A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression

Analytical Biochemistry 400 (2010) 163–172

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A reporter assay for G-protein-coupled receptors using a B-cell line suitable for stable episomal expression Satoshi Saeki, Hirofumi Kunitomo, Yoshiyasu Narita, Hideki Mimura, Tatsunari Nishi, Katsutoshi Sasaki * Drug Discovery Research Laboratories, Research Division, Kyowa Hakko Kirin, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan

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Article history: Received 27 August 2009 Received in revised form 3 December 2009 Accepted 26 January 2010 Available online 1 February 2010 Keywords: GPCR Reporter assay Chimeric G protein Estrogen-inducible expression Namalwa KJM-1 Episomal vector Constitutive activity Inverse agonist

a b s t r a c t We have established a cAMP response element (CRE)-mediated reporter assay system for G-protein-coupled receptors (GPCRs) using an oriP-based estrogen-inducible expression vector and the B-cell line (GBC53 or GBCC71) that expresses EBNA-1 and is adapted to serum-free culture. GBC53 harbors a GAL4–ER expression unit and a CRE–luciferase gene in the genome, and GBCC71 also harbors expression units for two chimeric Gas proteins (Gs/q and Gs/i). Introduction of a GPCR expression plasmid into GBC53 or GBCC71 creates polyclonal stable transformants in 2 weeks, and these are easily expanded and used for assays after induction of the GPCR expression. Using GBC53, we detected ligand-dependent signals of Gs-coupled GPCRs such as glucagon-like peptide 1 receptor (GLP1R) and b2 adrenergic receptor (b2AR) with high sensitivity. Interestingly, we also detected constitutive activity of b2AR. Using GBCC71, we detected ligand-dependent signals of Gq- or Gi-coupled GPCRs such as H1 histamine receptor and CXCR1 chemokine receptor in addition to Gs-coupled GPCRs. An agonist, antagonist, or inverse agonist was successfully evaluated in this system. We succeeded in constructing a 384-well high-throughput screening (HTS) system for GLP1R. This system enabled us to easily and rapidly make a large number of efficient GPCR assay systems suitable for HTS as well as ligand hunting of orphan GPCRs. Ó 2010 Elsevier Inc. All rights reserved.

G-protein-coupled receptors (GPCRs)1 attract much attention as drug targets because more than 30% of the approximately 500 clinically marketed drugs are targeting GPCRs [1]. Genome sequence analysis revealed the existence of approximately 360 nonolfactory GPCRs, leaving roughly 150 orphan GPCRs as of 2004 [2]. Functional assay systems for GPCRs are indispensable for drug screening and ligand hunting. As functional assay systems using mammalian cells, calcium flux assay, cAMP (30 ,50 -adenosine monophosphate) assay, and reporter assay are known to be useful and amenable to highthroughput screening (HTS) [3,4]. However, there is room for improvement in the areas of simpleness and efficiency. The first step in making a GPCR functional assay is heterologous expression of a GPCR in a host cell transiently or stably. Transient * Corresponding author. Fax: +81 55 986 7430. E-mail address: [email protected] (K. Sasaki). 1 Abbreviations used: GPCR, G-protein-coupled receptor; cAMP, cyclic 30 ,50 -adenosine monophosphate; HTS, high-throughput screening; GLP-1, glucagon-like peptide 1; IL-8, interleukin 8; db-cAMP, dibutyryl-cAMP; PMA, phorbol myristate acetate; GAL4–ER, chimeric transcription factor consisting of the hormone binding domain of estrogen receptor and the DNA-binding domain of yeast GAL4; GLP1R, glucagon-like peptide 1 receptor; b2AR, b2 adrenergic receptor; H1, H1 histamine receptor; CXCR1, CXC chemokine receptor 1; PCR, polymerase chain reaction; cDNA, complementary DNA; AT 1, angiotensin II type 1 receptor; bAT 1, bovine angiotensin type 1 receptor; CRE, cAMP response element; NECA, 50 -(N-ethylcarboxamide) adenosine; SE, standard errors; S/B, signal-to-background; S/N, signal-to-noise; SD, standard deviation; DMSO, dimethyl sulfoxide. 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.01.036

expression of GPCRs in COS-7 cells, CHO cells, or HEK293 cells is successfully used in ligand hunting and pharmacological analyses of agonists and antagonists of GPCRs. However, it is not suitable for large-scale screening in the areas of cost, labor, and consistency because the transient expression system requires repetitive preparation and transfection of plasmid DNAs. Stable expression systems are suitable for large-scale screening or HTS. However, it usually requires labor and 2–3 months to obtain a suitable stable transformant because stable expression is usually achieved by the introduction of a GPCR expression plasmid into the genome of host cells, such as CHO cells and HEK293 cells, followed by the selection of single clones highly expressing the GPCR. Furthermore, we should be careful to use a stable single clone in agonist screening as well as ligand hunting of orphan GPCRs. The biological responses of a single clone might not be mediated by the exogenously expressed GPCR because endogenously expressed GPCRs vary from clone to clone [5]. To overcome these drawbacks, we must use polyclonal assay cells such as stable transformants harboring an episomal-type expression plasmid or the above-described transient transformants. In this study, we have generated a GPCR assay system to overcome these problems. Previously, we generated a novel host–vector system using an oriP-based expression vector and a cell line, Namalwa KJM-1, which is a subline of the human Burkitt lymphoma cell line Namalwa and is adapted to serum-free suspension

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culture [6]. The vectors containing oriP, which is the replication origin of Epstein-Barr virus, are replicated and maintained stably in an episomal state in Namalwa KJM-1 that expresses the EBNA1 gene of Epstein-Barr virus. In this system, a large number of polyclonal stable transformants are easily obtained in 2 weeks. Serumfree culture is an advantage for high sensitivity and cost effectiveness. Suspension culture is easy to handle and suitable for a largescale culture. Based on this system, we generated a simple and efficient GPCR reporter assay system suitable for HTS and ligand hunting. Materials and methods Reagents b-Estradiol, glucagon-like peptide 1 (GLP-1) (7-36) amide, isoprenaline, propranolol, histamine, pyrilamine, forskolin, and A23187 were obtained from Sigma–Aldrich (St. Louis, MO). Recombinant human interleukin 8 (IL-8) was obtained from Bachem AG (Bubendorf, Switzerland). Dibutyryl-cAMP (db-cAMP) and phorbol myristate acetate (PMA) were purchased from Wako Pure Chemical Industries (Osaka, Japan). FuGENE 6 Transfection Reagent was purchased from Roche Diagnostics KK (Tokyo, Japan). Blasticidin S was supplied by Kaken Pharmaceutical (Tokyo, Japan). Hygromycin was obtained from Wako Pure Chemical Industries. Puromycin was obtained from Sigma–Aldrich. Geneticin G418 was obtained from Nacalai Tesque (Kyoto, Japan). Construction of GAL4–ER expression plasmid pGERbsrR2 Selection marker (the G418 resistance gene) of ERaAF2 in pM (a generous gift from Shigeaki Kato), which is an expression plasmid of GAL4–ER (a chimeric transcription factor consisting of the ligand-binding domain of estrogen receptor a and the DNAbinding domain of yeast GAL4), was replaced by the blasticidin S resistance gene to construct the GAL4–ER expression plasmid pGERbsrR2. A PvuII–EcoRI (blunted) fragment containing the blasticidin S resistance gene derived from the plasmid pSV2bsr (Kaken Pharmaceutical) and an AatII (blunted)–NdeI (blunted) fragment derived from ERaAF2 in pM were ligated. Orientation of the blasticidin S resistance gene was verified by sequencing. Construction of estrogen-inducible expression plasmids The plasmid pAGal9–luc (Fig. 1), which expresses the firefly luciferase gene under the control of the GAL4–ER-mediated promoter, was constructed as described elsewhere [7]. DNAs coding for human glucagon-like peptide 1 receptor (GLP1R) [8], human b2 adrenergic receptor (b2AR) [9], human H1 histamine receptor (H1) [10], and human CXC chemokine receptor 1 (CXCR1) [11] were obtained by polymerase chain reaction (PCR) using specific primers and the following templates: human pancreas complementary DNA (cDNA), human whole brain cDNA, human genomic DNA, and human leukocyte cDNA as templates, respectively. Forward and reverse PCR primers were designed to contain HindIII and NotI sites, respectively. The luciferase gene (HindIII–NotI DNA fragment) of pAGal9–luc was replaced by respective GPCR DNAs (HindIII–NotI DNA fragments) to make pAGal9–GLP1R, pAGal9–b2AR, pAGal9–H1, and pAGal9–CXCR1, respectively. Likewise, the luciferase gene (HindIII–NotI DNA fragments) of pAGal9–luc was replaced by the bovine angiotensin II type 1 receptor (AT 1) cDNA (HindIII–NotI DNA fragments) prepared from pAR1.8 [12] to make pAGal9–bAT 1 (bovine angiotensin II type 1 receptor). The empty vector pAGal9–nd was constructed by replacing the luciferase gene (HindIII–NotI DNA fragment) of pAGal9–luc

Fig. 1. Structure of the inducible expression plasmid pAGal9–luc. (A) pAGal9–luc consists of the following elements: the GAL4–ER-mediated inducible promoter containing five GAL4-binding motifs (UASg) derived from pFR–luc (Stratagene, La Jolla, CA), the firefly luciferase gene (luc) from pFR–luc, the G418 resistance gene (G418) expression unit, the ampicillin resistance gene (Amp) expression unit, and replication origin (oriP) of Epstein-Barr virus. To construct a GPCR expression plasmid, the luciferase gene was replaced by a DNA encoding a GPCR of interest. (B) The nucleotide sequence of the promoter region is shown. GAL4-binding motifs (bold), the TATA box (shadowed), the HindIII site (underlined), and the first methionine codon (italic) of luciferase are indicated.

with the stuffer sequence (2.5-kb HindIII–NotI DNA fragment) of pAMo [6]. Construction of CRE reporter plasmid pACREplucGI The plasmid pACREplucGI (Fig. 2) was constructed as described elsewhere [7]. pACREplucGI expresses the modified firefly luciferase gene luc+ derived from pGL3–Enhancer vector (Promega KK, Tokyo, Japan) under the control of the cAMP response element (CRE)-mediated promoter. Construction of chimeric Gas protein expression plasmid pAMopGsqMoGsiGI The plasmid pAMopGsqMoGsiGI (Fig. 3) was constructed as described elsewhere [7]. pAMopGsqMoGsiGI expresses the chimeric Gas proteins Gs/q and Gs/i in which C-terminal 5 amino acids of Gas are replaced with those of Gaq and Gai, respectively [13]. The DNA coding for human Gas4 protein [14] was obtained by PCR using specific primers and HL-60 cDNAs as templates. Forward and reverse PCR primers were designed to contain HindIII and Asp718 sites, respectively. The stuffer DNA (HindIII–Asp718 DNA fragment) of pAMoh [7] was replaced by the Gas4 DNA (HindIII– Asp718 DNA fragment) to make pAMoh-Gs. The C-terminal 5 amino acids of Gas4 encoded in pAMoh-Gs were replaced by those of Gaq using the synthesized DNA (50 -CACCTTCGTGAGTACAATCTG GTCTAACTCGAG-30 and 50 -GTACCTCGAGTTAGACCAGATTGTACTCA CGAAGGTGCATG-30 ) to make pAMoh-Gsq. The C-terminal 5 amino acids of Gas4 encoded in pAMoh-Gs were replaced by those of Gai

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Cell culture Namalwa KJM-1, a subline of the human Burkitt lymphoma cell line Namalwa, was maintained in a serum-free RPMI 1640–ITPSG medium as described previously [15]. RPMI 1640–ITPSG medium was prepared by adding 0.188% NaHCO3, 6 mmol/L L-glutamine, 10 mmol/L Hepes (pH 7.0), 3 mg/L insulin, 5 mg/L transferrin, 5 mmol/L sodium pyruvate, 125 nmol/L sodium selenite, 1 mg/ml galactose, and 100 U/ml penicillin–streptomycin to RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan). KJMGER8 cells were maintained in RPMI 1640–ITPSG medium containing 2 lg/ ml blasticidin S. GBC53 cells were maintained in RPMI 1640–ITPSG medium containing 2 lg/ml blasticidin S and 0.3 mg/ml hygromycin. GBCC71 cells were maintained in RPMI 1640–ITPSG medium containing 2 lg/ml blasticidin S, 0.3 mg/ml hygromycin, and 2 lg/ml puromycin. Establishment of host cell line KJMGER8 suitable for estrogen-inducible expression

Fig. 2. Structure of the CRE–luciferase plasmid pACREplucGI. (A) pACREplucGI consists of the following elements: the CRE-mediated promoter containing 16 repeats of synthetic cAMP response element (CRE), the modified firefly luciferase (luc+) gene, the hygromycin resistance gene (hyg) expression unit, and the ampicillin resistance gene (Amp) expression unit. (B) The nucleotide sequence of the promoter region is shown. CRE motifs (bold), the TATA box (shadowed), the HindIII site (underlined), and the first methionine codon (italic) of luciferase are indicated.

Fig. 3. Structure of the chimeric Gas-protein expression plasmid pAMopGsqMoGsiGI. pAMopGsqMoGsiGI consists of the following elements: the chimeric G-protein (Gs/q) expression unit, the chimeric G-protein (Gs/i) expression unit, the puromycin resistance gene (pur) expression unit, and the ampicillin resistance gene (Amp) expression unit. Expression of the chimeric G proteins is under the control of long terminal repeat promoters of Moloney murine leukemia virus (Pmo).

using the synthesized DNA (50 -CACCTTCGTGATTGTGGTCTCTTT TAAGCTTG-30 and 50 -GTACCAAGCTTAAAAGAGACCACAATCACGAA GGTGCATG-30 ) to make pAMoh-Gsi. In making pAMoh-Gsq and pAMoh-Gsi, the SphI–Asp718 DNA fragment (encoding C-terminal 8 amino acids of Gas4) of pAMoh-Gs was replaced by the abovesynthesized DNAs.

Namalwa KJM-1 cells were transfected with the GAL4–ER expression plasmid pGERbsrR2 by electroporation as described previously [6] and were selected in the presence of 2 lg/ml blasticidin S to obtain stable transformants. Many single clones were isolated and stably transfected with pAGal9–luc to check estrogeninducible expression of luciferase activities. Each single clone was transfected with pAGal9–luc by lipofection as described below and was selected in the presence of 0.5 mg/ml G418 and 2 lg/ml blasticidin S for 2 weeks to obtain polyclonal stable transformants. The transformants were cultured in the presence or absence of 10 nM b-estradiol for 24 h, and luciferase activities were examined. We selected the single clone KJMGER8 that showed higher induction rate and lower background activity. Establishment of host cell line GBC53 suitable for reporter assays of Gscoupled GPCRs KJMGER8 cells were transfected by electroporation with a FspIdigested pACREplucGI (Fig. 2), which contained a CRE luciferase reporter unit and the hygromycin resistance gene, and were selected in the presence of 0.3 mg/ml hygromycin and 2 lg/ml blasticidin S to obtain stable transformants. Many single clones were isolated, and their response to the adenosine agonist 50 -(N-ethylcarboxamide) adenosine (NECA) was examined. Because KJMGER8 cells express Gs-coupled adenosine A2A receptor endogenously, single clones with CRE–luciferase gene in the genome should respond to NECA with increased luciferase activities. Each clone was cultured in the presence or absence of 100 nM NECA for 6 h, and luciferase activities were examined. We selected the single clone GBC53 that showed higher induction rate and lower background activity. Establishment of the host cell line GBCC71 suitable for reporter assays of Gs-, Gq-, and Gi-coupled GPCRs GBC53 cells were transfected by electroporation with an AseIdigested pAMopGsqMoGsiGI (Fig. 3), which contained expression units of two chimeric Ga proteins (Gs/q and Gs/i), and were selected in the presence of 2 lg/ml puromycin, 0.3 mg/ml hygromycin, and 2 lg/ml blasticidin S to obtain stable transformants. Many single clones were isolated and stably transfected with pAGal9– bAT 1 to check their ability to detect Gq- and/or Gi-coupled GPCRs. Each single clone was transfected with pAGal9–bAT 1 by lipofection and was selected in the presence of 0.5 mg/ml G418, 2 lg/ ml puromycin, 0.3 mg/ml hygromycin, and 2 lg/ml blasticidin S for 2 weeks to obtain polyclonal stable transformants. The trans-

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formants were cultured in the presence of 10 nM b-estradiol for 24 h to induce expression of bovine AT 1, and luciferase activities were examined 6 h after cultivation in the presence or absence of 100 nM angiotensin II. We selected the single clone GBCC71 that showed higher induction rate and lower background activity. GPCR reporter assays Transfection of an oriP-based inducible GPCR expression plasmid to GBC53 or GBCC71 cells was performed using FuGENE 6 Transfection Reagent (Roche Diagnostics) according to the manufacturer’s instructions. In brief, the host cells were plated in 6-well plates at 2  106 cells/well in 2 ml of antibiotic-free RPMI 1640– ITPSG medium. Plasmid (1 lg) and FuGENE 6 (6 ll), which were dissolved separately in 100 ll of the same medium, were mixed and added to the cells. RPMI 1640–ITPSG medium (3 ml) and antibiotics were added to the cells 6 and 24 h later, respectively. Polyclonal stable transfectants were obtained in 2 weeks and were expanded by culture in the presence of the antibiotics described above. After cultivation in the presence or absence of 10 nM bestradiol for 24 or 48 h, cells were plated in 96-well plates at 50,000 cells/well in 90 ll of RPMI 1640–ITPSG medium and were added to compound solution (10 ll) for 6 h. Then 50 ll of reconstituted Bright-Glo (Promega KK) diluted with the same volume of lysis buffer (0.1% Triton X-100 and 11 mM KH2PO4, pH 7.4) was added to each well, and luminescence was counted 5 min later with TopCount NXT (PerkinElmer, Wellesley, MA, USA) for 1 s/well. In antagonist assays, cells were cultured with compounds for 15 min prior to the addition of agonists or ligands. In assays to detect constitutive activities, cells were cultured in the presence or absence of 10 nM b-estradiol for 24 h, and luciferase activities were compared. In inverse antagonist assays, luciferase assays were performed for 24 or 48 h after the concurrent addition of compounds and b-estradiol. In 384-well plate format assays, cells were plated in 384-well plates at 40,000 cells/well in 40 ll of RPMI 1640–ITPSG medium. Luminescence was measured with FDSS 6000 (Hamamatsu Photonics, Shizuoka, Japan) for 30 s/plate.

Host cell (GBCC71)

CRE-luc

Nucleus

Gs/q Gs/i pAGal9 vector + GPCR cDNA

Inducible GPCR expression plasmid

pAGal9-GPCR

Transfection Antibiotic selection 2 Weeks

Polyclonal stable transformant

Culture expansion

-Estradiol addition Reporter assay cell GPCR

Data analysis All data are presented as means ± standard errors (SE) of three independent experiments. EC50 values and IC50 values were calculated using SAS 9.1.3 software (SAS Institute, Heidelberg, Germany). Antagonist potency (KB value) was calculated by Schild plot analysis. The signal-to-background (S/B) ratio was calculated using the following equation: S/B = mean signal/mean background. The signal-to-noise (S/N) ratio was calculated using the following equation: S/N = (mean signal–mean background)/standard deviation (SD) of background. Z0 values were calculated using the following equation [16]: Z0 = 1  (3SD of positive control + 3SD of negative control)/(mean of positive control  mean of negative control).

Results Strategy to make a simple and efficient functional GPCR assay system Our assay system is outlined in Fig. 4. Based on the host–vector system using Namalwa KJM-1 and the oriP-based expression vector, we generated a CRE-mediated reporter assay system. We constructed host cells (GBC53 and GBCC71) suitable for inducible expression and CRE reporter assay from Namalwa KJM-1. GBC53 is applicable to reporter assays of Gs-coupled GPCRs, and GBCC71 is applicable to reporter assays of Gs-, Gq-, and Gi-coupled GPCRs.

Fig. 4. Schematic representation of the GPCR assay system using the inducible expression vector (pAGal9 vector: pAGal9–luc or pAGal9–nd) and GBCC71 cells. GBCC71 harbors a CRE–luciferase gene and expression units for two chimeric Gas proteins (Gs/q and Gs/i) in the genome. Transfection of an inducible GPCR expression plasmid (pAGal9–GPCR) into GBC7C1 cells creates polyclonal stable transformants in 2 weeks. The transformants are easily expanded for a large-scale assay. Assay cells expressing the GPCR are prepared by culture with b-estradiol for 24 or 48 h. GPCR assay using GBC53 cells is performed in the same way. GBC53 cells harbor the CRE–luciferase gene in the genome but not the expression units for the chimeric Gas proteins.

As estrogen-inducible expression vectors containing oriP, we constructed pAGal9–luc (Fig. 1) and pAGal9–nd. Calcium flux assays are suitable for Gaq- or Ga11-coupled GPCRs. In the case of GPCRs that do not couple to Gaq or Ga11, cotransfection of the GPCR with promiscuous Ga16 or chimeric Gaq proteins such as Gq/i and Gq/s, in which C-terminal several amino acids of Gaq are replaced with those of Gai and Gas, respectively, are known to be effective to detect activity [17]. However, signals of some Gs-coupled GPCRs are not detected by the above methods. Therefore, to compensate for calcium flux assay, we decided to make a reporter assay system suitable for detection of activity of Gs-coupled GPCRs using a CRE–luciferase gene. To de-

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tect signals of GPCRs that do not couple to Gas, we have used chimeric Gas proteins Gs/q and Gs/i, in which C-terminal 5 amino acids of Gas are replaced with those of Gaq and Gai, respectively [13]. Inducible expression system was used to keep normal cell growth and to avoid population change due to clone-to-clone differences in the expression level of GPCRs. Furthermore, it is expected that a reporter assay system capable of highly inducible expression of a GPCR enables us to detect constitutive activity of the GPCR. Constitutive activity of GPCRs is useful for screening of inverse agonists [18]. Construction of estrogen-inducible expression system using Namalwa KJM-1 cells A host cell line KJMGER8 suitable for estrogen-inducible expression [19,20] was constructed by integrating a GAL4–ER expression vector containing the blasticidin S resistance gene into the genome of Namalwa KJM-1 cells, followed by selection of highly responsive clones as described in Materials and methods. As an estrogeninducible expression plasmid, we constructed pAGal9–luc (Fig. 1), which expressed firefly luciferase under the control of a promoter consisting of five GAL4–ER-binding motifs and a TATA sequence. We are able to construct estrogen-inducible plasmids for GPCRs of interest by replacing the luciferase gene of pAGal9– luc with the GPCR gene (Fig. 4). We also constructed the empty vector pAGal9–nd by replacing the luciferase gene with the stuffer sequence. Transfection of pAGal9–luc into KJMGER8 cells generated polyclonal stable transformants in 2 weeks. When the transformants were cultured in the presence of 10 nM b-estradiol, luciferase activity increased in a linear fashion from 2 to 12 h, reached a maximal ( 18,000-fold) at 12 h, and was maintained until 48 h or more (Fig. 5). Luciferase activity increased according to the concentration of b-estradiol and was saturated at 1 nM (data not shown). Establishment of GBC53 cell suitable for reporter assays of Gs-coupled GPCRs

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clones that showed lower background activity and higher luciferase activity in response to agonist (NECA) of adenosine A2A receptor, which was a Gs-coupled GPCR endogenously expressed in KJMGER8. GBC53 cells showed 47-fold luciferase activity after stimulation by 100 nM NECA for 6 h (data not shown). Establishment of GBCC71 cell suitable for reporter assays of Gs-, Gq-, and Gi-coupled GPCRs To give GBC53 cells the ability to detect signals of Gq- and Gicoupled GPCRs, chimeric Ga protein (Gs/q and Gs/i, respectively) genes were transfected to GBC53 cells. A plasmid containing a Gs/q expression unit, a Gs/i expression unit, and the puromycin resistance gene was integrated into the genome of the GBC53 cell. From a number of single clones obtained, we screened the single clones by the ability to detect signals from bAT 1 (Gq- and Gi-coupled receptor). Each clone stably transfected with pAGal9–bAT 1 was cultured with 10 nM b-estradiol for 24 h to express bAT 1 and was subsequently stimulated by 100 nM angiotensin II. We selected the single clone GBCC71 that showed lower background and higher luciferase activity in response to the ligand. Without bestradiol treatment, GBCC71 cells stably transfected with pAGal9–bAT 1 showed no response to angiotensin II. Both the adenylylcyclase activator forskolin (100 lM) and the membrane-permeable cAMP analogue db-cAMP (10 mM) increased luciferase activity in GBCC71 cells (Fig. 6). On the other hand, neither the protein kinase C activator PMA (100 nM), the calcium ionophore A23187 (1 lM), nor simultaneous stimulation of PMA and A23187 increased luciferase activity in GBCC71 cells (Fig. 6). In addition, GBC53 cells expressing bAT 1 showed no response to angiotensin II addition (data not shown). These results indicate that detection of bAT 1 signals in GBCC71 cells is mediated by Gs/q and/or Gs/i. Reporter assays for Gs-coupled GPCRs using GBC53 or GBCC71 We constructed estrogen-inducible expression plasmids pAGal9–GLP1R that express Gs-coupled human GLP1R. Transfection

We constructed the GBC53 cell by integrating a CRE–luciferase reporter plasmid containing the hygromycin resistance gene into the genome of KJMGER8 cells, followed by selection of single

Fig. 5. Time course of induction rate in the Gal4–ER-based inducible expression system. KJMGER8 cells stably transfected with pAGal9–luc cells (50,000 cells/well) were cultured with or without 10 nM b-estradiol for the indicated time period prior to luciferase assay. Luciferase activity was measured as described in Materials and methods. Luciferase activities in the presence of b-estradiol are shown as induction rate (fold) over the controls (luciferase activities in the absence of b-estradiol). Data are presented as means ± SE of three independent experiments done in duplicate.

Fig. 6. Response of the host cell line GBCC71 to cAMP inducers and other compounds. GBCC71 cells (50,000 cells/well) were cultured with the indicated compounds for 6 h prior to luciferase assay. The following concentrations were used: forskolin, 100 lM; db-cAMP, 10 mM; PMA, 100 nM; and A23187, 1 lM. Luciferase activities are shown as induction rate (fold) over the control (luciferase activity in the absence of compounds). Data are presented as means ± SE of three independent experiments done in duplicate.

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of pAGal9–GLP1R (1 lg) into GBC53 or GBCC71 cells (2  106 cells) generated polyclonal stable transformants (GBC53/GLP1R or GBCC71/GLP1R, 5  107 cells) in 2 weeks. The transformants are grown in serum-free suspension culture with a doubling time of approximately 2 days. The transformants were cultured in the presence of 10 nM b-estradiol for 48 h to induce GLP1R expression. After 6 h of stimulation with GLP-1 in a 96-well plate (50,000 cells/ well), GBC53/GLP1R showed increased luciferase activity in response to GLP-1 in a dose-dependent manner, with an EC50 value of 275 pM and a maximal S/B ratio of 273 (Fig. 7A). Likewise, GBCC71/GLP1R showed increased luciferase activity in response to GLP-1 in a dose-dependent manner, with an EC50 value of 620 pM and a maximal S/B ratio of 131 (Fig. 7C). Without b-estra-

diol treatment, both of the transformants showed no response to GLP-1 (Figs. 7A and 7C). With or without b-estradiol treatment, empty vector-transfected cells showed no response to GLP-1 (data not shown). Likewise, polyclonal stable transformants (GBC53/b2AR or GBCC71/b2AR) were prepared by transfection of pAGal9–b2AR, which expresses Gs-coupled human b2AR, into GBC53 or GBCC71 cells. b-Estradiol-treated, but not -untreated, GBC53/b2AR responded to isoprenaline in a dose-dependent manner, with an EC50 value of 4.18 nM and a maximal S/B ratio of 35.8 (Fig. 7B). b-Estradiol-treated, but not -untreated, GBCC71/b2AR responded to isoprenaline in a dose-dependent manner, with an EC50 value of 10.8 nM and a maximal S/B ratio of 20.9 (Fig. 7D).

Fig. 7. Ligand dose–response curves of reporter assay cells for receptors (GLP1R, b2AR, H1, and CXCR1). (A and B) Using GBC53, assay cells for GLP1R (GBC53/GLP1R) (A) and b2AR (GBC53/b2AR) (B) were generated. (C–F) Using GBCC71, assay cells for GLP1R (GBCC71/GLP1R) (C), b2AR (GBCC71/b2AR) (D), H1 (GBCC71/H1) (E), and CXCR1 (GBCC71/ CXCR1) (F) were generated. The cells (50,000 cells/well) were cultured in the presence (j) or absence (h) of 10 nM b-estradiol for 48 h and then cultured with the indicated concentration of the ligand (GLP-1 amide, isoprenaline, histamine, or rIL-8) for 6 h prior to luciferase assay. Luciferase activities are shown as induction rate (fold) over the control (luciferase activity in the absence of b-estradiol and ligands). Data are presented as means ± SE of three independent experiments done in duplicate.

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Reporter assays for Gq- and/or Gi-coupled GPCRs using GBCC71 We constructed the plasmid pAGal9–H1 for estrogen-inducible expression of Gq-coupled human H1. Using practically the same method as for the GLP1R and b2AR receptors, we prepared polyclonal stable transformants (GBCC71/H1) by transfection of pAGal9–H1 into GBCC71 cells and examined whether GBCC71/H1 could detect signals from H1. The b-estradiol-treated GBCC71/H1 responded to histamine from 100 nM in a dose-dependent manner, with an S/B ratio of 37 at 100 lM (Fig. 7E). Likewise, polyclonal stable transformants (GBCC71/CXCR1) were prepared by transfection of pAGal9–CXCR1, which expresses Gi-coupled human CXCR1, into GBCC71 cells. The b-estradiol-treated GBCC71/CXCR1 responded to recombinant human IL-8 from 10 nM in a dose-dependent manner, with an S/B ratio of 67 at 1000 nM (Fig. 7F). EC50 values could not be calculated in both assays because the response did not reach a plateau at the maximum ligand concentration. Neither b-estradiol-untreated transformants nor empty vector-transfected cells showed any response to each ligand. Evaluation of GPCR antagonists To evaluate whether the GPCR reporter assay cells constructed using GBCC71 could be used for antagonist screening, antagonistic activities of the b2 antagonist propranolol and the H1 antagonist pyrilamine were examined using GBCC71/b2AR and GBCC71/H1, respectively. Because the assay tolerated dimethyl sulfoxide (DMSO) up to 1% with a slight change in activity (Fig. 8), the following assays were performed in the presence of 0.2% DMSO. Propranolol inhibited isoprenaline-induced luciferase activities in a dose-dependent manner to give a KB value of 766 pM (Fig. 9A). Pyrilamine inhibited luciferase activities induced by 1 lM histamine in a dose-dependent manner with an IC50 value of 2.95 nM (Fig. 9B). Detection of constitutive activity of b2AR

Fig. 9. Antagonism on ligand-mediated activation of Gs-coupled b2AR and Gqcoupled H1. After a 48-h culture with 10 nM b-estradiol, reporter assay cells (50,000 cells/well) for b2AR (GBCC71/b2AR) (A) or H1 (GBCC71/H1) (B) were incubated with antagonists (propranolol or pyrilamine) for 15 min, followed by a 6-h culture with ligands (isoprenaline or histamine) prior to luciferase assay. The antagonist concentrations used were 0 nM (d), 1 nM (s), 10 nM (j), and 100 nM (h). Luciferase activities are shown as percentages of the maximum activity for each receptor. Data are presented as means ± SE of three independent experiments done in duplicate.

b2AR, but not GLP1R, is known to show weak constitutive activity [21]. GBC53/b2AR cells were cultured with or without 10 nM bestradiol for 24 h, and luciferase activity was measured. b-Estradiol-treated GBC53/b2AR showed 7.9-fold luciferase activity compared with b-estradiol-untreated cells (Fig. 10A). When GBC53/ b2AR cells were cultured with 10 nM b-estradiol in the presence of the b2AR neutral antagonist propranolol or the b2AR inverse

agonist ICI-118551 [22] for 24 h, the increased luciferase activity was inhibited by ICI-118551, but not by propranolol, in a dosedependent manner (Fig. 10B). In contrast, GBC53/GLP1R showed nearly the same luciferase activity with or without b-estradiol treatment (Fig. 10A). These results clearly indicate that constitutive activity of b2AR is detected in this reporter assay system. Application of reporter assay systems using GBCC71 to HTS Using GBCC71/GLP1R, we evaluated the applicability of this GPCR assay system to HTS. For example, it is possible to obtain a large number of polyclonal stable transformants (GBCC71/GLP1R, 1  1010 cells) in 1 month by transfection of pAGal9–GLP1R (1 lg) into GBCC71 cells (2  106 cells). We succeeded in constructing an HTS system in a 384-well format (40,000 cells/well) with a Z0 value of 0.63, which was calculated from data of GLP-1 (3 nM)-stimulated (n = 704) and unstimulated (n = 704) wells (Fig. 11). The S/N ratio and S/B ratio were 138.0 and 40.9, respectively. The assay was performed in the presence of 0.2% DMSO. Plate-to-plate variability was hardly observed.

Fig. 8. DMSO tolerance of the GBCC71 assay cells. Reporter assay cells for GLP1R (GBCC71/GLP1R) were cultured with 10 nM b-estradiol for 48 h and seeded to a 96well plate (50,000 cells/well). The cells were cultured with DMSO at the indicated concentration in the presence () or absence (e) of 1 nM GLP-1 for 6 h prior to luciferase assay. Luciferase activities are shown as luminescence counts per second (cps). Data are presented as means ± SE of three independent experiments done in duplicate.

Discussion We have established a simple and efficient CRE reporter assay system for GPCRs using an inducible expression plasmid and a human B-cell line suitable for serum-free suspension culture as a host

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Fig. 10. Detection of constitutive activity of b2AR. (A) Reporter assay cells (50,000 cells/well) for b2AR (GBC53/b2AR) or GLP1R (GBC53/GLP1R) were cultured with or without 10 nM b-estradiol for 24 h prior to luciferase assay. Luciferase activities in the presence of b-estradiol are shown as induction rate (fold) over the controls (luciferase activities in the absence of b-estradiol). (B) Reporter assay cells (50,000 cells/well) for b2AR (GBC53/b2AR) were cultured with 10 nM b-estradiol in the presence of the neutral antagonist propranolol (s) or the inverse agonist ICI-118551 (d) for 24 h prior to luciferase assay. Luciferase activities are shown as percentages of the control (luciferase activity in the absence of compounds). Data are presented as means ± SE of three independent experiments done in duplicate.

the cells can be frozen after culture expansion and then thawed and used.

Merits of inducible expression of a GPCR

Fig. 11. Reporter assay for GLP1R in 384-well format. Reporter assay cells for GLP1R (GBCC71/GLP1R) were cultured with 10 nM b-estradiol for 48 h and plated to a 384well plate (40,000 cells/well). The cells were cultured in the presence (d, n = 704) or absence (s, n = 704) of 3 nM GLP-1 for 6 h prior to luciferase assay. The assay was performed in the presence of 0.2% DMSO. Luciferase activities are shown as luminescence counts per second (cps).

(Fig. 4). As host cells, we constructed GBC53 cells for Gs-coupled GPCRs and constructed GBCC71 cells for Gs-, Gq-, and Gi-coupled GPCRs. Because the host cells express the EBNA-1 gene and the expression plasmid contains oriP, transfection of the plasmid into the host cells yields a large number of polyclonal stable transformants in a short period. Because host cells are nonadherent cells, the assay system is easy to handle and suitable for a large-scale culture. Using this system, we detected signals of Gs-, Gq-, and Gi-coupled GPCRs, and we successfully evaluated an agonist, antagonist, or inverse agonist. The significant advantages of this assay platform are discussed below in detail. Rapid and mass preparation of assay cells Using this system, a large number of polyclonal stable transformants are generated in 2 weeks after transfection. The cells are easily expanded by serum-free suspension culture for large-scale screening. For example, 1  1010 cells (enough for 250,000 assays in a 384-well format) are obtained in 1 month by single transfection of a plasmid (1 lg) into the host cells (2  106 cells). Longer cultivation, multiple transfections, or scaling up the size of transfection is easily performed to obtain more assay cells. In addition,

Constitutive overexpression of a GPCR is likely to affect cell growth, causing a gradual loss of the overexpressing cells in a prolonged culture. In the case of polyclonal transformants, this is a major problem to maintain stability and high sensitivity of the assay system. We overcame the problem by making the host–vector system, allowing negligible basal expression and highly induced expression with an induction rate of 18,000-fold. The induction rate of this system is higher than the induction rates of other systems, including an ecdysone-inducible expression system [23–25] and a tetracycline system [26]. Because the basal (uninduced) expression level of GPCRs (b2AR, H1, GLP1R, and CXCR1) was very low, any ligand-dependent signals were not detected in the uninduced cells. As expected, growth inhibition was not observed in culture without induction. In addition, the inducible expression system is useful for avoiding desensitization and/or downregulation of the receptors by ligands that may exist in the culture medium [25,27]. The estrogen-inducible expression system is not commonly used because of potential side effects mediated by endogenous estrogen receptors. In the case of Namalwa-derived cell lines (KJMGER8, GBC53, and GBCC71), we did not observe side effects on reporter activity and cell growth. Because nuclear estrogen receptor (ERa) is not detected in Namalwa KJM-1 cells by Western blot analysis using anti-ER antibody (data not shown), side effects are negligible in our system.

Detection of signals from Gs-, Gq-, and Gi-coupled GPCRs The CRE reporter assay system using GBC53 cells was highly sensitive to Gs-coupled GPCRs such as b2AR and GLP1R. To make this assay system more versatile, GBCC71 was constructed by introducing expression units of chimeric Gas proteins (Gs/q and Gs/i) into GBC53. In the CRE reporter assay system using GBCC71 cells, we detected signals from Gq- and/or Gi-coupled receptors (H1 and CXCR1) in addition to Gs-coupled receptors (b2AR and GLP1R) under the same assay conditions. This is an advantage of our system because other assay platforms, including Xenopus melanophores [28] and a known reporter assay system [29], need to

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use different assay conditions to detect signals of Gs-, Gq-, and Gicoupled receptors even in the case of using the same assay cells.

verse agonists of constitutively active orphan GPCRs without ligand information.

Sensitivity and S/B ratio

Suitability for identification of real hits

The assay system using GBC53 detected signals from the Gscoupled receptors (GLP1R and b2AR) with high sensitivity comparable to that of well-validated GPCR assay systems. GBC53/GLP1R assay cells responded to GLP-1 with an EC50 value of 275 pM, which was comparable to the EC50 value (310 pM) in the intracellular cAMP measurement assay using GLP1R-transfected CHO cells [30]. GBC53/b2AR assay cells responded to isoprenaline with an EC50 value of 4.18 nM, which was comparable to the EC50 value (1.78 nM) in the intracellular cAMP measurement assay using b2AR-transfected CHO cells [31]. These results indicate that our assay system using GBC53 is comparable to well-validated GPCR assay systems using the GPCR-overexpressing CHO cell that was selected as a single clone to show high response to the ligand. Maximal S/B ratios of our system (131 in GBC53/GLP1R and 35.8 in GBC53/b2AR) were superior to those of other GPCR reporter assay systems [29,32]. The assay system using GBCC71 detected signals from the Gscoupled GPCRs (GLP1R and b2AR) with 2- to 3-fold higher EC50 values (620 pM in GBCC71/GLP1R and 10.8 nM in GBCC71/b2AR) than in the assays using GBC53. However, the sensitivity and S/B ratio were enough for screening of agonists and antagonists. Propranolol inhibited luciferase activities induced by isoprenaline in GBC71/ b2AR assay cells with a KB value of 766 pM (Fig. 9A), which was comparable to the KB value (398 pM) in the intracellular cAMP measurement assay using b2AR-transfected CHO cells [31]. The assay system using GBCC71 detected signals from the Gqand/or Gi-coupled GPCRs (H1 and CXCR1) with approximately 30-fold lower sensitivity than calcium flux assay systems [33,34]. The lower sensitivity is attributable to insufficient coupling between the GPCR and the chimeric Gas proteins. However, the assay system is especially useful for agonist screening or ligand hunting of orphan GPCRs without information of coupling G proteins. Pyrilamine inhibited luciferase activities induced by 1 lM histamine with an IC50 value of 2.95 nM (Fig. 9B), which was comparable to the IC50 value (10 nM when stimulated by 1 lM histamine) in the calcium flux assay using H1-transfected CHO cells [33]. These results indicate that the assay system using GBCC71 is also useful for antagonist screening of Gq- and/or Gi-coupled GPCRs. The substantially high S/B ratio in our assay system is attributable to the intrinsic amplifying property of the luciferase reporter, continuous serum-free culture causing low background, and inducible high expression of GPCRs.

In general, reporter gene assays give false-positive compounds that interact with signaling molecules other than a target GPCR itself. In our system, many GPCR assay systems are easily and simultaneously constructed in practically the same way. Using the other GPCR assay systems as controls, we are able to select compounds that interact only with the target GPCR. In addition, we should be careful to use a stable single clone in agonist screening as well as ligand hunting of orphan GPCRs as described in the introductory paragraphs of the article. Because our system is using polyclonal stable transformants, we are able to neglect difference of responsiveness based on single clones. Therefore, our assay system is very useful for ligand hunting of orphan GPCRs in addition to screening of agonists, antagonists, and inverse agonists of ligand-known GPCRs.

Detection of constitutive activity of GPCRs Some GPCRs show constitutive activities without ligand stimulation. It is known that inverse agonists inhibit constitutive activities and that some launched drugs show inverse agonist activities [35]. Therefore, compound screening using constitutive activities of GPCRs is attractive to find novel drugs. However, it was difficult to detect constitutive activities efficiently because the activities are very low, especially for wild-type b2AR [36]. Constitutive activities of GPCRs are sometimes detected by second messenger measurements or reporter assays in transient expression systems [37]. Our assay system using GBC53 clearly detected constitutive activity of b2AR with an S/B ratio of 7.9, which was enough for evaluation of the inverse agonist ICI-118551. Our assay system using GBC53 (for Gs-coupled GPCRs) and GBCC71 (for Gs-, Gq-, and Gicoupled GPCRs) would be very useful for detection of constitutive activities of GPCRs, leading to inverse agonist screening. In particular, our assay system is expected to make it possible to screen in-

Suitability for large-scale screening and HTS The GLP1R assay system constructed with GBCC71 was successfully applied to a 384-well plate format with a Z0 value of more than 0.6. In general, Z0 values of more than 0.5 indicate excellent assays [38] and are suitable for HTS and automation. Because the host cells are serum-free adapted nonadherent cells, our assay system is cost-effective, easy to handle, and suitable for a large-scale culture and automation. In this study, to overcome the problems of current GPCR assay systems using transient or stable expression system (see introductory paragraphs), we generated a CRE reporter assay system using polyclonal stable transformants capable of inducible expression of GPCRs based on the EBNA-1–oriP host–vector system. The system was designed to compensate for demerits (e.g., detection of signals from Gs-coupled GPCRs) of calcium flux assays. Furthermore, the system showed a unique feature to detect constitutive activities of GPCRs efficiently. Because constitutive activities are not detected by calcium flux assays, our system has advantages, especially in assays using constitutive activities. Our system would be widely useful for compound screening for ligand-known and -unknown GPCRs. Acknowledgment We thank Kikuko Funayama, Eri Suzuki, and Megumiko Magara for excellent technical assistance. References [1] A.L. Hopkins, C.R. Groom, The druggable genome, Nat. Rev. Drug Discov. 1 (2002) 727–730. [2] A. Wise, S.C. Jupe, S. Rees, The identification of ligands at orphan G-protein coupled receptors, Annu. Rev. Pharmacol. Toxicol. 44 (2004) 43–66. [3] P.A. Johnston, P.A. Johnston, Cellular platforms for HTS: Three case studies, Drug Discov. Today 7 (2002) 353–363. [4] M.J. Wigglesworth, L.A. Wolfe, A. Wise, Orphan seven transmembrane receptor screening, Ernst Schering Found. Symp. Proc. 2 (2006) 105–143. [5] P.G. Szekeres, Functional assays for identifying ligands at orphan G proteincoupled receptors, Receptors Channels 8 (2002) 297–308. [6] K. Sasaki, E. Watanabe, K. Kawashima, S. Sekine, T. Dohi, M. Oshima, N. Hanai, T. Nishi, M. Hasegawa, Expression cloning of a novel Galb1–3/1–4 GlcNAc a2, 3-sialyltransferase using lectin resistance selection, J. Biol. Chem. 268 (1993) 22782–22787. [7] K. Sasaki, K. Miura, S. Saeki, M. Yoshizawa, K. Kishimoto, H. Kunitomo, T. Nishi, M. Obinata, Endocrine cell line and method of using the same, international patent application PCT/JP2003/004840 (2003). [8] J.S. Dillon, Y. Tanizawa, M.B. Wheeler, X.H. Leng, B.B. Ligon, D.U. Rabin, H. YooWarren, M.A. Permutt, A.E. Boyd 3rd, Cloning and functional expression of the human glucagon-like peptide-1 (GLP-1) receptor, Endocrinology 133 (1993) 1907–1910.

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