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Analytical Biochemistry xxx (2013) xxx–xxx
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Contents lists available at ScienceDirect
Analytical Biochemistry
journal homepage: www.elsevier .com/locate /yabio
Monitoring G protein-coupled receptor activation usingan adenovirus-based b-arrestin bimolecular fluorescencecomplementation assay
0003-2697/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.ab.2013.12.017
⇑ Corresponding authors. Fax: +82 31 400 6232 (J.-Y. Jeong). Fax: +82 2 873 4740(W.-K. Huh).
E-mail addresses: [email protected] (J.-Y. Jeong), [email protected] (W.-K. Huh).1 Abbreviations used: GPCR, G protein-coupled receptor; MAPK, mitogen-activated
protein kinase; GFP, green fluorescent protein; BiFC, bimolecular fluorescencecomplementation; YFP, yellow fluorescent protein; AdHTS, adenovirus high-through-put system; TP, terminal protein; OS, osteosarcoma; VC, the C-terminal fragment ofVenus; VN, the N-terminal fragment of Venus; HCS, high-content screening; AdBiFC,adenoviral bimolecular fluorescence complementation; MEM, modified Eagle’smedium; FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium;WT, wild type.
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupled receptor activation using an adenovirus-based b-arrestin bimoleculrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1016/j.ab.2013.12.017
Yong Bhum Song a, Chul O. Park a, Jae-Yeon Jeong b,⇑, Won-Ki Huh a,c,⇑a Department of Biological Sciences and Research Center for Functional Cellulomics, Seoul National University, Seoul 151-747, Republic of Koreab Marine Biotechnology Research Division, Korea Institute of Ocean Science & Technology, Ansan 426-744, Republic of Koreac Institute of Microbiology, Seoul National University, Seoul 151-747, Republic of Korea
a r t i c l e i n f o a b s t r a c t
29303132333435363738394041
Article history:Received 13 August 2013Received in revised form 16 November 2013Accepted 10 December 2013Available online xxxx
Keywords:G protein-coupled receptor (GPCR)b-ArrestinBimolecular fluorescence complementation(BiFC)AdenovirusHigh-throughput screening
G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in avariety of pathological conditions including cancer and cardiovascular, metabolic, neurological, andautoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention,and the b-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here,we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescencecomplementation (BiFC) vectors and performing the b-arrestin BiFC assay in cells transduced with recom-binant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovi-rus-based b-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation inresponse to specific agonists or antagonists. Furthermore, the adenovirus-based b-arrestin BiFC assaywas able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that theadenovirus-based b-arrestin BiFC assay can serve as a simple and universal platform for studying GPCRactivation and thus will be useful for high-throughput screening of drugs that target GPCRs.
� 2013 Elsevier Inc. All rights reserved.
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G protein-coupled receptors (GPCRs)1 are seven-transmem-brane-domain receptors that respond to a wide range of signalsincluding light, odors, ions, neurotransmitters, chemoattractants,lipids, peptides, and hormones [1]. They are the largest class ofcell-surface receptors with almost 800 members identified in the hu-man genome, including more than 100 orphan receptors with noknown natural ligand or function [2]. GPCRs orchestrate all aspectsof human physiology including sensory and pain perception, neuro-transmission, cardiovascular function, digestion, development,growth, chemotaxis, and immune responses. Additionally, thesereceptors are also involved in various diseases. Not surprisingly,GPCRs are regarded as one of the more important families of drugtargets in the pharmaceutical industry and are the targets of at least
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40% of marketed prescription drugs [3]. Because of their importance,the development of robust and versatile drug screening assays thatmonitor GPCR activity in a high-throughput way has attracted greatinterest in the field of drug discovery.
On agonist binding, GPCR activates its associated heterotrimericG-proteins and initiates a cascade of intracellular signaling eventsthat depend mainly on its associated Ga subunit. There are 20known Ga subunits with characteristic signaling pathways, andeach GPCR interacts with specific types of Ga subunits, leadingto distinct physiological responses [4]. Shortly after activation,the GPCR is phosphorylated by GPCR kinases and signaling is ter-minated by the recruitment of b-arrestin to the phosphorylatedGPCR. Termination of GPCR signaling occurs at three levels. First,the recruitment of b-arrestin to ligand-bound GPCR sterically hin-ders the association of the receptor with its cognate G-proteins,leading to a termination of the intracellular signaling cascade.Immediately following desensitization, b-arrestin further targetsthe desensitized GPCR to clathrin-coated pits for endocytosis, andthe GPCR is either recycled back to the cell surface after dissocia-tion from its ligand in the endosomes or degraded in the lyso-somes. In addition to the G-protein uncoupling and GPCRendocytosis, b-arrestin can also efficiently reduce the signals gen-erated by activated receptors by degrading the second messengersdiacylglycerol and cAMP. This degradation is accomplished
ar fluo-
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2 Monitoring GPCR activation using an AdBiFC assayQ1 / Y.B. Song et al. / Anal. Biochem. xxx (2013) xxx–xxx
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through the recruitment of second messenger degrading enzymes,including diacylglycerol kinases and phosphodiesterases, to theactivated GPCRs [5,6]. b-Arrestin is not only involved in the termi-nation of GPCR signaling but also has roles in generating newsignaling by recruiting mitogen-activated protein kinases (MAPKs)to the activated GPCR and bringing MAPK, MAPK kinase, and MAPKkinase kinase into close proximity, resulting in the robust activa-tion of the MAPK signaling cascade [3].
Four types of arrestin are responsible for GPCR desensitizationin mammals [7]. Visual arrestins (arrestin 1 and 4) are localizedin the eye and are involved in the desensitization of photorecep-tors, whereas b-arrestin 1 (arrestin-2) and b-arrestin 2 (arrestin-3) are expressed ubiquitously and interact with most GPCRs.Depending on the interaction between GPCR and b-arrestin, GPCRsare grouped into two classes. Class A receptors bind b-arrestin 2with higher affinity than b-arrestin 1 and are recycled back tothe plasma membrane rapidly after transient association withb-arrestin in the clathrin-coated pits [8–10]. GPCRs includingsomatostatin receptor 3 (SSTR3), SSTR5, b2-adrenoceptor (ADRB2),vasopressin 1a receptor, dopamine D1A receptor, and CXC chemo-kine receptor 4 belong to this class. In contrast, class B receptorsbind both b-arrestins with high affinity and are targeted to theendosomes and lysosomes as stable complexes. Receptors suchas SSTR2, chemokine (C-C motif) receptor 5 (CCR5), neurotensinreceptor 1 (NTSR1), arginine vasopressin receptor 2 (AVPR2, alsoknown as vasopressin V2 receptor), thyrotropin-releasing hormonereceptor, and substance P receptor are included in this class. Thestability of the GPCR-b-arrestin complex depends on the presenceof a cluster of serine and threonine residues in the C-terminal tailof class B receptors [11] and the persistent ubiquitination ofb-arrestin associated with class B receptors [12].
Binding of b-arrestin to agonist-activated GPCR offers a univer-sal tool to measure GPCR activation because there is no need toconsider specific downstream signaling events depending on thetype of Ga protein. b-Arrestin 2 is preferred to b-arrestin 1 becauseit interacts with a broader range of GPCRs and has a higher affinityfor the clathrin adaptor complex AP-2, which is involved in endo-cytosis [8,10,13]. Several cell-based high-throughput drug screen-ing assays have been developed for monitoring and quantifyingGPCR activation using b-arrestin recruitment to activated GPCRs[10]. Earlier assays utilized the redistribution of either b-arrestinor GPCR tagged with green fluorescent protein (GFP) [14,15]. Moresophisticated assays, such as bioluminescence resonance energytransfer (BRET) and b-galactosidase complementation, monitorreal-time signals generated by the interaction between GPCRsand b-arrestin [16,17]. The bimolecular fluorescence complemen-tation (BiFC) assay was initially devised to detect protein–proteininteractions in living cells in which two complementary N- andC-terminal fragments of yellow fluorescent protein (YFP) reconsti-tute a fluorescent signal only when both fragments are close to-gether through interaction between the two different proteins towhich they are fused [18]. The BiFC assay provides informationnot only on the quantity but also on the subcellular localizationof protein interactions in live cells in the absence of any furtherstaining and without requiring the addition of expensivesubstrates or the use of special detection devices. Recently, theBiFC assay was adopted for detecting b-arrestin recruitment tob2-adrenoceptor and neuropeptide Y1 receptors [19–21].
One of the hurdles for high-throughput screening (HTS) of GPCRactivation is the need to establish stable cell lines expressing boththe b-arrestin and a specific type of GPCR, because plasmid-basedgene delivery systems have a low cotransfection efficiency. Adeno-viral vectors have great advantages over plasmid or other virus-based vectors in terms of gene delivery efficiency and available celltypes [22]. Adenoviruses exhibit the highest transductionefficiency as well as the highest viral titer. Because the adenoviral
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
genome does not integrate into the host genome, mutations due torandom integration do not arise. Adenoviruses can infect both rep-licating and terminally differentiated quiescent cells from variousorigins. Despite these advantages, construction of recombinantadenoviruses is considered to be difficult and laborious. Recently,a simple and robust method for constructing recombinant adeno-viruses, termed AdHTS (adenovirus high-throughput system), hasbeen developed using Gateway in vitro site-specific recombinationtechnology and terminal protein (TP)-coupled adenoviral vectors[23].
Here, we report the use of an adenovirus-based b-arrestin BiFCassay as a rapid and versatile platform for monitoring GPCR activa-tion. GPCRs were cloned into adenoviral BiFC vectors in a high-throughput manner using AdHTS. U-2 osteosarcoma (OS) cellswere cotransduced with GPCRs tagged with the C-terminal frag-ment of the YFP variant Venus (VC) and b-arrestin 2 tagged withthe N-terminal fragment of Venus (VN). The activation of SSTR2was then monitored by the adenovirus-based b-arrestin BiFC assay.The BiFC signals were increased by agonists and decreased by anantagonist in a dose-dependent manner, implying that the adeno-virus-based b-arrestin BiFC assay can be used for screening bothagonists and antagonists. The Z0 factor for SSTR2 b-arrestin BiFCin the presence of the SSTR2 agonist SRIF-14 was 0.55, suggestingthat the assay is suitable for high-content screening (HCS). In addi-tion, GPCRs belonging to various classes showed markedlyincreased BiFC signals in intracellular pits and vesicles on stimula-tion with the appropriate agonists, suggesting that this assay rep-resents a rapid and universal method for monitoring GPCRactivation.
Materials and methods
Reagents
Vectors containing BiFC fragments (pBiFC-VN173, pBiFC-VC155,pBiFC-bJun-VN173, pBiFC-bFos-VC155, and pBiFC-bFos(DZip)-VC155) were obtained from Chang-Deng Hu [18]. VectorspCS2+VN154, pCS2+VC155, and pCS2+VNm7�13 were obtainedfrom James Smith [24]. bJun, bFos, and bFos(DZip) were amplifiedby PCR and cloned into pCS2+. Human GPCR and b-arrestin 2(NM_004313.3) cDNAs were obtained from the Missouri S&T cDNAResource Center (Rolla, MO, USA). GPCR and b-arrestin 2 cDNAswere amplified by PCR with primers containing attB1 and attB2sites at each end and cloned into the pDONR201 vector via theattB-attP recombination reaction (Invitrogen, Carlsbad, CA, USA).GPCR agonists and antagonists were purchased from Sigma-Aldrich(St. Louis, MO, USA) and Tocris Bioscience (Bristol, UK).
Cell culture
HEK293 and U-2 OS cells were purchased from the AmericanType Culture Collection, and HEK293A cells were purchased fromInvitrogen (Carlsbad, CA, USA). HEK293 cells were transfected afterin vitro recombination between the TP-coupled adenoviral bimo-lecular fluorescence complementation (AdBiFC) vector and theGPCR entry clone, and HEK293A cells were used to amplify recom-binant adenoviruses. HEK293 cells were maintained in modifiedEagle’s medium (MEM; Hyclone, Logan, UT, USA) supplementedwith 10% fetal bovine serum (FBS), 100 units/ml of penicillin,100 lg/ml of streptomycin, 0.1 mM nonessential amino acids,and 1 mM sodium pyruvate at 37 �C in 5% CO2. HEK293A cells werecultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone,Logan, UT, USA) supplemented with 10% FBS, penicillin, and strep-tomycin, and U-2 OS cells were grown in McCoy’s 5A medium with10% FBS, penicillin, and streptomycin.
receptor activation using an adenovirus-based b-arrestin bimolecular fluo-016/j.ab.2013.12.017
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Production of recombinant BiFC adenoviruses
pAdBiFC-VN and pAdBiFC-VC vectors were constructed usingthe AdEasy vector system according to the manufacturer’s instruc-tions (Qbiogene, Carlsbad, CA, USA); vector maps are presented inFig. S1. To obtain TP-coupled AdBiFC vectors, pAdBiFC-VN/VC puri-fied from DH10B bacterial cells were linearized with PacI (NewEngland Biolabs, Beverly, MA, USA) and transfected to HEK293 cells[23]. Recombinant adenoviruses containing BiFC cassettes wereamplified in HEK293A cells by repeated infection to a final roundof 2.5 � 108 cells in twenty-five 15-cm dishes and a multiplicityof infection (MOI) of 10. After 2 days, cell lysates were obtainedby three rounds of rapid freezing and thawing and recombinantadenoviruses were purified by two consecutive CsCl gradientultracentrifugations. Capsid proteins were denatured using 6 Mguanidine HCl, and TP-coupled AdBiFC vectors were purified usingthe Montage Plasmid Miniprep96 kit (Millipore, Bedford, MA, USA).TP-coupled AdBiFC vectors were digested with SwaI (New EnglandBiolabs, Beverly, MA, USA) for 4 h at 25 �C. After heat inactivationat 65 �C for 20 min, the digestion mixture was used directly inattL-attR (LR) recombination reaction or stored in aliquots at�80 �C for later use (Fig. 1A). Single preparation of TP-coupled Ad-BiFC vectors provided sufficient material for about 5000 recombi-nation reactions.
GPCR and b-arrestin 2 cDNAs were cloned into adenoviral BiFCvectors as described previously [23]. In brief, cDNA in an entry vec-tor (20 ng), SwaI-digested TP-coupled AdBiFC vector (10–20 ng),2 ll of LR recombinase (Lambda Integrase/Excisionase; ELPISBiotech, Daejeon, Korea) were added to a final volume of 10 ll LRbuffer (40 mM Tris-Cl, pH 7.4, 5 mM spermidine, 2 mM EDTA,2 mM DTT, 100 lg/ml BSA) in a well of a 96-well PCR plate, andsite-specific recombination was performed for 12 h at 25 �C. Afterinactivation of LR recombinase for 20 min at 65 �C, HEK293 cellswere transfected with the recombination mixture. Recombinantviruses were released from the cells 7–10 days after transfectionby three cycles of rapid freezing and thawing and subsequentlyamplified in HEK293A cells by successive infection. The recombi-nation efficiency and production of recombinant adenoviruseswere examined by PCR using AdBiFC-F (50-GGTCTATATAAGCA-GAGCTG-30) and AdBiFC-R (50-GTATGGCTGATTATGATCAG-30)primers (Fig. 1A).
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Adenovirus-based b-arrestin BiFC assay
U-2 OS cells were seeded at a density of 3000 cells per well in ablack 96-well clear-bottom plate in 100 ll of McCoy’s 5A mediumsupplemented with 10% FBS. On the following day, the cells weretransduced with 30 MOI each of GPCR-VC and b-arrestin2-VNrecombinant adenoviruses and allowed to grow for 3 days. Afterpreequilibration with serum-free medium for 1 h, the cells weretreated with the indicated amounts of agonist or antagonist for1 h at 37 �C, fixed with 2% formaldehyde, and stained with Hoechst33342 (Invitrogen, Carlsbad, CA, USA).
BiFC and nuclear images were captured using IN Cell Analyzer2000 (GE Healthcare, Waukesha, WI, USA) using 20� objectiveand 350/455 nm (for Hoechst) and 500/535 nm (for BiFC) excita-tion/emission filters with a polychroic mirror (QUAD2). The IN CellDeveloper ToolBox was used to define the cell and granule bound-ary and analyze the number and fluorescence intensity of granuleas described previously [25]. In brief, cell boundary was identifiedby thresholding the nuclear area stained with Hoechst, and parti-cles with 0.2–5 lm in diameter inside the cells were classified asBiFC-positive granules (Fig. S2). Totals of 200–300 cells from 6fields per well were analyzed and the average number or intensityof granule per field was calculated.
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
Data analysis
The pEC50 and pIC50 values were calculated from curve fits ofthe pooled data using GraphPad Prism (version 5; GraphPad Soft-ware, San Diego, CA, USA). The t1/2 values were calculated by OnePhase Exponential Association analysis using GraphPad Prism.The Z0 factor was calculated as described by Zhang et al. [26] usingthe following equation: Z0 = 1 � 3(SD positive + SD negative)/(mean positive �mean negative). Z0 values between 0.5 and 1 wereregarded as evidence of an assay suitable for HCS.
Results
Selection of Venus fragments for BiFC assay
We used a YFP variant, Venus, that was developed for rapid andefficient maturation at 37 �C and increased resistance to acidicenvironments such as endosomes [27]. To reduce the backgroundsignal from VN and VC self-assembly, we compared Venusfragment VN154 and VN154 variants (VNm7 to VNm13) [24] withVN173, which has an overlapping b-strand repeat (155–172) whencomplemented with VC155 [28], by introducing VN and VC155constructs in combination into HEK293 cells. Compared withVN173, VN154 and its derivatives showed reduced self-assemblysignals when coexpressed with VC155 at 24 h after transfection;however, the fluorescence signal increased markedly in most com-binations at 48 h posttransfection (Fig. S3A). Out of nine combina-tions, the VNm10-VC155 pair was chosen for further analysisbecause it had the lowest background BiFC signal even at 48 hposttransfection (Table S1, Fig. S3B).
To determine whether the VNm10 and VC155 combination pro-vides a sufficient BiFC signal to detect protein–protein interactions,VN and VC fragments were fused with the basic leucine zipper do-mains of bFos and bJun, respectively, and introduced into HEK293cells. VNm10-bJun produced a BiFC signal that was comparable tothat of bJun-VN173 when cointroduced with VC155 fused towild-type bFos; however, it produced a very weak signal whencointroduced with VC155 fused to mutant bFos(DZip) (Fig. S4). Incontrast, the VN173 and VC155 combination produced a distinctBiFC signal even when bJun was coexpressed with bFos(DZip),which is indicative of VN173 and VC155 self-assembly. Therefore,VNm10 and VC155 fragments were selected for further BiFCstudies.
Adenovirus-based b-arrestin BiFC assay
To facilitate coexpression of both VN and VC fusion proteinswith high efficiency and to reduce the time and labor requiredfor making stable cell lines, we used an adenoviral gene deliverysystem. Gene cloning into adenoviral vectors was also simplifiedby using the AdHTS technique, which is characterized by universaland simple cloning based on the Gateway system and high adeno-virus productivity based on the adenoviral DNA-TP complex [23].Entry clones containing human GPCRs or b-arrestin 2 were mixedwith TP-coupled fragmented AdBiFC vector in 96-well plates, andthe cDNAs were C-terminally tagged with either VN or VC throughattL and attR recombination in vitro (Fig. 1A). Owing to thepresence of TP at the ends of the adenoviral vector, which protectsthe viral DNA from endonuclease digestion and enhances virusreplication inside the packaging cells, recombinant adenoviruseswere produced efficiently by transfecting the recombination reac-tion mixture directly into HEK293 packaging cells. At 7 to 10 daysafter transfection, recombinant adenoviruses were harvested andamplified by successive infection into HEK293A cells. If GPCR-VCand b-arrestin-VN are coexpressed in mammalian cells by
receptor activation using an adenovirus-based b-arrestin bimolecular fluo-016/j.ab.2013.12.017
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Fig. 1. Schematic overview of the adenovirus-based b-arrestin BiFC assay. (A) High-throughput production of recombinant adenovirus using AdHTS. An entry clonecontaining GPCR cDNA was mixed with fragmented TP-coupled AdBiFC vector, and site-specific recombination between attL and attR was performed in vitro. The reactionmixture was transfected directly into HEK293 cells seeded in a 96-well plate, and recombinant adenoviruses were produced 7 to 10 days after transfection. Therecombination efficiency and the production of recombinant adenoviruses were examined by PCR using AdBiFC-F and AdBiFC-R primers. Adenoviruses were further amplifiedby repetitive infection of HEK293A cells on a larger scale. For the adenovirus-based b-arrestin BiFC assay, U-2 OS cells were cotransduced with equal amounts of recombinantGPCR-VC and b-arrestin-VN adenoviruses. The cells were allowed to express the recombinant proteins for 3 days. (B) Cells transduced with recombinant adenoviruses weretreated with the appropriate agonist and/or antagonist for 1 h, and the BiFC signal was visualized using an automated fluorescence image analyzer. B1, attB1; B2, attB2; F,AdBiFC-F; L1, attL1; L2, attL2; LITR, left inverted terminal repeat; R, AdBiFC-R; R1, attR1; R2, attR2; RITR, right inverted terminal repeat; pA, poly-adenylation site; PCMV, CMVpromoter; TP, terminal protein.
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adenoviral transduction, the BiFC signal will not be detected untilb-arrestin is recruited to the activated GPCR on agonist treatment(Fig. 1B). Therefore, the appearance of fluorescence signal on ago-nist stimulation allows clear interpretation of the results and sim-plifies the quantification of the response.
To compare the expression levels of VN and VC fusion proteinsachieved by adenoviral transduction (Fig. S5A) with those achievedby plasmid transfection (Fig. S5B), we monitored the BiFC signalderived from the interaction between bJun-VN and bFos-VC. The
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
proportion of cells expressing the BiFC signal was increased in adose-dependent manner in both methods. However, adenoviralgene transduction gave more BiFC-positive cells with a broaderrange of DNA doses (Fig. S5A–C). Although both gene deliverymethods caused dose-dependent cell death, adenoviral transduc-tion resulted in significantly less cell toxicity compared to plasmidtransfection (Fig. S5D). Furthermore, adenoviral transductioncaused less cell-to-cell variation in the BiFC signal intensity thanplasmid transfection at all doses tested. In the case of plasmid
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transfection, bright BiFC signals derived from dying cells, as judgedby cell morphology in the bright-field images, were observed evenat the lowest plasmid concentration (Fig. S5B, arrow). These resultsdemonstrate the advantage of adenovirus-mediated gene transferin terms of coexpression efficiency, cell toxicity, and the homoge-neity of gene expression.
To optimize adenovirus-based BiFC assay, the expression ofBiFC partners was detected using an anti-GFP antibody aftercotransducing U-2 OS cells with bJun-VN and bFos-VC adenovi-ruses (Fig. S6A). The expression of each fusion protein was in-creased up to 30 MOI and was slightly decreased at higher dosesprobably due to cell toxicity as observed in Fig. S5. Interestingly,the bJun-VN band was much more intense than the bFos-VC band,presumably because the reactivity of VC against the anti-GFP anti-body used in this study is lower than that of VN. Consistent withthis, when cells were cotransduced with 30 MOI each of SSTR2-VC and b-arrestin-VN adenoviruses, the protein band intensity ofb-arrestin-VN appeared much higher than that of SSTR2-VC(Fig. S6B). Poor solubility of GPCR may also contribute to the lowband intensity of SSTR2-VC.
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Fig. 2. Treatment of cells cotransduced with SSTR2 and b-arrestin adenoviruses with SRIGFP (A), SSTR2-VN and b-arrestin-VC (B), SSTR2-VC and b-arrestin-VN (C), or SSTR2-VN athe absence of serum and treated with vehicle alone (control), 100 nM SRIF-14, or 100 nMmembrane SSTR2-GFP into intracellular vesicles (A) or reconstitution of Venus fluorescaddition of agonist or antagonist. (E) Images obtained using the IN Cell Analyzer 2000 wmethods. The data were analyzed by Student’s t test and are represented as the mean ±
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
We next investigated the effect of different MOI ratios of SSTR2-VC: b-arrestin-VN viruses on the pharmacology of agonist-inducedBiFC response using an SSTR2 agonist SRIF-14. When transducedwith 30 MOI each of SSTR2-VC and b-arrestin-VN adenoviruses,cells exhibited the highest response against SRIF-14 (Fig. S7). Cellstransduced with 30 MOI of SSTR2-VC and 10 MOI of b-arrestin-VNadenoviruses exhibited a lower response than cells transducedwith 10 MOI of SSTR2-VC and 30 MOI of b-arrestin-VN adenovi-ruses, suggesting that the amount of b-arrestin-VN may be the lim-iting factor for BiFC response. Notably, different MOI ratios ofSSTR2-VC: b-arrestin-VN viruses resulted in similar pEC50 values,indicating that this assay gives a consistent result across a broadrange of expression levels of GPCR and b-arrestin.
Finally, we examined the possibility of background signal de-rived from self-assembly of VN and VC when they were overex-pressed. Background signals derived from self-assembly of VNand VC started to appear at 50 MOI each of VN and VC viruses(Fig. S8), implying that cotransduction with more than 50 MOIeach of viruses may cause nonspecific BiFC responses. Taken to-gether, we selected the MOI of 30:30 (SSTR2-VC:b-arrestin-VN)
postCYN 1548064
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SSTR2-VC +β-arrestin-VN
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F-14 specifically induced the BiFC signal. U-2 OS cells were transduced with SSTR2-nd HA-VC adenoviruses (D). The cells were incubated with Hoechst 33342 for 1 h inCYN 154806 at 37 �C for 1 h. Activation of SSTR2 was monitored by redistribution of
ence (B–D). Images were obtained immediately before (Pre) or 1 h after (Post) theere analyzed with the IN Cell Developer ToolBox as described under Materials andSEM (n = 3). Scale bar, 20 lm; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
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as the optimal transduction condition for BiFC assay on the bases ofthe proportion of BiFC-positive cells, cell toxicity, the pharmaco-logical response to agonist, and background signal from VN andVC self-assembly.
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Monitoring GPCR activation via the b-arrestin BiFC assay in cellstransduced with recombinant adenoviruses
To investigate whether the BiFC signal is indicative of GPCR andb-arrestin interaction, U-2 OS cells were plated in a 96-well plateand transduced with C-terminally labeled SSTR2 and b-arrestin.The presence of C-terminal tags did not influence the signaling ofSSTR2 against SRIF-14 when inhibition of cAMP production wasmeasured, implying that SSTR2-VN and SSTR2-VC can be used inb-arrestin recruitment assay (Fig. S9). When SSTR2-GFP was ex-pressed in U-2 OS cells, the GFP signal was mainly localized inthe plasma membrane (Fig. 2A). In contrast, very low, if any, fluo-rescence signal was detected in cells coexpressing SSTR2-VN andb-arrestin-VC (Fig. 2B) or SSTR2-VC and b-arrestin-VN (Fig. 2C)when the cells were treated with vehicle alone (control) or just be-fore agonist treatment (pre). A strong BiFC signal appeared whenthese cells were stimulated with SRIF-14, the natural agonist ofSSTR2, manifesting as bright dots of various sizes (post). This resultindicates the reconstitution of functional Venus as a result ofligand-dependent association between SSTR2 and b-arrestin. TheBiFC signal did not appear when the cells were treated with theantagonist CYN 154806, further supporting that the BiFC signal isspecifically induced by agonist binding to SSTR2. In the case ofSSTR2-GFP, only part of the GFP signal was redistributed into intra-cellular compartments on agonist stimulation, whereas the SSTR2/b-arrestin BiFC signal was more distinct and easily distinguishable
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Fig. 3. Functional pharmacology of the SSTR2 receptor. (A) U-2 OS cells were cotranadenoviruses and stimulated with increasing concentrations of SRIF-14 at 37 �C for 1 h.intensity (right panel). (B, C) Dose–response curves for SSTR2/b-arrestin BiFC complex fovarious concentrations of the SSTR2 agonists SRIF-28 and octreotide (B) or with various co37 �C for 1 h. The data are represented as the mean ± SEM (n = 3). pEC50 and pIC50 values win GraphPad Prism.
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
as intracellular vesicles and clathrin-coated pits owing to the lackof any background fluorescence signal (Fig. 2B and C). To deter-mine whether any BiFC signal is derived from VN and VC self-assembly, SSTR2-VN was cotransduced with hemagglutinin-VCand the cells were treated with SRIF-14 or CYN 154806. No fluores-cence signal was detected even when these cells were stimulatedwith SRIF-14 (Fig. 2D), suggesting that the BiFC signal detected incells coexpressing SSTR2 and b-arrestin is due to the recruitmentof b-arrestin to agonist-bound SSTR2.
The BiFC images were analyzed by counting the number ofgranules with diameters of 0.2-5 lm or measuring the intensityof granules per field. The BiFC signal was not increased in cellstreated with CYN 154806 compared with those treated with vehi-cle alone (control) when quantified by granule number or intensity(Fig. 2E). The BiFC signal was also negligible in cells expressingSSTR2-VN and hemagglutinin-VC regardless of whether the cellswere stimulated with agonist or antagonist. On the contrary, boththe average number and the intensity of granules per field weresignificantly increased by treatment of cells coexpressing SSTR2and b-arrestin 2 with SRIF-14, implying that this assay is suitablefor monitoring GPCR activation.
Quantitative analysis of GPCR activation via the adenovirus-basedb-arrestin BiFC assay
To examine whether the adenovirus-based b-arrestin BiFC assayis suitable for quantifying GPCR activation, we monitored theSSTR2/b-arrestin BiFC signal in response to various doses of SRIF-14 1 h after stimulation. Both SSTR2-VN/b-arrestin-VC andSSTR2-VC/b-arrestin-VN exhibited dose-dependent increases inBiFC signal with pEC50 values of 7.686 ± 0.104 and 7.265 ± 0.049,
C
sduced with either SSTR2-VN and b-arrestin-VC or SSTR2-VC and b-arrestin-VNBiFC responses were analyzed by measuring granule number (left panel) or granulermation in cells coexpressing SSTR2-VC and b-arrestin-VN. Cells were treated withncentrations of the antagonist CYN 154806 in the presence of 100 nM SRIF-14 (C) atere determined by nonlinear regression using the sigmoidal curve-fitting algorithm
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respectively, by granule number and 7.711 ± 0.105 and7.333 ± 0.046, respectively, by granule intensity (Fig. 3A). As shownin Fig. 2E, we obtained a higher maximum BiFC response withSSTR2-VC/b-arrestin-VN than with SSTR2-VN/b-arrestin-VC interms of granule number and intensity. Treatment of cells coex-pressing SSTR2-VC and b-arrestin-VN with SRIF-28 or octreotidealso resulted in a dose-dependent increase in the BiFC signal(Fig. 3B). The pEC50 values of SRIF-28 and octreotide were deter-mined to be 7.673 ± 0.038 and 7.829 ± 0.066, respectively, by gran-ule number. As described previously, the natural agonists SRIF-14and SRIF-28 displayed pEC50 values that were similar to that ofthe synthetic agonist octreotide, and these values are similar tothe published pEC50 values obtained using a b-arrestin transloca-tion assay [29]. These results suggest that the adenovirus-basedb-arrestin BiFC assay can be used to investigate the pharmacolog-ical properties of agonists against GPCRs.
Dose-dependent inhibition of the BiFC signal was also demon-strated with the SSTR2-specific antagonist CYN 154806 in the pres-ence of 100 nM SRIF-14 (Fig. 3C). The pIC50 for CYN 154806 wascalculated to be 6.835 ± 0.123. Based on the pEC50 of SRIF-14, thepKb of CYN154806 was calculated to be 7.3417. Taken together,these results imply that the adenovirus-based b-arrestin BiFC assaycan be used to monitor GPCR response to both agonists and antag-onists in quantitative pharmacological studies.
Fig. 4. Time–response curves for SSTR2/b-arrestin BiFC complex formation. Cellstransiently coexpressing SSTR2-VC and b-arrestin-VN were challenged with 100 nMSRIF-14, SRIF-28, or octreotide at 37 �C for the indicated time periods. The cellswere fixed immediately, and the response to different ligands was examined bygranularity analysis as described in Fig. 2. The data are represented as themean ± SEM (n = 3).
Fig. 5. Validation of the adenovirus-based b-arrestin BiFC assay for use in HCS. U-2OS cells were seeded in black 96-well clear-bottom tissue culture plates at a densityof 3000 cells/well and cotransduced with 30 MOI each of SSTR2-VC and b-arrestin-VN adenoviruses on the following day. The cells were allowed to express therecombinant proteins for 3 days and were preincubated with serum-free media for
Kinetics of the adenovirus-based b-arrestin BiFC assay
The formation of a BiFC complex involves a fast refolding stepand a subsequent slow maturation step [18]. Immediately afterthe association of two interacting fusion proteins, the N- andC-terminal fragments of the fluorescent protein come into proxim-ity and fold into a b-barrel structure. The refolded chromophoremust be oxidized to generate a functional fluorescence signal[27,30]. Although the slow maturation of the chromophore delaysthe onset of the BiFC signal, the first refolding step is known to befast enough to detect the transient interactions that are involvedin various signaling processes [19,31]. In addition, the irreversiblenature of the maturation step may confer sensitivity for the detec-tion of transient or weak interactions.
To understand the kinetics of the b-arrestin BiFC assay and toidentify an optimal time window for agonist induction, the recon-stitution of the BiFC signal was monitored in cells cotransducedwith SSTR2-VC and b-arrestin-VN adenoviruses at different timepoints after agonist treatment. The SSTR2/b-arrestin BiFC signalin response to 100 nM SRIF-14, SRIF-28, and octreotide exhibiteda time-dependent increase with half-time of maximum response(t1/2) of 59.79, 55.01, and 48.35 min, respectively, when measuredby granule number and 36.73, 34.25, and 30.49 min, respectively,when measured by granule intensity (Fig. 4). The kinetics of thisassay reflect slow chromophore formation between VN and VC,as previously reported [18].
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1 h. Half the cells were treated with PBS, and the other half were stimulated with100 nM SRIF-14 at 37 �C for 1 h. Activation of SSTR2 was accessed by analyzing theBiFC response, and the Z0 score was calculated.
Adenovirus-based b-arrestin BiFC as a universal platform formonitoring GPCR activation
Given that GPCR is one of the most popular targets for drugdevelopment, we examined whether the adenovirus-basedb-arrestin BiFC assay can serve as an HCS platform for GPCR ago-nists. SSTR2-VC and b-arrestin-VN were transiently coexpressedin U-2 OS cells by transducing the cells with both adenoviruses.SRIF-14 at 100 nM was dispensed into half of the wells of a 96-wellplate, and an equal amount of PBS was dispensed into the otherhalf. When the individual BiFC values for all the tested wells weredetermined 1 h after treatment, the Z0-factor for the assay was 0.55(Fig. 5). This result indicates that the adenovirus-based b-arrestin
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
BiFC assay for SSTR2 is of high quality and is suitable for HCS (Z0
score > 0.5) [26].We next examined whether the adenovirus-based b-arrestin
BiFC assay can be used as a universal system for investigating GPCRactivation. Various GPCR cDNAs were tagged with VC using AdHTSas described in Fig. 1A, and GPCR-VC and b-arrestin-VN adenovi-ruses were cotransduced into U-2 OS cells. GPCRs have been groupedinto two classes depending on their affinity for b-arrestin [8–10].Cells cotransduced with GPCR-VC and b-arrestin-VN did not exhibita noticeable fluorescence signal before agonist stimulation (Pre),
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ADCYAP1R1VIP
VIPR2VIP
GRM3NAAG
Class A
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tADRB2
isoproterenolSSTR3SRIF-14
SSTR5SRIF-14
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CHRM1Desmethylclozapine
CCR7CCL19
CNR2CP55,940
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Rhodopsin-like Secretin-like Metabotropicglutamate-like
Fig. 6. Monitoring the activation of various GPCRs on agonist stimulation via the adenovirus-based b-arrestin BiFC assay. (A) Activation of class A GPCRs, which interacttransiently with b-arrestin, as well as class B GPCRs, which form stable complexes with b-arrestin, was successfully monitored by the adenovirus-based b-arrestin BiFC assay.U-2 OS cells were cotransduced with GPCR-VC and b-arrestin-VN and allowed to express the proteins for 3 days. The cells were treated with isoproterenol (1 lM), SRIF-14(50 nM), CCL7 (50 nM), neurotensin (100 nM), or AVP (100 nM) at 37 �C for 1 h. BiFC images were obtained before (Pre) and after (Post) agonist treatment, and images of thesame cells are presented. (B) GPCRs belonging to the rhodopsin-like family (CNR2, CHRM1, CCR7), secretin-like family (ADCYAP1R1, VIPR2), and metabotropic glutamate-likefamily (GRM3) were stimulated with CP55,940 (1 lM), desmethylclozapine (1 lM), CCL19 (50 nM), VIP (100 nM), or NAAG (1 lM). BiFC images were obtained before (Pre)and after (Post) agonist treatment, and images of the same cells are presented. Scale bar, 20 lm.
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whereas the same cells showed a distinct fluorescence signal incoated pits and vesicles 1 h after treatment with the appropriateagonist regardless of GPCR class (Post) (Fig. 6A). These results sug-gest that all the GPCR-VCs were properly expressed on the cell sur-face and the BiFC signals were specifically initiated on GPCR bindingwith the appropriate agonist rather than by altering the trafficking ofpreexisting GPCR-b-arrestin complexes.
GPCRs can also be grouped into three major classes according totheir structural characteristics: the rhodopsin-like, the secretin-like, and the metabotropic glutamate-like family [32]. To examinewhether GPCRs belonging to three major structural classes can beanalyzed by the adenovirus-based b-arrestin BiFC assay, weassessed recruitment of b-arrestin and formation of the agonist-induced BiFC complex in cells coexpressing different GPCRs ofthe rhodopsin-like family (cannabinoid receptor 2 (CNR2), musca-rinic acetylcholine receptor M1 (CHRM1), and CCR7), the secretin-like family (adenylate cyclase activating polypeptide 1 receptortype 1 (ADCYAP1R1) and vasoactive intestinal peptide receptor 2(VIPR2)), and the metabotropic glutamate-like family (metabotro-pic glutamate receptor 3 (GRM3)). GPCRs belonging to all threemajor structural classes displayed markedly enhanced BiFCresponses on treatment with the appropriate agonist (Fig. 6B).Furthermore, cells treated with the proper agonists efficiently
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
induced distinct intracellular BiFC signals for 33 GPCRs, includingthe above-noted GPCRs, coupled with Gas, Gai, and Gaq withoutany background signal before stimulation (Table S2). The validityof the BiFC response for a broad spectrum of GPCRs suggests thatthe adenovirus-based b-arrestin BiFC assay can be used as a gen-eral platform to monitor GPCR activation.
Discussion
In this study, we described the systematic cloning of GPCRcDNAs into adenoviral BiFC vectors and the establishment of ab-arrestin BiFC assay in cells cotransduced with GPCR-VC andb-arrestin-VN adenoviruses (Fig. 1). The appearance of a BiFCsignal on agonist treatment enabled the activation of GPCR to beeasily monitored both qualitatively and quantitatively (Fig. 2).The adenovirus-based b-arrestin BiFC assay was suitable for mon-itoring GPCR activity in response to both agonists and antagonists(Fig. 3) and worked well with a broad spectrum of GPCRs (Fig. 6).Although slow maturation of the chromophore between VNm10and VC155 hampered real-time detection of GPCR and b-arrestininteraction (Fig. 4), the initial refolding of the chromophoreappeared to be fast and converted a transient interaction between
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GPCRs and b-arrestin into a stable fluorescent signal (Fig. 6). Byevaluating the Z0-factor, we showed that transient coexpressionof GPCR-VC and b-arrestin-VN by adenoviral transduction is suffi-cient for HCS of GPCR agonists (Fig. 5). Unlike in other assays forreal-time monitoring of GPCR and b-arrestin interaction, the signalderived from the b-arrestin BiFC assay allows end-point measure-ment. Furthermore, the adenovirus-based b-arrestin BiFC assayrequires minimal manipulation and instruments, and reagentsare not required for reading the signal.
The key methodological advance of this assay system is the useof adenovirus-based transient gene expression to implement theBiFC assay. Compared with plasmid-mediated BiFC, adenovirus-based BiFC produces more uniform BiFC response in a larger num-ber of cells (Fig. S5). Cell toxicity is also markedly reduced whenpartner proteins are expressed by adenoviral transduction. Theadenovirus-based b-arrestin BiFC assay has several advantagesover other stable cell-based assays for GPCR activation. The con-struction of stable cell lines requires time and labor for optimiza-tion, limiting the number of GPCRs and cell types available fordrug screening. By contrast, the high cotransduction efficiencyand uniform protein expression of adenovirus circumvent the needto make stable cell lines and ensure the availability of a broad spec-trum of GPCRs for monitoring GPCR activity in various cell types. Inaddition, the wide host range of adenovirus enables the examina-tion of GPCR ligand pharmacology in physiologically relevant celltypes.
The major limitation of using adenovirus is the requirement ofhands-on skills for constructing recombinant adenovirus. TheAdHTS technology still needs experience with adenovirus, but asingle preparation of TP-coupled AdBiFC vector is sufficient forthousands of one-step in vitro recombination, allowing high-throughput construction of recombinant adenoviruses [23]. Theuse of premade recombinant adenovirus only requires the amplifi-cation of virus by simply infecting HEK293 or HEK293A cells withthe virus.
A pharmacological approach using a b-arrestin BiFC assay hasbeen reported for neuropeptide Y receptors, ADRB2, and l-opioidreceptors in stably transfected cells [19,33,34]. Using anadenovirus-based b-arrestin BiFC assay, we validated the pharma-cological responses of 33 GPCRs. These GPCRs include receptorslinked to different classes of Ga proteins, activated by a varietyof ligand types (e.g., neurotransmitters, chemokines, peptides,and hormones), and belonging to three major structural classes:the rhodopsin-like, the secretin-like, and the metabotropic gluta-mate-like receptors (Table S2). Some of the GPCRs included inour list have few selective ligands, although they play importantphysiological roles and are involved in pathological conditions.Furthermore, like other b-arrestin recruitment assays, the adenovi-rus-based b-arrestin BiFC assay can be used to identify ligands fororphan GPCRs.
In addition to GPCRs, b-arrestin 2 is also known to regulate theendocytosis of the unconventional seven-transmembrane recep-tors frizzled and smoothened as well as non-seven-transmem-brane receptors such as the LDL receptor, nephrin, the TGF-btype III receptor, and the Na+/H+ exchanger NHE5 [3]. With theuse of AdHTS, which allows the rapid and simple cloning of a cDNAin an entry vector into an adenoviral vector [23], the adenovirus-based b-arrestin BiFC assay can be readily applied to the identifica-tion of other proteins that interact with b-arrestin 2.
In summary, this assay represents a rapid, simple, and univer-sal tool for monitoring GPCR activity in various cell types. Theadenovirus-based b-arrestin BiFC assay may be especially suit-able for screening the limited number of chemical or naturalproduct libraries for drug candidates with pharmacological sig-nificance that target GPCRs or for screening ligands for orphanGPCRs.
Please cite this article in press as: Y.B. Song et al., Monitoring G protein-coupledrescence complementation assay, Anal. Biochem. (2013), http://dx.doi.org/10.1
Acknowledgments
We thank Chang-Deng Hu and James Smith for generously pro-viding the plasmids containing the VN and VC sequences. Thiswork was supported by a National Research Foundation of Korea(NRF) grant (2012R1A2A2A01047175), the SRC/ERC Program(2011-0006426) funded by the Ministry of Education, Science,and Technology, Republic of Korea, and the KIOST in-houseprogram.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ab.2013.12.017.
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