion Bioassay

An agar diffusion bioassay (halo assay) was performed to measure growth inhibition in response to signal-induced cell-cycle arrest [36]. Cells were grown in YPDA media overnight at 30°C. Sterilized paper filter disks (6 mm in diameter) were placed on a square Petri dish, and various amounts of α-factor pheromone (Zymo Research, Orange, CA, USA) were spotted onto the disks. YPDA medium containing 20 g/l agar (maintained at 50°C) was inoculated with the grown cells to give an initial optical density of 5×10−4 at 600 nm (OD600 = 5×10−4), and the suspension was immediately poured into the dish. The plates were incubated at 30°C for 1 to 2 days.

Evaluation of Receptor Dimerization

Cub and NubG fusion constructs (Table S2) were co-transformed into yeast strains. Cells were grown in SD media lacking leucine and tryptophan overnight at 30°C on a rotatory shaker set at 150 rpm and then harvested to evaluate receptor dimerization by growth assay and β-galactosidase assay.

Growth Assay

Harvested cells were washed with distilled water, and cell suspensions were prepared to give an OD600 of 10. Seven microliters of serial dilutions of cell suspensions (1∶10) were spotted on SD agar plates lacking leucine, tryptophan, adenine and histidine. The plates were incubated at 30°C.

β-D-galactosidase Activity Assay

β-D-galactosidase activity was determined by using chlorophenol red β-D-galactopyranoside (CPRG) (Roche Applied Science, Indianapolis, IN, USA) as the chromogenic substrate. The procedure basically followed the method described in the Yeast Protocols Handbook (Clontech Laboratories, Inc./Takara Bio Company). Harvested cells were washed once with buffer 1 [100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 4 mM L-aspartic acid hemimagnesium salt hydrate, 10 g/l bovine serum albumin (BSA) and 0.05% polyoxyethylene sorbitan monolaurate (Tween 20); pH 7.25–7.30] and resuspended in buffer 1 to give an OD600 of 10. Four microliters of chloroform and 7 µl of 0.1% SDS were added to 100 µl of cell suspension, the mixtures were agitated with a vortex, and then buffer 1 (700 µl) containing 2.23 mM CPRG was added to the mixtures. After incubation for 10 min at room temperature, 500 µl of 3 mM ZnCl2 was added to stop the enzyme reaction. After centrifugation, the OD578 of supernatants were measured with a spectrophotometer. β-Gal units were calculated as 1,000×OD578/(10 min×0.1 ml×OD600).

Ligand Assay

Harvested cells were inoculated into 5 mL of fresh SD media containing ligand to give an initial OD600 of 0.03. They were incubated at 30°C with shaking at 150 rpm for up to 18 h. Afterwards, the β-D-galactosidase activity was performed.

Model Screening

A small-sized prey GPCR library (Table S3) was transformed into yeast strain NMY63 harboring pBT3-AGTR1 by using the lithium acetate method [31]. Transformants were selected on SD medium lacking leucine, tryptophan, adenine and histidine for bait-prey interaction. Prey plasmids were isolated from 30 positive clones, amplified in Escherichia coli, and analyzed by sequencing analysis.

Supporting Information

Figure S1.

Effect of promoter on detection of dimerization of yeast Ste2p receptor in NMY62 strain. NMY62 yeast strain was transformed with the plasmids expressing indicated protein pairs and grown on SD –Leu, Trp, Ade and His dropout plate at 30°C. (A) CYC1 promoter. (B) PHO5 promoter and TPI1 promoter. (C) TDH3 promoter.

https://doi.org/10.1371/journal.pone.0066793.s001

(TIF)

Figure S2.

Effect of removal of C-terminal tail on detection of dimerization of yeast Ste2p receptor in NMY62 strain. NMY62 yeast strain was transformed with plasmids expressing indicated protein pairs. Quantitative β-galactosidase assays for full-length Ste2p receptor under the control of TPI1 promoter (A) and Ste2p receptor that lacks the C-terminal tail (Ste2ΔC) under the control of TPI1 promoter (B).

https://doi.org/10.1371/journal.pone.0066793.s002

(TIF)

Figure S3.

Detection of homodimerization of human SSTR5 receptor in NMY62 or NMY63 strains. NMY63 yeast strain was transformed with plasmids expressing indicated protein pairs. Quantitative β-galactosidase assays for the NMY62 strain (A) and NMY63 strain (B).

https://doi.org/10.1371/journal.pone.0066793.s003

(TIF)

Figure S4.

Optimization of promoter to detect homodimerization of human GPCRs in NMY63 strain. NMY63 yeast strain was transformed with plasmids expressing the indicated protein pairs. (A) Quantitative β-galactosidase assays. (B–F) Growth assays on SD –Leu, Trp, Ade and His dropout plate at 30°C. (A) GABAB2 receptor (GABBR2) (B) AT1 angiotensin receptor (AGTR1) (C) MT1 melatonin receptor (MTNR1A) (D) somatostatin receptor 2 (SSTR2) (E) β2-adrenergic receptor (ADRB2) (F) 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A). The control prey plasmid was pPR3-C mock vector.

https://doi.org/10.1371/journal.pone.0066793.s004

(TIF)

Figure S5.

Detection of homo- and hetero-dimerization of human GABAB1a receptor with GABAB2 receptor in NMY63 strain. Quantitative β-galactosidase assay. NMY63 yeast strain was transformed with plasmids expressing indicated protein pairs. Error bars represent the standard deviations (n = 3). The control prey plasmid was pPR3-C mock vector.

https://doi.org/10.1371/journal.pone.0066793.s005

(TIF)

Document S1.

Supplementary Materials and Methods (Plasmid constructions for supporting information).

https://doi.org/10.1371/journal.pone.0066793.s009

(PDF)

Author Contributions

Conceived and designed the experiments: YN JI AK. Performed the experiments: YN. Analyzed the data: YN JI. Wrote the paper: YN JI AK.

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