Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
Title page:
Purinergic receptor transactivation by the β2-adrenergic receptor increases intracellular Ca2+ in non-excitable cells.
Wayne Stallaert, Emma T van der Westhuizen, Anne-Marie Schönegge, Bianca Plouffe, Mireille Downloaded from
Hogue, Viktoria Lukashova, Asuka Inoue, Satoru Ishida, Junken Aoki, Christian Le Gouill &
Michel Bouvier. molpharm.aspetjournals.org
Department of Biochemistry, Université de Montréal, Montréal, QC, Canada (WS, ETvdW, A-
MS, BP, MB). Institute for Research in Immunology and Cancer, Université de Montréal,
Montréal, QC, Canada (WS, ETvdW, A-MS, BP, MH, VL, CLG, MB). Monash Institute for at ASPET Journals on September 30, 2021
Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia (ETvdW). Graduate
School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (AI, SI, JA).
Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and
Technology (PRESTO), Kawaguchi, Saitama, Japan (AI). Japan Agency for Medical Research
and Development, Core Research for Evolutional Science and Technology (AMED-CREST),
Chiyoda-ku, Tokyo, Japan (JA).
1 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Running title page: a) Running title: β2AR transactivation of purinergic receptors b) Corresponding author: Michel Bouvier, IRIC - Université de Montréal, P.O. Box 6128
Succursale Centre-Ville, Montréal, Qc. Canada, H3C 3J7. Tel: +1-514-343-6319. Fax: +1-
514-343-6843. Email: [email protected] c) Number of: text pages: 37 Downloaded from tables: 0
figures: 8
references: 58 molpharm.aspetjournals.org
Number of words in: abstract: 238
introduction: 572 (max 750)
discussion: 1029 (max 1500) at ASPET Journals on September 30, 2021 d) Non standard abbreviations: 2-APB, 2-aminoethoxydiphenyl borate; AC, adenylyl cylase;
β2AR, β2 adrenergic receptor; BAPTA-AM, 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-
tetraacetic acid tetrakis(acetoxymethyl ester); BRET, bioluminescent resonance energy
transfer; Cch, Carbamylcholine chloride; CTX, cholera toxin; EGFR, epidermal growth factor
receptor; FRET, fluorescent resonance energy transfer; HB-EGF, heparin-binding epidermal
growth factor; HEK293, human embryonic kidney 293cells; TG, thapsigargin;
2 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Abstract:
2+ The 2 adrenergic receptor (2AR) increases intracellular Ca in a variety of cell types. By
combining pharmacological and genetic manipulations, we reveal a novel mechanism through
2+ which the β2AR promotes Ca mobilization (pEC50 = 7.32 ± 0.10) in non-excitable human
embryonic kidney (HEK)-293S cells. Down-regulation of Gs with sustained cholera toxin pre-
treatment and the use Gs-null HEK293 (∆Gs-HEK293) cells generated using the CRISPR/Cas9 Downloaded from system, combined with pharmacological modulation of cAMP formation revealed a Gs-
2+ dependent but cAMP-independent increase in intracellular Ca following β2AR stimulation. The
increase in cytoplasmic Ca2+ was inhibited by P2Y purinergic receptor antagonists as well as a molpharm.aspetjournals.org
dominant negative mutant form of Gq, a Gq-selective inhibitor and an IP3 receptor antagonist,
suggesting a role for this Gq-coupled receptor family downstream of the 2AR activation.
Consistent with this mechanism, β2AR stimulation promoted the extracellular release of at ASPET Journals on September 30, 2021 2+ adenosine triphosphate (ATP) and pre-treatment with apyrase inhibited the β2AR-promoted Ca
mobilization. Together, these data support a model whereby the 2AR stimulates a Gs-dependent
release of ATP, which transactivates Gq-coupled P2Y receptors through an “inside-out”
2+ mechanism, leading to a Gq- and IP3-dependent Ca mobilization from intracellular stores.
Given that 2AR and P2Y receptors are co-expressed in various tissues, this novel signalling
paradigm could be physiologically important and have therapeutic implications. In addition, this
study reports the generation and validation of HEK293 cells deleted of Gs using the
CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-
dependent signalling.
3 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Introduction:
The β2 adrenergic receptor (β2AR) has been shown to regulate a vast signalling network, leading
to the activation of key cellular effectors such as adenylyl cyclase (AC), ERK1/2 and Akt (De
Blasi, 1990; Daaka et al., 1998; Bommakanti et al., 2000) to control a variety of physiological processes, including the regulation of cardiac function, smooth muscle tone, immunological responses, fat metabolism as well as both central and peripheral nervous system activity Downloaded from (Guimaraes and Moura, 2001; Collins et al., 2004; Sitkauskiene and Sakalauska, 2005; Pérez-
Schindler et al., 2013). Although much is known about the signalling repertoire of the β2AR, new
insights into its full signalling capabilities and its role in cellular biology continue to be molpharm.aspetjournals.org discovered (Stallaert et al., 2012; van der Westhuizen et al., 2014).
In addition to the abovementioned signalling pathways, the β2AR can also stimulate an
increase in intracellular Ca2+. This signalling response is well established in excitable cells, such at ASPET Journals on September 30, 2021 as cardiomyocytes, via a mechanism involving cAMP-mediated regulation of plasma membrane
L-type Ca2+ channels (Zhang et al., 2001; Christ et al., 2009; Benitah et al., 2010). However, increases in intracellular Ca2+ levels have also been observed in non-excitable cells, such as
alveolar epithelial cells (Keller et al., 2014) and in human embryonic kidney cells (HEK293)
endogenously or stably overexpressing the β2AR (Schmidt et al., 2001; Stallaert et al., 2012; van
der Westhuizen et al., 2014). Previous observations from our laboratory indicated that such
2+ 2AR-mediated increases in intracellular Ca in HEK293 cells are inhibited by an IP3 receptor antagonist, 2-aminoethoxydiphenyl borate (2-APB) (Stallaert et al., 2012), suggesting the release of Ca2+ from intracellular stores, which points to a mechanism distinct from the plasma
membrane Ca2+ channel activation described in excitable cells.
4 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Several studies have reported that activation of the β2AR can also stimulate the release of extracellular mediators as part of its signalling repertoire. Activation of the β2AR in mouse
skeletal muscle, vascular smooth muscle cells or cardiac fibroblasts promotes the extracellular
release of cAMP, which is converted to AMP by ecto-phosphodiesterases and then to adenosine
by ecto-5’-nucleotidase on the extracellular surface of the cells (Dubey et al., 1996; 2001; Duarte
et al., 2012). In addition, extracellular adenosine can be produced by the release of intracellular Downloaded from adenine nucleotides to the extracellular space, which are converted to adenosine by ecto-ATPase,
ecto-ADPase and ecto-5’-nuclotidase (Jackson et al., 1996). Stimulation of human erythrocytes
with isoproterenol (ISO) increases extracellular ATP levels (Montalbetti et al., 2011), suggesting molpharm.aspetjournals.org
that βARs can also be coupled to ATP release. In some cases, this release of mediators into the
extracellular milieu can lead to the subsequent transactivation of other GPCRs or receptor tyrosine kinases. Previously, an “inside-out” signalling mechanism was demonstrated for the at ASPET Journals on September 30, 2021 β2AR, leading to the transactivation of adenosine receptors through the production of
extracellular adenosine as described above (Sumi et al., 2010), as well as the epithelial growth
receptor (EGFR) through the membrane shedding of heparin-binding epithelial growth factor
(HB-EGF) (Kim et al., 2002).
In this study, we describe a new “inside-out” pathway activated by the β2AR in non-excitable
cells, involving the Gs-dependent, but cAMP-independent increase in intracellular Ca2+ through the release of extracellular ATP and the subsequent transactivation of Gq-coupled P2Y purinergic receptors, providing a previously unrecognised link between adrenergic and purinergic receptors. This study also provides the first description and use of CRISPR/Cas9 genomic editing to knockout Gs in human cells, providing a powerful new tool to explore the signalling events downstream of this G subunit.
5 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Materials and Methods:
Reagents: (-)-isoproterenol hydrochloride (ISO), carbamoylcholine chloride (Cch), thapsigargin (TG), 8-bromo-cAMP (8-br-cAMP), forskolin, cholera toxin (CTX), , suramin and apyrase were purchased from Sigma Aldrich (St. Louis, MO, USA). ICI118,551, 1,2-Bis(2- aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM),
2-aminoethoxydiphenyl borate (2-APB), 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine Downloaded from (SQ22536), , 8,8'-[Carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1- phenylene)carbonylimino]]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF157), 4,4'-
(Carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic molpharm.aspetjournals.org acid) tetrasodium salt (NF340), 8,8'-[Carbonylbis(imino-4,1-phenylenecarbonylimino-4,1- phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF279), N-
Cyano-N"-[(1S)-1-phenylethyl]-N'-5-quinolinyl-guanidine (A-804598), 5-(3-Bromophenyl)-1,3- at ASPET Journals on September 30, 2021 dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD), 9-Chloro-2-(2-furanyl)-
[1,2,4]triazolo[1,5-c]quinazolin-5-amine (CGS 15943) and 4,4',4'',4'''-[Carbonylbis(imino-5,1,3- benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic acid octasodium salt (NF449) were obtained from R&D Systems (Minneapolis, MN, USA). Coelenterazine 400a and coelenterazine h were purchased from Nanolight technologies (Pinetop, AZ, USA) and coelenterazine cp was purchased from Biotium (Hayward, CA, USA). Arginine-vasopressine
(AVP) and 5-(Methylamino)-2-[[2R,3R,6S,8S,9R,11R) -3,9,11-trimethyl-8-[(1S)-1-methyl-2- oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187) were from Tocris (Bio-Techne, Minneapolis, MN, USA). Cell culture reagents were from Wisent Incorporated (Montreal, QC, Canada). The Gq selective inhibitor, FR900359
(Schrage et al., 2015) was obtained from Dr. Eve Kostenis and Dr. Stefan Kehraus from the
6 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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University of Bonn. The Gq and Gq(Q209L/D277N) constructs were purchased from Missouri
S&T cDNA Resource Center (Rolla, MO, USA). Hap II, Pvu II and Hae II restriction enzymes
were from Takara Bio (Japan). All other reagents were obtained from Sigma-Aldrich unless
otherwise stated.
Cell culture and transfections: Parental HEK293S cells or HEK293S cells stably expressing
an amino-terminal tagged human 2AR (3.17 ± 0.32 pmol/mg protein; HA-2AR-HEK293S Downloaded from
cells; Galandrin et al. 2006) were grown at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 5% fetal bovine serum. HEK293S and HA-2AR-
HEK293S cells were transiently transfected (600 000 cells/well) in 6 well plates with biosensors molpharm.aspetjournals.org
(950 ng/1106 cells) for Ca2+ measurements (Obelin luminescence), bioluminescence resonance
energy transfer (BRET) and fluorescence resonance energy transfer (FRET) assays using linear
polyethylenimine (PEI; 1 mg/ml; Polysciences Inc., Warrington, PA) diluted in NaCl (150 mM, at ASPET Journals on September 30, 2021 pH 7.0) (PEI:DNA ratio 3:1) as previously described (Reed et al., 2006). Twenty four hours
post-transfection, cells were re-plated (50,000 cells/well) onto white 96-well CulturePlates
(Perkin-Elmer, Woodbridge, ON, Canada). Six hours after re-plating, growth medium was
replaced with fresh starvation media consisting of DMEM supplemented with 0.5% FBS for the
next 18 h.
Parental HEK293 and ∆Gs-HEK293 cells were cultured and transfected as described for the
HEK293S cells, with the following modification: medium was supplemented with 10% FBS, PEI
was diluted in Dulbecco's Phosphate-Buffered Saline (DPBS) and added to cells in
suspension (300,000-350,000 cells/ml). Cells were then transferred into white 96-well
CulturePlates (30,000-35,000 cells/well). Cells were cultured with 10% FBS for the following 48
hrs prior to experimentation.
7 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Generation of HEK293 cells devoided of Gs (∆Gs-HEK293): The two members (Gs and
Golf, encoded by the GNAS and the GNAL genes, respectively; both were expressed in
HEK293 cells) of the Gs family were simultaneously targeted by a CRISPR/Cas9 system to introduce frame shift into the coding sequence as described previously (Ran, et al., 2013) with some modifications. Briefly, GNAS-targeting sgRNA sequence (5’-
CTACAACATGGTCATCCGGG-3’) was inserted into the Bbs I site of the pSpCas9(BB)-2A- Downloaded from GFP vector (PX458; a gift from Feng Zhang, Broad Institute; Addgene plasmid # 48138) using two synthesized oligonucleotides (5’-CACCGCTACAACATGGTCATCCGGG-3’ and 5’-
AAACCCCGGATGACCATGTTGTAGC-3’; FASMAC, Japan). Similarly, GNAL-targeting molpharm.aspetjournals.org sgRNA sequence #1 (5’-TGTTTGATGTTGGTGGCCAG-3’), #2 (5’-
GTAATGTTTGCCGTCACCGG-3’) and #3 (5’-ATTGTGCACAGTCAATCAGC-3’) were inserted using three sets of oligonucleotides (for #1, 5’- at ASPET Journals on September 30, 2021 CACCGTGTTTGATGTTGGTGGCCAG-3’ and 5’-AAACCTGGCCACCAACATCAAACAC-
3’; for #2, 5’-CACCGTAATGTTTGCCGTCACCGG-3’ and 5’-
AAACCCGGTGACGGCAAACATTAC-3’; for #3, 5’-
CACCGATTGTGCACAGTCAATCAGC-3’ and 5’-AAACGCTGATTGACTGTGCACAATC-
3’). Correct insertion of the sgRNA sequences was verified by sequencing using the Sanger
method (FASMAC, Japan). HEK293 (200,000 cells/well) were seeded into 12 well plates and
transfected with a mixture of the GNAS-targeting PX458 vectors and either of the GNAL-
targeting PX458 vectors (0.25 µg each/well) with Lipofectamine® 2000 (Life Technologies, CA,
USA). Twenty four hours later, cells were detached and GFP-positive cells (approximately 30%
of cells) were isolated using a cell sorter (SH800, Sony, Japan). After growing clonal cell
colonies, clones were analyzed for mutations in the GNAS and the GNAL genes by PCR and
8 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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restriction enzyme digestion, using the primer and restriction enzyme combinations shown in
Supplemental Table 1. PCR was performed with an initial denaturation cycle of 95ºC for 2 min,
followed by 35 cycles of 95ºC for 15 sec, 64ºC for 30 sec and 72ºC for 30 sec. The resulting
PCR product was digested with the corresponding restriction enzyme and analyzed by agarose
gel electrophoresis. Candidate positive clones were further analyzed by genomic DNA
sequencing using a TA cloning method. The lack of functional Gs was also confirmed by Downloaded from assessing GPCR-stimulated cAMP production as described below. For the TA cloning, PCR-
amplified genomic DNA fragments using an ExTaq polymerase (Takara Bio, Japan) were gel-
purified (Promega, WI, USA) and cloned into a pMD20 T-vector (Takara Bio, Japan). Ligated molpharm.aspetjournals.org
products were introduced into SCS1 competent cells (Stratagene, CA, USA) and transformed
cells were selected on an ampicillin-containing LB plate. At least twelve colonies were picked
and inserted fragments were PCR-amplified using the ExTaq polymerase and primers (5’- at ASPET Journals on September 30, 2021 CAGGAAACAGCTATGAC-3’ (M13 Primer RV) and 5’-GTTTTCCCAGTCACGAC-3’ (M13
Primer M4)) designed to anneal the pME20 T-vectors. PCR products of the transformed pMD20
T-vector were sequenced using the Sanger method (FASMAC, Japan) and the M13 Primer RV.
Whole transcriptome expression profiling was performed using the Clariom S assay (human,
analysis using the Transcriptome Analysis Console from Affymetrix) to assess differences in
gene expression between the parental HEK293 and ∆Gs-HEK293 cell lines (Supplemental
Figure 5 and Supplemental Tables 2 and 3).
6 mRNA expression: Total RNA was isolated from 3x10 HA-2AR-HEK293S cells using the
RNAeasy Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol for
animal cells. Microarray analysis of mRNA samples was performed using Illumina’s HumanRef-
8 v3.0 expression bead chips at McGill University and the Génome Québec Innovation Centre.
9 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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The gene expression microarray data was analyzed and normalized by the bioinformatics
platform at the Institute for Research in Immunology and Cancer (IRIC) at the University of
Montreal. The raw fluorescent values were then normalized using a quantile normalization
method (Bolstad et al., 2003; Lemieux, 2006), which re-ranks the sampled based on the raw
fluorescent values obtained for each probe. Using this method of normalization, the distribution
of the samples becomes the reference point, therefore, no internal control is required. For the Downloaded from microarray dataset analyzed, the distribution of the data is defined by the median value 7.162;
the minimum value 5.851 and the maximum value 15.760, the first quartile of 6.578 and the third
quartile of 9.215. The normalized values were plotted alongside known housekeeping genes, β- molpharm.aspetjournals.org
actin and α-tubulin, which were used for comparison purposes to represent genes that are highly
expressed in the cells. The inhibitory G protein, Gαo, was also plotted because it is not expressed
in HEK293 cells (Law et al., 1993) and therefore represents an appropriate baseline background at ASPET Journals on September 30, 2021 value.
Western blot analysis: The parental HEK293 cells and the ∆Gs-HEK293 cells were harvested
and approximately 1 x 106 cells were lysed in 500 µL of SDS-PAGE sample buffer (62.5 mM
Tris-HCl (pH 6.8), 50 mM dithiothreitol, 2% SDS, 10% Glycerol and 4 M urea) containing 1
mM EDTA and 1 mM phenylmethylsulfonyl fluoride. Cell lysates were homogenized with a
handy ultrasonic homogenizer (Microtech) and boiled at 95ºC for 5 min. Western blots were performed according to standard procedures using an anti-Gαs/olf mouse monoclonal antibody
(clone E-7, cat. no. sc55546, Santa Cruz Biotechnology) or anti--tubulin mouse monoclonal antibody (clone DM1A, cat. no. sc-32293, Santa Cruz Biotechnology) (both at 1 µg/mL in Tris- buffered saline, 1% BSA and 0.05% Tween 20), followed by horse radish peroxidase-linked anti- mouse IgG secondary antibody (cat. no. NA9310, GE Healthcare; 1:2000 diluted with Tris-
10 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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buffered saline, 5% skim milk, 0.05% Tween 20). Chemiluminescence signals were detected
using a LAS-4000 (FujiFilm) and visualized with Multi Gauge ver. 3.0 (FujiFilm).
Ca2+ measurements: Ca2+ measurements were carried out as described previously (van der
Westhuizen et al., 2014). Briefly, HEK293S, HA-2AR-HEK293S or ∆Gs-HEK293 cells were
transiently transfected with the mCherry-obelin or GFP2-obelin-based Ca2+-sensitive biosensors.
The day of experimentation, cells were washed twice and pre-incubated in stimulation buffer Downloaded from (Modified Hank’s Balances Salt Solution (HBSS): 137 mM NaCl, 5.4 mM KCl, 0.25 mM
Na2HPO4, 0.44 mM KH2PO4, 1.8 mM CaCl2, 0.8 mM MgSO4, 4.2 mM NaHCO3, 0.2% (w/v) D-
glucose, pH 7.4) containing the obelin substrate, coelenterazine cp (1 M), at 25°C for 2 h in the molpharm.aspetjournals.org
dark. Compounds diluted in stimulation buffer were injected into the wells and luminescence readings were taken every 0.3 seconds using the SpectraMax L (Molecular Devices, Sunnyvale,
CA). Luminescence was monitored for a total of 60 seconds post-stimulation and expressed as at ASPET Journals on September 30, 2021 relative luminescence units (RLU). The area under the resulting curves (AUC) and the peak response to stimulation were calculated. Where indicated, the AUC was transformed as % peak
ISO or A23187 response.
cAMP production: cAMP production measurements were carried out as described previously
(van der Westhuizen et al., 2014). Briefly, cells were transfected as indicated with either the
FRET-based GFP10-EPAC1-vYFP or the BRET-based GFP10-EPAC1-RlucII cAMP-sensitive
biosensors in which binding of cAMP induces a conformational change that leads to a decrease
in FRET or BRET. Compounds were diluted in stimulation buffer and added to cells at 37°C for
30 minutes prior to measurement. The light emitted from FRET donor and acceptor proteins was
measured with the FlexStation II (Molecular Devices, Sunnyvale, CA, USA) using the 510 nm
(GFP10) and 533 nm (vYFP) emission filters. BRET was measured with the Mithras LB 940
11 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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(Berthold Technologies, Bad Wildbad, Germany) using 410±70 nm (RlucII) and 515±20 nm
(GFP10) emission filters or the SynergyNeo microplate reader from BioTek using 41080 nm
and 51530 nm emission filters. FRET or BRET ratios were calculated (acceptor/donor) and
∆FRET and ∆BRET were determined by subtracting the FRET or BRET signal generated in vehicle treatment control conditions.
Measurements of cAMP for Figure 4A were performed using the HTRF-cAMP dynamic kit (Cisbio) Downloaded from as described previously (Stallaert et al., 2012).
Gs activation assay: HA-2AR-HEK293S cells were co-transfected with a three-component
G protein activation biosensor composed of Gs-67-RlucII (50 ng/well), G1 (100 ng/well) and molpharm.aspetjournals.org
GFP10-G1 (100 ng/well) in which Gs activation results in the separation between the G and
G subunits, thus leading to a decrease in BRET (Gales et al., 2005). The day of the experiment, cells were washed twice with stimulation buffer and pre-treated with various at ASPET Journals on September 30, 2021 inhibitors diluted in stimulation buffer for the indicated times at 37°C. Coelenterazine 400a, diluted in stimulation buffer (5 µM) was added to the wells for 5 min, then increasing concentrations of ISO were added for 2 min. Plates were read on the Mithras LB 940 with
410±70 nm (RlucII) and 515±20 nm (GFP10) emission filters and BRET ratios calculated as
above.
Quantification of extracellular ATP: Extracellular ATP was measured using the luciferin-
luciferase-based ENLITEN® ATP Assay System (Promega, Madison, WI, USA) according to the
manufacturer’s instructions. Briefly, cells were grown to 90-100% confluence in a 96-well plate.
Cells were washed with stimulation buffer, then pre-treated with 10 µM ICI 118,551 or
stimulation buffer (vehicle) for 30 min at 37°C. Cells were stimulated with 10 µM ISO and the
cell supernatant (conditioned buffer) was collected at 15-120 sec post stimulation. Luciferin-
12 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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luciferase reagent (100 µl) was injected into 10 µl of conditioned buffer, and luminescence was
measured on the Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). ATP concentration of each sample was determined by comparing the luminescence of samples with
those of standards in the concentration range of 10-6 to 10-10 M.
Data Analysis: Data analysis was performed using Microsoft Excel (Microsoft, Redmond,
WA, USA) and GraphPad Prism (versions 5 and 6, GraphPad Software, La Jolla, CA, USA). Downloaded from Statistical analyses were performed as indicated in Figure legends.
molpharm.aspetjournals.org at ASPET Journals on September 30, 2021
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Results:
2+ 2AR activation leads to an increase in intracellular Ca in HEK293S cells: Isoproterenol
(ISO) stimulation of the endogenously expressed β2ARs in HEK293S cells induces a rapid and
transient increase in intracellular Ca2+ (Figure 1A). The increase in intracellular Ca2+ was detected as early as 5 seconds following the addition of ISO, reaching a maximum at approximately 12 seconds followed by a slow decrease thereafter and returning to basal levels 60 Downloaded from seconds post-stimulation, demonstrating a functional Ca2+ response in non-excitable cells that
endogenously express the receptor. Stable overexpression of the β2AR in HEK293S cells (HA- molpharm.aspetjournals.org 2AR-HEK293S cells) led to acceleration in the onset (≈ 2 sec) and increased magnitude of the
response (Figure 1B). This increase in intracellular Ca2+ was inhibited in both cell lines
following pre-treatment with the 2AR-selective antagonist, ICI 118,551, confirming the 2AR- specificity of the response (Figure 1C). The response was concentration-dependent, yielding at ASPET Journals on September 30, 2021 similar pEC50 values for ISO in both cell lines (7.32±0.10 and 7.05±0.16 in HEK293S and HA-
2AR-HEK293S cells, respectively) (Figure 1D).
2+ 2AR promotes a Gs-dependent but cAMP-independent Ca response: To probe the
2+ mechanism underlying the 2AR-mediated Ca response, we first examined the Gs/cAMP
2+ pathway in the HA-2AR-HEK293S cell line, which produces a larger Ca response following stimulation, thus facilitating its mechanistic dissection. To confirm the involvement of Gs, the primary G protein-coupling partner of the 2AR, we tested the effect of overnight pre-treatment
with cholera toxin (CTX) to down-regulate Gs signalling (Levis and Bourne, 1992), as well as
the effect of acute pre-treatment with a small molecule inhibitor of Gs, NF449 (which prevents
GDP/GTP exchange (Hohenegger et al., 1998)). We found that the ISO-stimulated Ca2+ response
was significantly inhibited by both CTX and NF449 pre-treatment (Figure 2A), suggesting the
14 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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2+ involvement of Gs in the β2AR-mediated Ca response. However, while chronic pre-treatment
with CTX led to the expected inhibition of Gs activation measured using a bioluminescence resonance energy transfer (BRET)-based biosensor that monitors the separation between Gs and G1 as a reporter for G protein activation (Gales et al., 2005), acute pre-treatment with
NF449 had no impact on Gs activation (Figure 2B), indicating that its effect on the 2AR-
promoted Ca2+ response resulted from another mechanism of action. This is consistent with the Downloaded from
fact that, although a potent and selective inhibitor of Gs, NF449 contains multiple negative
charges (El-Ajouz et al., 2012) that impede its cell permeability and likely restrict its effect to molpharm.aspetjournals.org extracellular target(s) in cell-based assays. Observations that NF449 is also a potent antagonist of
purinergic P2X and P2Y receptors (Braun et al., 2001; Kassack et al., 2004) provided valuable
2+ insights into additional mechanistic components underlying the 2AR-stimulated Ca response,
as discussed below. However, the inhibitory effect of sustained CTX treatment supported a role at ASPET Journals on September 30, 2021 for Gs in the Ca2+ response.
To confirm the role of Gs in the cytoplasmic Ca2+ increase, we genetically deleted functional
Gs from HEK293 cells using the CRISPR/Cas9 system (ΔGs-HEK293). The genetic
characterization of three ∆Gs cell clones is presented in Supplemental Figures 1-4. Loss of both
the long (GαsL) and the short (GαsS) forms of Gαs was confirmed in 3 ΔGs-HEK293 clones
(Figure 2C).
∆Gs cells were then tested for a loss of function using the BRET-based EPAC biosensor to
monitor the effect of Gs knockout on the β2AR-mediated cAMP response. In ΔGs-HEK293 cells,
the cAMP response to ISO stimulation was significantly reduced compared to parental HEK293
cells (Figure 2D, Supplemental Figure 4A). Similarly, stimulation of another Gs-coupled
receptor, the vasopressin type 2 receptor, with AVP failed to promote cAMP accumulation in all
15 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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3 ΔGs-HEK293 selected clones (Supplemental Figure 4B). Furthermore, the ISO-promoted
increase in intracellular Ca2+ was also found to be inhibited in ΔGs-HEK293 cells (Figure 2E,
Supplemental Figure 4C), while the response to the Ca2+ ionophore, A23187, was similar to that
observed in parental HEK293 cells (Figure 2F, Supplemental Figure 4C), thus confirming the
2+ 2+ essential role of Gs in the 2AR-promoted Ca response. The loss of Ca responses to 2AR
stimulation was observed in all 3 ΔGs-HEK293 clones (Supplemental Figure 4C), excluding a Downloaded from possible clonal effect. Furthermore, a gene array analysis (Supplemental Figure 5) of the parental
and one of the ΔGs-HEK293 cells (clone 1) revealed that none of the other G protein subunit (,
or ) were among the 489 genes (out of 21,448 genes detected) found to be significantly up or molpharm.aspetjournals.org
down regulated with a minimum threshold of 2 fold in the ∆Gs cells (Supplemental Table 2),
excluding the possible implication of other G protein expression changes in the observed
responses. The expression level of the G protein subunits detected in the microarray for parental at ASPET Journals on September 30, 2021 and ∆Gs cells are presented in Supplemental Table 3.
We next assessed the role of the Gs-, and adenylate cyclase (AC)-dependent second
messenger cAMP in the ISO-promoted Ca2+ response. Pre-treatment with SQ22536, a
pharmacological inhibitor of AC, significantly inhibited ISO-promoted cAMP production (by
43%; Figure 3A), yet it failed to block the Ca2+ response (Figure 3B), producing a response
higher than in the absence of the inhibitor. To further confirm this surprising result, we directly
assessed whether increases in intracellular cAMP alone were capable of generating a Ca2+
response. Stimulation with forskolin (Fsk), which increases cAMP independently of β2AR
activation through the direct activation of AC (Seamon et al., 1981), increased the intracellular
cAMP to a similar level as obtained for ISO (Figures 2D and 3C), yet elicited only a marginal
Ca2+ response (Figure 3D). Further supporting these results, the cell permeable cAMP analog, 8-
16 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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bromo-cAMP, elicited a similarly weak Ca2+ response (Figure 3E). Taken together, our data
2+ indicate the 2AR-promoted increase in intracellular Ca occurs predominantly independently of
cAMP production.
2+ Involvement of purinergic receptors in the 2AR-promoted Ca response: To further
investigate the above observation that NF449, a small molecule inhibitor of Gs and a purinergic
receptor antagonist (Braun et al. 2001; Kassack et al. 2004), blocked the ISO-promoted Ca2+ Downloaded from response (see Figure 2A), we assessed additional purinergic receptor antagonists to test the potential role of this receptor family. Pre-treatment with a pan-purinergic receptor antagonist, suramin, significantly inhibited the ISO-promoted increase in intracellular Ca2+ (Figure 4A), molpharm.aspetjournals.org
while the pan-adenosine receptor antagonist CGS 15943 had no effect (Figure 4B). Gene
expression microarray analysis of HA-β2AR-HEK293S cells indicated the expression of several
P2X purinergic ion channels and P2Y purinergic GPCRs in this cell line (Supplemental Figure at ASPET Journals on September 30, 2021 6). To determine the identity of the purinergic receptor(s) involved in the Ca2+ response, we
selectively inhibited the most highly expressed subtypes. NF279 (P2X1-selective), 5-BDBD
(P2X4-selective) and A804598 (P2X7-selective) were without effect, while NF157
(P2Y11/P2X1-selective) and NF340 (P2Y11-selective) significantly inhibited the ISO-promoted
Ca2+ response (Figure 4C), suggesting a role for the P2Y11 purinergic receptor. The effect of
NF449 (Figure 2A), NF340 and NF157 (Figure 4C) was specific to the 2AR-stimulated
response since these compounds did not significantly inhibit the carbamylcholine chloride (Cch)-
stimulated increase in Ca2+ via endogenously expressed Gq-coupled muscarinic receptors in HA-
β2AR-HEK293S cells (Figures 4D). The effect of the P2Y11-selective antagonist, NF340, was
2+ concentration-dependent and resulted in a decrease in the Emax of the ISO-promoted Ca
response, with no significant effect on the EC50 (Figure 4E), confirming that its effect does not
17 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
occur directly through a competitive blockade of the 2AR but instead suggesting a non- competitive antagonism of the response. Furthermore, both NF340 and NF157 were found to progressively decrease the ISO-promoted Ca2+ response in a dose-dependent manner, with
potencies compatible with their affinity for the purinergic receptors (Figure 4F).
A previous study demonstrated that β2AR activation could induce the release of adenine
2+ nucleotides from HEK293 cells (Sumi et al., 2010). To determine if the 2AR promotes a Ca Downloaded from response through the release of an extracellular mediator, leading to the subsequent
transactivation of a purinergic receptor, we performed co-culture experiments in which parental
HEK293S cells transfected with the obelin Ca2+ biosensor (obelin-HEK293S responder cells) molpharm.aspetjournals.org
were co-cultured at a 1:1 ratio with either parental HEK293S cells or with HA-β2AR-HEK293S
cells not expressing the biosensor (releaser cells). When obelin-HEK293S responders were co-
cultured with releaser cells overexpressing the β2AR (HA-β2AR-HEK293S), a significant at ASPET Journals on September 30, 2021 potentiation in the Ca2+ response was observed following ISO treatment compared to co-culture
with parental HEK293S releaser cells (Figure 5A), suggesting a role for the 2AR-dependent
release of an extracellular mediator acting in a paracrine manner. As a control experiment, we
examined the Ca2+ response to Cch stimulation in both co-culture conditions and found no
significant difference (Figure 5B), further supporting the β2AR-specificity of this paracrine
signalling response.
Since both ATP and ADP adenine nucleotides act as agonists for the P2Y11 receptor (van der
Weyden et al., 2000), we assessed the role of extracellular adenine nucleotides in the β2AR- promoted Ca2+ response, by pre-treating cells with apyrase, a cell-impermeable enzyme that
hydrolyses both ATP and ADP to AMP. Apyrase treatment decreased both the efficacy (34%
-8 -6 decrease in Emax) and potency (increase in EC50 from 9.2x10 M to 1.7x10 M) of the ISO-
18 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
promoted response (Figure 5C), consistent with the depletion of an extracellular mediator released upon β2AR activation and acting in trans on the P2Y11 receptor. The Cch-mediated
Ca2+ response, on the other hand, was not inhibited and instead was increased following apyrase treatment (Figure 5D). Consistent with adenine nucleotides being the paracrine mediator responsible for the transactivation, ISO treatment promoted the release of ATP from HA-β2AR-
HEK293S cells (Figure 6A), with kinetics similar to those of the ISO-promoted Ca2+ response Downloaded from (reaching a maximum at 15 sec and returning to basal levels by 60 seconds). This ATP release
was blocked by the β2AR-selective antagonist, ICI 118,551, supporting a role for β2AR in the
response (Figure 6B). These results are consistent with the hypothesis that β2AR activation leads molpharm.aspetjournals.org
to the release of adenine nucleotides, which can act in trans on P2Y11 receptors to activate a
Ca2+ response.
P2Y11 transactivation elicits a Gq-dependent increase in Ca2+ from intracellular stores: To at ASPET Journals on September 30, 2021 investigate the potential role of Gq activation following P2Y11 transactivation (Qi et al., 2001),
we tested the effects of overexpressing either WT Gq or a dominant-negative mutant form of
Gq (Gq-Q209L/D277N). Overexpression of WT Gq in HA-2AR-HEK293S cells increased the magnitude of the ISO-promoted Ca2+ response, whereas overexpression of Gq-Q209L/D277N or
pre-treatment with the Gq-selective inhibitor FR900359 (Schrage et al., 2015) completely
abolished the response (Figure 7). These results further support the involvement of a Gq-coupled
2+ receptor downstream of the β2AR in the Ca response.
We next assessed whether the increase in intracellular Ca2+ occurs via release from
intracellular stores, as is typical for Gq-mediated responses. For this purpose, cells were pre-
2+ treated with the IP3 receptor antagonist, 2-APB, the cell-permeable Ca chelator, BAPTA-AM,
or the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (TG). Upon stimulation
19 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419 with ISO, the Ca2+ response was completely blocked by all three inhibitors (Figure 8A). These inhibitors had a similar effect on the Cch-mediated Ca2+ response, which activates a well- characterized IP3-dependent pathway via activation of Gq-coupled muscarinic receptors
(Caulfield, 1993) (Figure 8B). Together these data are consistent with the proposed transactivation of the Gq-coupled P2Y11 receptor upon activation of the 2AR.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 30, 2021
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MOLPHARM/2016/106419
Discussion:
In this study, we describe a new node in the β2AR signalling network involving a Gs- dependent but cAMP-independent release of ATP, which transactivates P2Y11 purinergic receptors and subsequently initiates a Gq-dependent mobilization of intracellular Ca2+. This discovery is consistent with the accumulating reports that receptor transactivation, via the
production and/or release of an extracellular mediator acting on a distal receptor, represents a Downloaded from bona fide signalling event for many GPCRs (Ostrom et al., 2000; Gschwind et al., 2001; Lee et al., 2002; Sumi et al., 2010). Given that the β2AR and the P2Y receptors are co-expressed in molpharm.aspetjournals.org various non-excitable tissues, including the brain, gastrointestinal tract, lung, adipocytes, kidney, skeletal muscle, heart and liver (Andre et al., 1996; Nicholas et al., 1996; Moore et al., 2001), the results obtained in the current study could provide additional insight into how β2AR and P2Y receptors might collaborate in normal conditions and pathophysiology. at ASPET Journals on September 30, 2021
In addition to characterizing a new signalling modality for the 2AR, the present study introduces a new genetically engineered HEK293 cell line lacking both members of Gs (Gs and
Golf), offering a powerful tool to directly assess the contribution of Gs to cellular event. Current methods to investigate the role of Gs involve either overnight treatment with CTX, which initially causes chronic activation of Gs to stimulate its downregulation, the use of the few currently available small molecule inhibitors, which provide poor target specificity, and/or inhibition of the downstream effectors AC or protein kinase A. Genetic knockout of Gs provides a useful tool to specifically investigate the role of Gs and, as was the case in the present study, allow the discovery of Gs-dependent responses that do not require cAMP production. These cells should prove useful in future studies to explore additional non-canonical Gs signalling pathways.
21 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Although the β2AR-mediated release of adenine nucleotides has been described previously
(Baker et al., 2004; Sumi et al., 2010), the functional consequences of this signalling event are
just beginning to be explored. P2Y11 mRNA was the most abundant in HEK293S cells and it
has the highest affinity for ATP compare to other purinergic receptors expressed in these cells
(von Kügelgen et al., 2006). Yet, the capacity of the 2AR to stimulate the release of ATP may in
fact lead to the activation of other purinergic receptor subtypes in cells expressing a different Downloaded from repertoire or spatial organization of receptors.
2+ The mechanism described in this study differs from previous reports of β2AR-mediated Ca
responses in non-excitable HEK293 cells, which suggested a cAMP-dependent increase in molpharm.aspetjournals.org
intracellular Ca2+, through a mechanism involving exchange protein directly activated by cAMP
(EPAC) and phospholipase Cε or calcium release activated calcium (CRAC) channels (Schmidt
et al., 2001; Keller et al., 2014). Although we cannot exclude a contribution of these mechanisms at ASPET Journals on September 30, 2021 2+ to the Ca response studied here, our data clearly indicate that β2AR activation in HEK293 cells
can elicit a Ca2+ response independently of cAMP since blocking approximately 50% of the cAMP production did not affect the Ca2+ response (Figure 3A,B). Moreover, direct elevation of
cAMP following treatment with forskolin or the cAMP analogue 8-Br-cAMP elicited only
2+ marginal Ca responses compared to 2AR stimulation (Figure 3D,E). Whether CRAC could
subsequently contribute to the response following the initial Ca2+ mobilisation from the ER pool
described in the current study has not been investigated. Interestingly, these previous studies
used the Fura-2 indicator dye to measure intracellular Ca2+, which is dissolved in the non-ionic
detergent pluronic F127. This detergent inhibits the multi-drug resistant proteins (ABC-B
transporters), which have been implicated in ATP transport to the extracellular space (Guan et
al., 2011). The use of the genetically encoded obelin Ca2+ biosensor in the current study should
22 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
not interfere with this ATP release mechanism and may have aided in the elucidation of the
mechanism described in the current work.
This collaboration between the β2AR and purinergic receptors may be of particular relevance
in the context of the airway epithelium. Indeed, the β2AR plays an important role in the cAMP-
dependent secretion of airway surfactants, ciliary beating and regulation of mucosal clearance
(Wright and Dobbs, 1991; Salathe, 2002), properties that contribute to the therapeutic success of Downloaded from
β2AR agonists for the treatment of asthma (Giembycz and Newton, 2006). Interestingly,
activation of Gq-coupled receptors and increases in intracellular Ca2+ promote undesirable
inflammatory responses in pulmonary tissues (Barnes, 1998; Rider et al., 2011). Furthermore, an molpharm.aspetjournals.org increase in purine nucleotide release from the airway epithelium is a hallmark of inflammatory diseases in the lungs (Burnstock et al., 2012) and purinergic signalling has been proposed to participate in asthmatic airway inflammation (Basoglu et al., 2005; Idzko et al., 2007) as well as at ASPET Journals on September 30, 2021 in the pathogenesis of chronic obstructive pulmonary disorder (COPD) (Adriaensen and
Timmermans, 2004; Mortaz et al., 2010). Given the beneficial effects of cAMP production and deleterious consequences of increases in intracellular Ca2+ in airway epithelia, the identification
2+ of functionally selective 2AR ligands that stimulate cAMP production without promoting Ca
mobilization could represent a desirable class of 2AR agonists for the treatment of pulmonary
disorders. Salmeterol, a clinically effective 2AR agonist used in the treatment of asthma,
exhibits less side effects than what is typically associated with chronic use of other 2AR
agonists (Cazzola et al., 2013). Biased signalling studies have also shown that salmeterol is
highly efficacious towards cAMP production but does not increase intracellular Ca2+ (van der
Westhuizen et al., 2014). Given the results obtained in the current work, future studies examining
the influence of purinergic receptor transactivation in the context of β2AR-targeted treatment of
23 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419 pulmonary disorders could be of great interest. Indeed, it is tempting to speculate that optimal therapy for such pulmonary disorders might involve harnessing the therapeutic benefit of - adrenergic-mediated cAMP/PKA activity while avoiding the pro-inflammatory effect of purinergic transactivation and subsequent Ca2+-dependent signalling.
Our study demonstrates a novel “inside-out” pathway for the β2AR, adding to its signalling repertoire the transactivation of purinergic receptors and stimulation of Ca2+ mobilization. Given Downloaded from the preponderance of data supporting such transactivation mechanisms for both the β2AR and other GPCR family members, we propose that such “inside-out” signalling events are not rare occurrences and instead represent a common signalling motif for GPCRs that may provide molpharm.aspetjournals.org tissue-specific signalling in cells expressing a given repertoire of receptors.
at ASPET Journals on September 30, 2021
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MOLPHARM/2016/106419
Acknowledgements: a) The authors would like to thank Dr. Monique Lagacé for critical reading of the manuscript.
We would also like to thank Dr. Kumiko Makide and Yuji Shinjo for technical assistant of
flow cytometry cell isolation and Kouki Kawakami for Western blot analysis.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 30, 2021
25 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
Authorship contributions:
Participated in research design: Stallaert, van der Westhuizen, Le Gouill, Bouvier
Conducted Experiments: Stallaert, van der Westhuizen, Schonegge, Plouffe, Hogue,
Lukashova
Contributed to new reagents or analytical tools: Inoue, Ishida, Aoki, Le Gouill Downloaded from
Performed data analysis: Stallaert, van der Westhuizen, Schonegge, Plouffe, Hogue,
Lukashova, Inoue, Bouvier molpharm.aspetjournals.org
Wrote or contributed to the writing of the manuscript: Stallaert, van der Westhuizen, Inoue,
Bouvier
at ASPET Journals on September 30, 2021
26 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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33 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
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Footnotes:
a) This work was supported, in part, by grants from the Canadian Institutes for Health Research
(CIHR) [MOP 11215] to MB, PRESTO, JST to AI and AMED-CREST, AMED to JA. WS
was supported by the Vanier Canada Graduate Scholarship from the CIHR. ETvdW was
supported by post-doctoral research fellowships from the CIHR, the Canadian Hypertension
Society, the Fonds de la Recherche en Santé du Quebec (FRSQ) and the National Health and Downloaded from Medical Research Council Australia (NHMRC) [GNT-1013819]. A-MS was supported by
post-doctoral research fellowships from the FRSQ while BP was supported by post-doctoral
research fellowships from the CIHR. MB holds the Canada Research Chair in Signal molpharm.aspetjournals.org
Transduction and Molecular Pharmacology. b) c) Reprint requests to: Michel Bouvier, IRIC - Université de Montréal, P.O. Box 6128 at ASPET Journals on September 30, 2021 Succursale Centre-Ville, Montréal, Qc. Canada, H3C 3J7. Email:
d) WS and ETvdW contributed equally to this work.
34 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
Figure legends:
2+ Figure 1 – Isoproterenol increases intracellular Ca via the 2AR. HEK293S (endogenously
expressing β2AR at 18.74±3.14 fmol/mg protein) or HA-β2AR-HEK293S (stably expressing
β2AR at 3 174±320 fmol/mg protein) cells were transiently transfected with mCherry-obelin. (A-
B) Cells were treated with vehicle or isoproterenol (ISO; 10 μM) and increases in intracellular Downloaded from Ca2+ (increases in obelin relative luminescence unit, RLU) were measured. (C) Cells were pre- treated with vehicle (Ctl) or ICI 118,551 (ICI, 100 nM) for 1 h, then treated with ISO (10 μM).
Data represent ISO-promoted peak Ca2+ responses. (D) The Ca2+ response was measured with molpharm.aspetjournals.org increasing concentrations of ISO. EC50 values: 7.32±0.10 for HEK293S and 7.05±0.16 for HA-
β2AR-HEK293S. Data are expressed as the mean ± S.E.M. of 3-4 independent experiments, each
performed in triplicate. Data in column graphs were analyzed by two-tailed paired Student’s t- at ASPET Journals on September 30, 2021 test, where p<0.05 (*) was considered significant.
2+ Figure 2 – The ISO-promoted increase in intracellular Ca is Gs-dependent. (A) HA-β2AR-
HEK293S cells transiently transfected with mCherry-obelin were pre-treated with cholera toxin
(CTX) (200 ng/ml; 18 h) or with NF449 (10 µM; 1 h). Cells were treated with isoproterenol
(ISO; 10 μM) and increases in intracellular Ca2+ were measured. Inset: The area under the curve
(AUC) was calculated for each treatment. (B) HA-β2AR-HEK293S cells were transiently
transfected with Gαs-67-RlucII and GFP10-Gγ1. Cells were pre-treated with vehicle (Ctl), CTX or NF449, as indicated above, followed by ISO treatment, and Gs activation was measured as a decrease in the BRET response. Protein extracts from parental HEK293 and three clones of ∆Gs-
HEK293 cells were separated on SDS-PAGE and immunoblotted with anti-Gαs/olf antibody (top
35 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
panel) or anti-α-tubulin antibody (bottom panel). Note that the parental HEK293 cells express
the long (GsL) and short (GαsS) Gαs isoforms while they were both undetectable in the mutant
clones. (D) The parental HEK293 or Gs-HEK293 (Clone1) cells were transiently transfected
with GFP10-EPAC-RlucII. Cells were then treated with either isoproterenol (ISO; 10 μM) or
forskolin (Fsk; 10 μM) for 20 min. ISO increased intracellular cAMP levels in HEK293 cells at a
similar level to Fsk while the response to ISO was significantly blocked in Gs-HEK293 cells. Downloaded from
(E-F) The parental HEK293 or Gs-HEK293 (Clone1) cells were transiently transfected with
β2AR and Obelin-GFP2, and treated with ISO (10M, C) or the Ca2+ ionophore A23187 (10 molpharm.aspetjournals.org M, D). ISO increased intracellular Ca2+ in the parental HEK293 cells while the response was
blocked in ∆Gs-HEK293 cells (E). The response to the Ca2+ ionophore A23187 was comparable
in both cells lines (F). cAMP and Ca2+ data for the 2 other clones are shown in Supplemental
Figure 4. Data are expressed as the mean ± S.E.M. of 3-6 independent experiments, each at ASPET Journals on September 30, 2021
performed in duplicate or triplicate. Data in panel A were analyzed by one-way ANOVA, with a
Dunnett’s multiple comparison post-hoc test, where p<0.05 (*) was considered significant. Data
in planel D were analyzed by two-way ANOVAbfollowed by a Sidak’s multiple comparison
post-hoc test, where p<0.05 (*) and p<0.001 (***)was considered significant.
Figure 3 – The ISO-promoted increase in Ca2+ is independent of cAMP production. (A)
HA-β2AR-HEK293S cells were pretreated with vehicle or the adenylyl cyclase inhibitor,
SQ22536 (100 mM, 1 h), then treated with isoproterenol (ISO; 10 μM) and cAMP measured using the Cisbio kit. (B-E) HA-β2AR-HEK293S cells were transiently transfected with either
2+ GFP10-EPAC-vYFP (C) or mCherry-obelin (B, D-E). (B) Cells were treated as in A and Ca response measured. Inset: Ca2+ response was plotted using the area under the curve (AUC).
36 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
SQ22536 significantly inhibited ISO-promoted cAMP production, but did not attenuate the ISO-
induced Ca2+ response. (C-D) Cells were treated with either forskolin (Fsk; 100 μM, 30 min) or
ISO and cAMP (C) and Ca2+ (D) responses were measured. Fsk activates cAMP production to a
similar extent as ISO (C), but stimulates only a marginal Ca2+ response compared to ISO (D) and
over vehicle (D, inset). (E) Cells were stimulated or not (vehicle) with ISO or the non-
hydrolysable cAMP analog, 8-bromo-cAMP (100 μM) and Ca2+ response measured. Inset: Ca2+ Downloaded from response was plotted using the area under the curve (AUC). Data are expressed as the mean ±
S.E.M. of 3-6 independent experiments, each performed in duplicate or triplicate. Column graphs
were analyzed by two-tailed paired Student’s t-tests, where p<0.05 (*) was considered molpharm.aspetjournals.org significant.
Figure 4 – The ISO-promoted Ca2+ response is inhibited upon purinergic receptor at ASPET Journals on September 30, 2021
blockade. (A-E) HA-β2AR-HEK293S cells transiently transfected with mCherry-obelin were
pretreated with the pan-adenosine receptor antagonist CGS15943 (1 M, 30 min) (B) or the purinergic receptor antagonists: suramin (500 μM; 1 h) (A, C), NF279 (1 μM, 30 min) (C), 5-
BDBD (10 μM, 30 min) (C), A804598 (1 μM, 30 min) (C), NF449 (10 μM, 30 min) (D), NF157
(10 μM, 30 min) (C-D) or NF340 (10 μM, 30 min) (C-D). Cells were then stimulated with either
ISO (10 μM) (A-C, E) or Carbamylcholine chloride (Cch, 100 μM) (D), and the Ca2+ response
was measured. Inset in A and B shows the Ca2+ response plotted using the area under the curve
(AUC). (E) Cells were pre-treated with vehicle (Ctl) or the indicated concentration of NF340,
followed by stimulation with increasing concentrations of ISO and the Ca2+ response was
measured. (F) Cells were pretreated with increasing concentration of either NF157 or NF340,
2+ followed by stimulation with ISO at an EC80 concentration (150 nM). Ca response was then
37 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
measured. IC50 values: 4.36 1.91 M for NF157 and 50.4 27.5 M for NF340. Data are
expressed as the mean ± S.E.M. of 3-4 independent experiments, each performed in triplicate.
Area under the curve (AUC) data presented in column graphs were analyzed by two-tailed
unpaired Student’s t-tests, where p<0.05 (*) was considered significant.
Downloaded from Figure 5 – ISO stimulates the releases of an extracellular mediator involved in the Ca2+ response. Parental HEK293S cells were transiently transfected with mCherry-obelin and co- cultured with HEK293S or HA-β2AR-HEK293S cells not expressing the biosensor. (A-B) Cells molpharm.aspetjournals.org
were treated with either ISO (10 μM) (A) or Cch (100 M) (B) and the Ca2+ response was
measured. (C-D) Cells were pre-treated with apyrase (1 UI/ml; 1 h) followed by stimulation with
increasing concentration of ISO (C) or Cch (100 μM) (D) and the Ca2+ response was measured.
Inset for A, B and D: Ca2+ response plotted using the area under the curve (AUC). Data are at ASPET Journals on September 30, 2021
expressed as the mean ± S.E.M. of 3 independent experiments, each performed in triplicate. Area
under the curve (AUC) data presented in column graphs were analyzed by two-tailed paired t-
tests, where p<0.05 (*) was considered significant.
Figure 6 – ISO promotes ATP release in HA-β2AR-HEK293S cells. HA-β2AR-HEK293S
cells were stimulated with ISO (10 M) for different time points and ATP was measured in the
bulk media. (A) ISO promoted a rapid and transient release of ATP over a period of 60 sec, with
peak release measured at 30 sec. (B) The response to ISO was blocked in both HEK293S and
HA-β2AR-HEK293S cells pre-treated with the β2AR-selective antagonist ICI 118551. Data are
the mean ± S.E.M. of 2-4 independent experiments performed in duplicate.
38 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
MOLPHARM/2016/106419
Figure 7 – The ISO-promoted Ca2+ response is modulated by wild-type or dominant
negative Gq overexpression. HA-β2AR-HEK293S were transiently transfected or not (Mock)
with either wild-type Gq or a dominant negative mutant of Gq (Q209L/D277N) (Gq-DN), or pre- treated with the Gq-selective inhibitor FR900359, and the Ca2+ response was measured. Data are
expressed as the mean ± S.E.M. of 4-5 independent experiments, each performed in triplicate. Downloaded from
Figure 8 – Ca2+ originating from the intracellular stores is involved in the ISO and Cch- molpharm.aspetjournals.org mediated responses. HA-β2AR-HEK293S cells were pre-treated with the sarco/endoplasmic
reticulum Ca2+-ATPase inhibitor thapsigargin (Tg; 5 μM, 1 h), the cell-permeable Ca2+ chelator
BAPTA-AM (20 μM, 1 h), or the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB;
200 μM, 1 h), followed by stimulation with ISO (10 μM) (A) or Cch (100 μM) (B) and the Ca2+ at ASPET Journals on September 30, 2021
response was measured. Inset for A and B: Ca2+ response plotted using the area under the curve
(AUC). Data are expressed as the mean ± S.E.M. of 3-6 independent experiments, each performed in triplicate. Area under the curve (AUC) data in column graphs were analyzed by one-way ANOVA, with a Dunnett’s multiple comparison post-hoc test, where p<0.05 (*) was considered significant.
39 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 1
AB ) ) 5 2.5 HEK293S 5 20 HA-2AR-HEK293S
2.0 vehicle 15 vehicle 1.5 ISO ISO 10 1.0 5
response ( RLU x 10 0.5 response ( RLU x 10 2+ 2+ Ca Ca 0 0
in j 10 20 30 40 50 60 in j 10 20 30 40 50 60 Downloaded from Time (sec) Time (sec)
C D
6.0 * 8.0 molpharm.aspetjournals.org HEK293S ) 5 ) 5 6.0 HA-2AR-HEK293S 4.0 4.0 response
response * 2+ 2+ 2.0
Ca 2.0 Ca (peak RLU x 10 (peak RLU x 10
0 0 ISO ISO + ICI ISO ISO + ICI -11 -10 -9 -8 -7 -6 -5 -4 at ASPET Journals on September 30, 2021 veh HEK293S HA-2AR-HEK293S log[ISO] (M) Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 2
AB2,500 2,000 0.02 100 1,500
ISO AUC 1,000 ISO + CTX 0.00 80 500 * *
ISO + NF449 0 60 ISO -0.02 + CTX + NF449 response
40 BRET) ISO 2+ ∆
s activatrion -0.04 (
ISO + CTX Ca (% peak ISO) 20 G ISO + NF449 -0.06 0 -0.08 in j 10 20 30 40 50 60 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 Time (sec) Log[ISO] (M) Downloaded from
CD *** 0.20 ∆Gs-HEK293 * HEK293 CL1 CL2 CL3 0.15
Fsk molpharm.aspetjournals.org GsL ISO GsS
response 0.10
BRET) ∆ ( -tubulin
cAMP 0.05
0.00 HEK293 ∆Gs-HEK293
E F ISO A23187 at ASPET Journals on September 30, 2021 200 5,000
4,000 150 3,000 100 HEK293 2,000 HEK293 response response (RLU) ∆Gs-HEK293 2+ 2+ 50 ∆Gs-HEK293 1,000 Ca Ca (% peak A23187) 0 0
inj 10 20 30 40 50 60 inj 10 20 30 40 50 60 Time (sec) Time (sec) Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 3
AB C
ISO 8,000 40 200 0.08 * ISO + SQ22536 6,000
150 4,000 30 FRET) 0.06 AUC 2,000 100
response 20 0 0.04
response ISO + SQ 2+ (pmol/well) 50 response ( ∆
(% peak ISO)
10 Ca cAMP 0.02 Downloaded from 0 cAMP 0 0.00 inj 10 20 30 40 50 60 Fsk -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 ISO Time (sec) log[ISO] (M)
+ SQ22536 DE 10 2,000 Fsk molpharm.aspetjournals.org 100 8 100 vehicle 1,500 ISO 6 ISO 80 80 4 Fsk 8-br-cAMP 1,000
2 AUC 60 (% peak ISO) 60 0 500
response inj 10 20 30 40 50 60 40 response 40 2+ Time (sec) 0 2+ (% peak ISO) Ca Veh ISO 8-br (% peak ISO) 20 Ca 20
0 0
in j 10 20 30 40 50 60
inj 10 20 30 40 50 60 at ASPET Journals on September 30, 2021 Time (sec) Time (sec) Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 4
A B 1,600 1,600 125 125 ISO 1,400 ISO 1,400 1,200 1,200 ISO + Suramin ISO + CGS15943 100 1,000 100 1,000 800 800 AUC 600 AUC 600 * 75 ISO) 75 ISO) 400 400 200 200
response 50 0 response 50 0 2+ 2+ ISO ISO + CGS (% peak (% peak Ca 25 + Suramin Ca 25
0 0
inj 10 20 30 40 50 60 inj 10 20 30 40 50 60 Time (sec) Time (sec) Downloaded from C D 6 15 molpharm.aspetjournals.org ) 3 4 10 response response 2+ 2+ 2 5 (AUC x 10 (AUC x 100) Ca Ca * * * 0 0
ISO ISO ISO ISO ISO ISO Cch Cch Cch
+ NF279 + NF157 + NF340 + NF449 + NF157 + NF340 + Suramin + 5-BDBD + A804598 at ASPET Journals on September 30, 2021
E ISO F 150 120 ISO + NF340 (0.1 mM) 100 ISO + NF340 (1 mM) ISO + NF340 (10 mM) 80 100 ISO)
60 response 2+ 40 (% max)
(% peak 50 Ca ISO + NF157
20 response at ISO IC80
2+ ISO + NF340 0 Ca 0 -11 -10 -9 -8 -7 -6 -5 -4 -3 -9 -8 -7-6 -5 -4 -3
basal log[ISO] (M) log[antagonist] (M) Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 5
ISO Cch A 3,500 B 1,000 200 3,000 * 125 HEK293S HEK293S 800 2,500 HA-2AR-HEK293S 100 150 2,000 HA-2AR-HEK293S 600 AUC AUC 1,500 75 400 100 1,000 200
response 50 500 response 2+ 50 0 2+ 0 Ca (% peak ISO) 25 WT 2AR Ca WT 2AR (% peak Cch)
0 0
inj 10 20 30 40 50 60 inj 10 20 30 40 50 60 Time (sec) Time (sec) Downloaded from
C D 1,500 HA-2AR-HEK293S HA-2AR-HEK293S molpharm.aspetjournals.org 150 200 ISO Cch * ISO + apyrase Cch + apyrase 1,000 100 150 AUC 500 50 100 response response
2+ 0 2+ 50 0 Ca Ca (% peak ISO) (% peak Cch) Cch 0 -50 + apyrase
-11 -10 -9 -8 -7 -6 -5 -4 -3 inj 10 20 30 40 50 60 at ASPET Journals on September 30, 2021
basal Log[ISO] (M) Time (sec) Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 6
A B 150 600 HA-2AR-HEK293S
400 100
200 50 ATP release (nM) ATP release (% control) 0 0 Downloaded from 0 204060 ISO + ICI ISO + ICI Time (sec) HEK293S HA-β2AR-HEK293S molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 7
400 ISO
300 Mock
200 + Gq response + Gq-DN 2+ + FR900359 Ca 100
(% peak ISO for Mock) 0
inj 10 20 30 40 50 60 Time (sec) Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 Molecular Pharmacology Fast Forward. Published on March 9, 2017 as DOI: 10.1124/mol.116.106419 This article has not been copyedited and formatted. The final version may differ from this version.
FIGURE 8
ABISO 6,000 600 100 ISO + Tg 100 Cch ISO + BAPTA-AM Cch + Tg 4,000 400 80 ISO + 2-APB 80 Cch + BAPTA-AM AUC AUC Cch + 2-APB 200 60 2,000 60
*** response *** 0 40 0 response 40
2+ Cch ISO + Tg 2+ + Tg + 2-APB + 2-APB Ca (% peak ISO) Ca 20 (% peak Cch) 20 + BAPTA-AM + BAPTA-AM Downloaded from 0 0
in j 10 20 30 40 50 60 in j 10 20 30 Time (sec) Time (sec) molpharm.aspetjournals.org at ASPET Journals on September 30, 2021 Supplemental Data
Article's title: Purinergic receptor transactivation by the β2-adrenergic receptor increases intracellular Ca2+ in non-excitable cells
Author's names: Wayne Stallaert, Emma T van der Westhuizen, Anne-Marie Schönegge, Bianca Plouffe, Mireille Hogue, Viktoria Lukashova, Asuka Inoue, Satoru Ishida, Junken Aoki, Christian Le Gouill & Michel Bouvier
Journal title: Molecular Pharmacology
1 Supplemental Table 1 – Primer sequences and restriction enzyme combinations to screen for mutants.
Target Primer 1 Primer 2 Enzyme
GNAS 5’-TCAACGGTAGGATGCTGTGG-3’ 5’-CTACAAGAAGGGAGGCCGTG-3’ Hap II
GNAL #1 5’-AAGCACGTTTGCCATTGTCC-3’ 5’-CTTCAGGTTATCCGCCCTCC-3’ Hae III
GNAL #2 5’-ACTGTCACCAAAGCCTCCAG-3’ 5’-TACTGCTTGAGGTGCATCCG-3’ Hap II
GNAL #3 5’-ACACTAAACATAGAGTGGGTGC-3’ 5’-TGAACAAAACTTTCTGGTTGTCAG-3’ Pvu II
2 Supplemental Table 2: List of genes found to be significantly up or down regulated in the ΔGs cells (clone 1) with a minimum of 2 fold change (<-2 or >2) vs the parental cells. Significance was established using unpaired one-way ANOVA between-samples, using the transcriptome analysis console from Affymetrix (p < 0.05 was considered statistically significant). The false discovery rate (FDR) adjustment has been calculated for each gene and is indicated.
Fold Change FDR Gene Description (linear) p-value Symbol (parental vs. ∆Gs) (parental vs ∆Gs) SHISA2 shisa family member 2 11.41 0.110671 PADI3 peptidyl arginine deiminase, type III -2.03 0.110671 ANKRD1 ankyrin repeat domain 1 (cardiac muscle) -16.70 0.110671 CNTFR ciliary neurotrophic factor receptor 2.82 0.110671 CD44 CD44 molecule (Indian blood group) -3.47 0.112203 MPPED2 metallophosphoesterase domain containing 2 2.11 0.112203 TCF24 transcription factor 24 5.14 0.112203 KCTD12 potassium channel tetramerization domain containing 12 36.44 0.112203 EFNB2 ephrin-B2 6.82 0.112203 RPS6KA3 ribosomal protein S6 kinase, 90kDa, polypeptide 3 -2.14 0.112203 GDF11 growth differentiation factor 11 2.09 0.112203 ERVMER34-1 endogenous retrovirus group MER34, member 1 4.41 0.112203 GJB2 gap junction protein beta 2 28.92 0.112203 MRPL50 mitochondrial ribosomal protein L50 2.46 0.112203 OSMR oncostatin M receptor -2.81 0.112203 EPHA7 EPH receptor A7 12.08 0.112203 GPC5 glypican 5 2.29 0.112203 ODF2 outer dense fiber of sperm tails 2 3.02 0.112203 LYST lysosomal trafficking regulator -2.05 0.112203 GREM1 gremlin 1, DAN family BMP antagonist -8.63 0.112203 SRPX2 sushi-repeat containing protein, X-linked 2 -7.93 0.112203 MMP2 matrix metallopeptidase 2 -4.43 0.112203 EMP3 epithelial membrane protein 3 -2.41 0.112203 SERPINF1 serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 4.50 0.112203 SLC6A9 solute carrier family 6 (neurotransmitter transporter, glycine), member 9 2.01 0.112203 SP100 SP100 nuclear antigen -2.58 0.112203 FAXDC2 fatty acid hydroxylase domain containing 2 -2.01 0.112203 CPS1 carbamoyl-phosphate synthase 1 -3.74 0.112203 NR6A1 nuclear receptor subfamily 6, group A, member 1 2.92 0.112203 NME5 NME/NM23 family member 5 5.70 0.112203 NALCN sodium leak channel, non selective -4.25 0.112203 IL2RG interleukin 2 receptor, gamma -6.77 0.112203 ISOC1 isochorismatase domain containing 1 2.04 0.112438 MLLT11 myeloid/lymphoid or mixed-lineage leukemia; translocated to, 11 -3.38 0.112438 TMEM154 transmembrane protein 154 -2.47 0.113586 GGT1 gamma-glutamyltransferase 1 -4.26 0.113586 STX3 syntaxin 3 -2.01 0.113586 MXRA8 matrix-remodelling associated 8 3.07 0.113586 ALPPL2 alkaline phosphatase, placental like 2 2.41 0.114411 TLR6 toll-like receptor 6 -2.53 0.114411 TRIB2 tribbles pseudokinase 2 -2.50 0.114411 DNAJC3 DnaJ (Hsp40) homolog, subfamily C, member 3 -2.04 0.114411 PAX7 paired box 7 -2.05 0.114411 HHIP hedgehog interacting protein 2.08 0.114411 BMP2 bone morphogenetic protein 2 -5.67 0.114411 ROR2 receptor tyrosine kinase-like orphan receptor 2 5.05 0.114411 F10 coagulation factor X -2.02 0.114411 PARP14 poly(ADP-ribose) polymerase family member 14 -8.88 0.114411 PDGFD platelet derived growth factor D -2.12 0.114411 NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha -3.53 0.114411 KNOP1 lysine-rich nucleolar protein 1 2.26 0.114411 H1F0 H1 histone family, member 0 -2.67 0.114771 TINAGL1 tubulointerstitial nephritis antigen-like 1 -2.86 0.115278 RIT1 Ras-like without CAAX 1 -2.19 0.115278 RIBC2 RIB43A domain with coiled-coils 2 2.08 0.115927 GBP2 guanylate binding protein 2, interferon-inducible -2.20 0.115927 TOR2A torsin family 2, member A 3.30 0.115927 CA2 carbonic anhydrase II 8.75 0.115927 IQGAP2 IQ motif containing GTPase activating protein 2 2.20 0.115992 WDR34 WD repeat domain 34 3.83 0.115992 SULT1C4 sulfotransferase family 1C member 4 10.70 0.115992 FHL2 four and a half LIM domains 2 -2.29 0.115992 GABRA3 gamma-aminobutyric acid (GABA) A receptor, alpha 3 -5.18 0.115992 ABCC2 ATP binding cassette subfamily C member 2 -2.25 0.115992 HOXB-AS3 HOXB cluster antisense RNA 3 20.44 0.119418 CITED1 Cbp/p300-interacting transactivator, with Glu/Asp rich carboxy-terminal domain, 1 4.71 0.121838 TNFRSF9 tumor necrosis factor receptor superfamily, member 9 -16.22 0.122868 FILIP1L filamin A interacting protein 1-like -2.09 0.122868 AMBN ameloblastin 5.54 0.122868 GGT2 gamma-glutamyltransferase 2 -2.08 0.125061 OLFM1 olfactomedin 1 -15.15 0.125061 VRK1 vaccinia related kinase 1 2.43 0.125061 YPEL3 yippee like 3 -2.03 0.125061 CNKSR1 connector enhancer of kinase suppressor of Ras 1 3.26 0.125061 TBC1D13 TBC1 domain family, member 13 2.95 0.125061 GPRC5C G protein-coupled receptor, class C, group 5, member C -2.09 0.125061 ZBTB7B zinc finger and BTB domain containing 7B -2.15 0.125061 GREM1 gremlin 1, DAN family BMP antagonist [Source:HGNC Symbol;Acc:HGNC:2001] -5.89 0.125061 EDIL3 EGF-like repeats and discoidin I-like domains 3 -2.76 0.125061 Supplemental Table 2: continue
MYO5B myosin VB -2.24 0.125061 TCIRG1 T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3 -2.03 0.125061 LINC00649 long intergenic non-protein coding RNA 649 7.92 0.125061 MXD1 MAX dimerization protein 1 -2.65 0.125061 SMAD7 SMAD family member 7 -2.05 0.125061 FAM111A family with sequence similarity 111, member A -3.67 0.125061 SPINT1 serine peptidase inhibitor, Kunitz type 1 -3.30 0.125061 MYL9 myosin light chain 9 7.77 0.125061 LGALS1 lectin, galactoside-binding, soluble, 1 -4.66 0.125061 TAPBP TAP binding protein (tapasin) -2.07 0.129017 LRRN1 leucine rich repeat neuronal 1 -3.64 0.129017 MOB3B MOB kinase activator 3B 3.46 0.129017 TGM2 transglutaminase 2 -2.11 0.129017 SLC27A4 solute carrier family 27 (fatty acid transporter), member 4 2.63 0.129017 INA internexin neuronal intermediate filament protein, alpha 6.31 0.129017 CXCL10 chemokine (C-X-C motif) ligand 10 -2.64 0.129017 C1QTNF1 C1q and tumor necrosis factor related protein 1 -3.53 0.129017 DOLK dolichol kinase 2.54 0.129017 TRUB2 TruB pseudouridine (psi) synthase family member 2 2.66 0.129017 DDR1; discoidin domain receptor tyrosine kinase 1; -2.07 0.129017 MIR4640 microRNA 4640 FAM174B family with sequence similarity 174, member B -2.20 0.129179 RGS9 regulator of G-protein signaling 9 -3.45 0.129920 KDM5B lysine (K)-specific demethylase 5B -2.04 0.130577 ZNF711 zinc finger protein 711 2.40 0.131949 HSPB8 heat shock 22kDa protein 8 -2.52 0.133236 MFAP2 microfibrillar associated protein 2 4.33 0.133506 NFKB2 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) -3.33 0.133506 NDUFAF2 NADH dehydrogenase (ubiquinone) complex I, assembly factor 2 2.09 0.133506 TFAP2A transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha) -2.02 0.133506 ADAM28 ADAM metallopeptidase domain 28 -2.55 0.133506 FAM27E3 family with sequence similarity 27, member E3 5.35 0.133506 CAV1 caveolin 1 -2.69 0.133506 COQ4 coenzyme Q4 3.77 0.133506 ACSM3 acyl-CoA synthetase medium-chain family member 3 2.08 0.135266 EFNA1 ephrin-A1 -2.24 0.135266 PCYT1B phosphate cytidylyltransferase 1, choline, beta -2.08 0.136233 KIAA1217 KIAA1217 -2.36 0.136233 DTX4 deltex 4, E3 ubiquitin ligase 2.29 0.136366 HBP1 HMG-box transcription factor 1 -2.06 0.137726 POU6F2 POU class 6 homeobox 2 3.42 0.138586 ACTBL2 actin, beta-like 2 -6.53 0.138788 NEK6 NIMA-related kinase 6 2.27 0.139162 RPP25L ribonuclease P/MRP 25kDa subunit-like 2.72 0.140728 FEZF1 FEZ family zinc finger 1 -3.16 0.140857 SPTLC3 serine palmitoyltransferase, long chain base subunit 3 -8.29 0.140857 PHF14 PHD finger protein 14 2.33 0.141430 SV2A synaptic vesicle glycoprotein 2A -2.33 0.141430 PRDM6 PR domain containing 6 3.28 0.141430 SDC4 syndecan 4 -3.34 0.141482 RUNX3 runt-related transcription factor 3 2.50 0.141482 NEFL neurofilament, light polypeptide 2.83 0.142040 DDT D-dopachrome tautomerase 2.10 0.142188 ACADVL acyl-CoA dehydrogenase, very long chain -2.01 0.143238 TNFRSF12A tumor necrosis factor receptor superfamily, member 12A -2.43 0.143238 KRT17 keratin 17, type I -2.56 0.143625 SERPINB8 serpin peptidase inhibitor, clade B (ovalbumin), member 8 -4.11 0.146230 TNFAIP3 tumor necrosis factor, alpha-induced protein 3 -3.62 0.146230 METTL7A methyltransferase like 7A 2.09 0.146230 IER3 immediate early response 3 -7.50 0.149465 URM1 ubiquitin related modifier 1 3.77 0.149465 CASP4 caspase 4 -10.88 0.149833 IL13RA2 interleukin 13 receptor, alpha 2 -2.55 0.149863 FLT1 fms-related tyrosine kinase 1 2.48 0.150351 ETV5 ets variant 5 -5.05 0.151176 PAMR1 peptidase domain containing associated with muscle regeneration 1 -3.90 0.153221 PLTP phospholipid transfer protein 7.87 0.153221 NUAK2 NUAK family, SNF1-like kinase, 2 -2.95 0.153221 LAMP1 lysosomal-associated membrane protein 1 -2.23 0.153221 CCL2 chemokine (C-C motif) ligand 2 -12.25 0.153262 TMCO3 transmembrane and coiled-coil domains 3 -2.13 0.154608 NCKAP1L NCK-associated protein 1-like -2.74 0.154608 SLAIN1 SLAIN motif family member 1 3.82 0.155068 GLE1 GLE1 RNA export mediator 4.51 0.155068 EYA1 EYA transcriptional coactivator and phosphatase 1 -2.62 0.155068 MAL2 mal, T-cell differentiation protein 2 (gene/pseudogene) -3.04 0.155068 SPON1 spondin 1, extracellular matrix protein 2.27 0.155068 MAPKAP1 mitogen-activated protein kinase associated protein 1 2.43 0.155068 YDJC YdjC homolog (bacterial) 2.01 0.155068 TP53INP1 tumor protein p53 inducible nuclear protein 1 -2.46 0.155068 GPR160 G protein-coupled receptor 160 -2.01 0.155068 RINL Ras and Rab interactor like -3.72 0.155068 EID2B EP300 interacting inhibitor of differentiation 2B 2.02 0.155068 SP140L SP140 nuclear body protein-like -4.04 0.155068 SVIL supervillin -2.24 0.155068 GCOM1; MYZAP; POLR2M GRINL1A complex locus 1; myocardial zonula adherens protein; polymerase (RNA) II (DNA directed) polypeptide M -2.01 0.155124 FABP5 fatty acid binding protein 5 (psoriasis-associated) 2.22 0.155124 DUSP9 dual specificity phosphatase 9 2.03 0.155124 TENM1 teneurin transmembrane protein 1 4.35 0.155124 PPP1R15A protein phosphatase 1, regulatory subunit 15A -2.55 0.155124 ZER1 zyg-11 related, cell cycle regulator 2.64 0.155124 Supplemental Table 2: continue
GUCY1A3 guanylate cyclase 1, soluble, alpha 3 7.58 0.155124 RALGPS1 Ral GEF with PH domain and SH3 binding motif 1 2.01 0.155144 ATF3 activating transcription factor 3 -3.08 0.155596 SYT11 synaptotagmin XI -4.12 0.155596 NOV nephroblastoma overexpressed -5.79 0.155773 GALNT3 polypeptide N-acetylgalactosaminyltransferase 3 -2.73 0.155773 PRKAR2A protein kinase, cAMP-dependent, regulatory, type II, alpha 2.03 0.155773 RELB v-rel avian reticuloendotheliosis viral oncogene homolog B -3.11 0.155773 CD83 CD83 molecule -2.24 0.155773 GPNMB glycoprotein (transmembrane) nmb -4.50 0.155773 RAC2 ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2) -3.67 0.155824 MYD88 myeloid differentiation primary response 88 -7.29 0.155824 SPATA16 spermatogenesis associated 16 16.93 0.155959 IQCK IQ motif containing K 2.09 0.156641 MYOF myoferlin -3.62 0.156641 FAM129A family with sequence similarity 129, member A -3.39 0.156641 REXO1L4P REX1, RNA exonuclease 1 homolog-like 4, pseudogene -2.33 0.156641 RNF122 ring finger protein 122 2.78 0.157160 TMEM129 transmembrane protein 129, E3 ubiquitin protein ligase 2.71 0.157433 OPTN optineurin -3.04 0.157570 GABRE; MIR224; MIR452 gamma-aminobutyric acid (GABA) A receptor, epsilon; microRNA 224; microRNA 452 -2.90 0.157570 GREM1 gremlin 1, DAN family BMP antagonist [Source:HGNC Symbol;Acc:HGNC:2001] -10.02 0.157570 CD109 CD109 molecule -3.07 0.157570 LOXL4 lysyl oxidase-like 4 -2.23 0.157570 CYBRD1 cytochrome b reductase 1 -2.28 0.157570 ZBED8 zinc finger, BED-type containing 8 2.65 0.157570 DUSP5 dual specificity phosphatase 5 -2.15 0.157570 FLVCR2 feline leukemia virus subgroup C cellular receptor family, member 2 2.03 0.157570 CPA4 carboxypeptidase A4 -4.72 0.157570 CAPN6 calpain 6 -4.49 0.157845 TFPI2 tissue factor pathway inhibitor 2 -3.61 0.157845 QPCT glutaminyl-peptide cyclotransferase -2.51 0.158768 TRPC6 transient receptor potential cation channel, subfamily C, member 6 -2.10 0.158782 AIFM2 apoptosis-inducing factor, mitochondrion-associated, 2 -2.07 0.158984 DERL3 derlin 3 3.09 0.160890 PSAT1 phosphoserine aminotransferase 1 2.38 0.160890 CNN1 calponin 1, basic, smooth muscle -2.60 0.161490 MALSU1 mitochondrial assembly of ribosomal large subunit 1 2.24 0.162899 LPAR5 lysophosphatidic acid receptor 5 -2.32 0.162957 GPX3 glutathione peroxidase 3 -2.67 0.162957 RGS7BP regulator of G-protein signaling 7 binding protein 2.59 0.162957 IRX4 iroquois homeobox 4 -2.35 0.162957 DACH1 dachshund family transcription factor 1 3.85 0.162957 JUNB jun B proto-oncogene -3.22 0.162957 DHRS2 dehydrogenase/reductase (SDR family) member 2 -3.84 0.162999 S100A3 S100 calcium binding protein A3 -4.88 0.162999 LIN7A lin-7 homolog A (C. elegans) 2.01 0.164273 DCBLD2 discoidin, CUB and LCCL domain containing 2 -2.12 0.164354 PTDSS2 phosphatidylserine synthase 2 2.11 0.165015 PARD6G par-6 family cell polarity regulator gamma 2.65 0.165351 TXLNG taxilin gamma 2.14 0.165737 CDC42EP3 CDC42 effector protein (Rho GTPase binding) 3 -2.11 0.167513 SPRY4 sprouty RTK signaling antagonist 4 -3.05 0.167513 IRS2 insulin receptor substrate 2 2.49 0.167513 SYTL5 synaptotagmin-like 5 -6.40 0.167769 PLCXD1 phosphatidylinositol-specific phospholipase C, X domain c ontaining 1 2.77 0.168301 GRHPR glyoxylate reductase/hydroxypyruvate reductase 2.07 0.168301 MAP2 microtubule associated protein 2 -2.66 0.168423 ABHD13 abhydrolase domain containing 13 -2.21 0.168423 TSPAN18 tetraspanin 18 2.19 0.168423 MVP; PAGR1 major vault protein; PAXIP1 associated glutamate-rich protein 1 -4.83 0.168423 KCNH2 potassium channel, voltage gated eag related subfamily H, member 2 -3.66 0.168423 C12orf56 chromosome 12 open reading frame 56 5.71 0.168423 RCAN2 regulator of calcineurin 2 10.42 0.168423 PCDH9 protocadherin 9 3.51 0.168696 SFN stratifin -2.68 0.168775 RNASE1 ribonuclease, RNase A family, 1 (pancreatic) -2.02 0.169077 HLA-DMA major histocompatibility complex, class II, DM alpha -2.95 0.169077 NR2F1 nuclear receptor subfamily 2, group F, member 1 2.14 0.169077 TMEM80 transmembrane protein 80 2.27 0.169077 selenoprotein M; selenoprotein M [Source:EntrezGene; Acc:140606]; Salzman2013 ANNOTATED, CDS, coding, OVCODE, OVERLAPTX, OVEXON, UTR3 best transcript NM_080430; Salzman2013 ANNOTATED, CDS, coding, OVCODE, SELM -2.36 0.169077 OVERLAPTX, OVEXON, UTR3, UTR5 best transcript NM_080430; Transcript Identified by AceView, Entrez Gene ID(s) 140606 HLA-DRB1 major histocompatibility complex, class II, DR beta 1 -2.47 0.169077 S100A16 S100 calcium binding protein A16 -6.34 0.169077 SEL1L3 sel-1 suppressor of lin-12-like 3 (C. elegans) -2.79 0.169077 SLC16A4 solute carrier family 16, member 4 -2.14 0.170338 CD68 CD68 molecule -2.74 0.170338 RAB26 RAB26, member RAS oncogene family 2.56 0.170338 HOXA4 homeobox A4 2.50 0.170400 HLA-DPA1 major histocompatibility complex, class II, DP alpha 1 -4.10 0.170400 PRKCB protein kinase C, beta 2.86 0.170400 HOXC8 homeobox C8 2.49 0.170611 TCEAL3 transcription elongation factor A (SII)-like 3 -2.89 0.170793 GPC3 glypican 3 2.22 0.171328 COL4A1 collagen, type IV, alpha 1 -2.11 0.172224 CD163L1 CD163 molecule-like 1 -8.28 0.172224 GBP3 guanylate binding protein 3 -2.11 0.172739 PNMAL1 paraneoplastic Ma antigen family-like 1 -2.13 0.172779 DSEL dermatan sulfate epimerase-like -5.03 0.172930 Supplemental Table 2: continue
C11orf70 chromosome 11 open reading frame 70 2.09 0.173044 CDK9 cyclin-dependent kinase 9 2.07 0.173832 GLIPR1 GLI pathogenesis-related 1 -4.11 0.173832 KRT222 keratin 222, type II -2.40 0.174048 PUDP pseudouridine 5-phosphatase 4.97 0.174048 SLC16A7 solute carrier family 16 (monocarboxylate transporter), member 7 -3.40 0.174229 SPANXB1 SPANX family, member B1 -2.60 0.174229 ALS2CR11 amyotrophic lateral sclerosis 2 chromosome region candidate 11 2.60 0.175651 CCDC102B coiled-coil domain containing 102B -2.32 0.175738 GUCY1B3 guanylate cyclase 1, soluble, beta 3 2.96 0.175976 TNFRSF11B tumor necrosis factor receptor superfamily, member 11b -2.20 0.175976 PLCXD1 phosphatidylinositol-specific phospholipase C, X domain containing 1 2.72 0.175976 TRPV3 transient receptor potential cation channel, subfamily V, member 3 -4.30 0.175976 AIMP2 aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 2.02 0.175976 NPTX2 neuronal pentraxin II 2.66 0.175976 SARM1 sterile alpha and TIR motif containing 1 2.48 0.176147 LOXL1 lysyl oxidase-like 1 -2.22 0.178033 DPM2 dolichyl-phosphate mannosyltransferase polypeptide 2, regulatory subunit 2.68 0.179237 SYNGR4 synaptogyrin 4 -2.64 0.179237 RPA3 replication protein A3 2.07 0.179296 STXBP1 syntaxin binding protein 1 2.21 0.179296 LIPG lipase, endothelial -2.72 0.179296 KLRG1 killer cell lectin-like receptor subfamily G, member 1 2.40 0.179296 NUDCD2 NudC domain containing 2 2.08 0.179296 FAM84B family with sequence similarity 84, member B 3.16 0.179296 IRF1 interferon regulatory factor 1 -2.36 0.179296 ZNF334 zinc finger protein 334 4.81 0.179883 SLC25A1 solute carrier family 25 (mitochondrial carrier; citrate transporter), member 1 2.20 0.180060 MAN2A2 mannosidase, alpha, class 2A, member 2 -2.01 0.180101 UNC5C unc-5 netrin receptor C 2.78 0.181190 KCNT2 potassium channel, sodium activated subfamily T, member 2 -3.69 0.181454 CCBL1 cysteine conjugate-beta lyase, cytoplasmic 2.93 0.181636 MARCH3 membrane associated ring finger 3 2.64 0.182556 TMEM40 transmembrane protein 40 -4.92 0.182630 KCNK1 potassium channel, two pore domain subfamily K, member 1 -2.23 0.183459 ITGA3 integrin alpha 3 -2.05 0.183675 RABEPK Rab9 effector protein with kelch motifs 2.75 0.183675 PTX3 pentraxin 3, long -2.63 0.183693 CLDN1 claudin 1 -3.24 0.184224 PCDHB9 protocadherin beta 9 -2.90 0.185098 ASRGL1 asparaginase like 1 2.17 0.185098 NOVA1 neuro-oncological ventral antigen 1 -3.47 0.185098 SLC1A4 solute carrier family 1 (glutamate/neutral amino acid transporter), member 4 -2.15 0.185098 PLAU plasminogen activator, urokinase -2.16 0.185098 SLC43A1 solute carrier family 43 (amino acid system L transporter), member 1 2.51 0.185361 Homo sapiens DiGeorge syndrome critical region gene 6 (DGCR6), mRNA.; protein DGCR6; Homo sapiens DiGeorge DGCR6; LOC102724770 syndrome critical region gene 6, mRNA (cDNA clone MGC:54086 IMAGE:5229172), complete cds.; Protein DGCR6 2.03 0.185639 [Source:UniProtKB/Swiss-Prot;Acc:Q14129] SLC13A4 solute carrier family 13 (sodium/sulfate symporter), member 4 -2.94 0.185696 SPANXN3 SPANX family, member N3 -3.62 0.186944 SLC35F1 solute carrier family 35, member F1 4.41 0.186944 MCTP2 multiple C2 domains, transmembrane 2 -3.56 0.187292 OSTN osteocrin 3.26 0.187429 GALNT14 polypeptide N-acetylgalactosaminyltransferase 14 4.35 0.187521 SWI5 SWI5 homologous recombination repair protein 2.84 0.187521 AFF2 AF4/FMR2 family, member 2 3.80 0.187557 FAIM2 Fas apoptotic inhibitory molecule 2 -2.35 0.187846 HLA-DRB5 major histocompatibility complex, class II, DR beta 5 -3.91 0.188078 SFXN3 sideroflexin 3 -2.12 0.188313 DDR2 discoidin domain receptor tyrosine kinase 2 2.02 0.189055 POLR3K polymerase (RNA) III (DNA directed) polypeptide K, 12.3 kDa 2.28 0.189702 ZMYM5 zinc finger, MYM-type 5 -2.12 0.189702 GABRQ gamma-aminobutyric acid (GABA) A receptor, theta -3.47 0.189935 TTC39B tetratricopeptide repeat domain 39B 2.17 0.190122 IFI44L interferon-induced protein 44-like -4.29 0.190453 COLEC12 collectin sub-family member 12 2.19 0.190889 FLII flightless I actin binding protein -2.04 0.191209 PCDHB6 protocadherin beta 6 -4.55 0.191353 ARPC5L actin related protein 2/3 complex subunit 5-like 2.04 0.192061 LAMB3; MIR4260 laminin, beta 3; microRNA 4260 -7.53 0.192103 SMAD3 SMAD family member 3 -2.46 0.192233 TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) -3.55 0.192353 GSTO2 glutathione S-transferase omega 2 -2.69 0.192431 DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 -2.37 0.193172 HAND1 heart and neural crest derivatives expressed 1 3.77 0.194813 ADAP1 ArfGAP with dual PH domains 1 2.24 0.194813 ADCYAP1R1 adenylate cyclase activating polypeptide 1 (pituitary) receptor type I 4.75 0.194813 ADGRA2 adhesion G protein-coupled receptor A2 2.06 0.194813 GALC galactosylceramidase -3.94 0.194839 BTN3A2 butyrophilin, subfamily 3, member A2 -2.05 0.195830 C16orf91 chromosome 16 open reading frame 91 2.05 0.196597 APOBEC3B apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B -2.45 0.196597 PDIA4 protein disulfide isomerase family A, member 4 -2.03 0.196618 USP17L7 ubiquitin specific peptidase 17-like family member 7 -3.34 0.197758 SETSIP SET-like protein 2.96 0.197988 GYPC glycophorin C (Gerbich blood group) 2.96 0.197988 SCIN scinderin 2.05 0.199238 PCDHB16 protocadherin beta 16 -2.71 0.199555 CATSPER1 cation channel, sperm associated 1 -2.12 0.199555 ANKFN1 ankyrin-repeat and fibronectin type III domain containing 1 -2.35 0.200357 DLX5 distal-less homeobox 5 2.99 0.200747 Supplemental Table 2: continue
SEMA3A sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3A -2.50 0.201399 TUFT1 tuftelin 1 -2.03 0.202677 ANK1 ankyrin 1, erythrocytic -2.41 0.203372 FAM214A family with sequence similarity 214, member A -2.21 0.203372 IL32 interleukin 32 -6.65 0.203372 SEMA4B sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4B -2.02 0.203372 UGGT2 UDP-glucose glycoprotein glucosyltransferase 2 -2.29 0.203372 CCNA1 cyclin A1 2.29 0.203372 MAP3K14 mitogen-activated protein kinase kinase kinase 14 -2.12 0.203380 BIRC3 baculoviral IAP repeat containing 3 -3.25 0.203380 AKR1B10 aldo-keto reductase family 1, member B10 (aldose reductase) -2.06 0.203380 RASGRF2 Ras protein-specific guanine nucleotide-releasing factor 2 2.36 0.203530 KHDRBS3 KH domain containing, RNA binding, signal transduction associated 3 4.20 0.203732 HIST1H3I histone cluster 1, H3i 2.83 0.204472 CDR1 cerebellar degeneration related protein 1 -2.02 0.204472 RUNX1 runt-related transcription factor 1 -2.34 0.204472 MR1 major histocompatibility complex, class I-related -2.51 0.204472 MYRF myelin regulatory factor -2.10 0.205442 SLC35D2 solute carrier family 35 (UDP-GlcNAc/UDP-glucose transporter), member D2 -6.37 0.205971 IL7R interleukin 7 receptor -3.62 0.206261 MARC1 mitochondrial amidoxime reducing component 1 2.15 0.206410 LRRC49 leucine rich repeat containing 49 -2.18 0.206552 TCFL5 transcription factor-like 5 (basic helix-loop-helix) 2.05 0.206700 TRIM10 tripartite motif containing 10 -2.06 0.207171 TGFB1 transforming growth factor beta 1 -2.57 0.207319 FURIN furin (paired basic amino acid cleaving enzyme) -2.33 0.208054 LIPH lipase, member H -2.11 0.208054 ANKRD18A; FAM95C ankyrin repeat domain 18A; family with sequence similarity 95, member C 2.07 0.208054 PAQR4 progestin and adipoQ receptor family member IV 2.23 0.208054 DPYD dihydropyrimidine dehydrogenase -3.99 0.209838 STS steroid sulfatase (microsomal), isozyme S 2.91 0.211094 HIST1H1T histone cluster 1, H1t -3.12 0.211155 CARD6 caspase recruitment domain family, member 6 -2.23 0.211783 LUM lumican -2.78 0.212696 SYT1 synaptotagmin I -2.54 0.212696 LBH limb bud and heart development -2.60 0.212696 DMRTA1 DMRT-like family A1 -2.69 0.214015 SERINC2 serine incorporator 2 -2.12 0.214149 ZCCHC10 zinc finger, CCHC domain containing 10 2.10 0.214175 PRKAR1B protein kinase, cAMP-dependent, regulatory, type I, beta 2.32 0.214304 VGLL3 vestigial-like family member 3 -2.92 0.216748 CYP1B1 cytochrome P450, family 1, subfamily B, polypeptide 1 -2.08 0.217849 LRP2 LDL receptor related protein 2 3.22 0.218946 TSHZ2 teashirt zinc finger homeobox 2 3.35 0.220753 EMILIN2 elastin microfibril interfacer 2 -2.58 0.220772 IL6R interleukin 6 receptor -2.85 0.220898 BICC1 BicC family RNA binding protein 1 -2.21 0.221128 RTN4RL1 reticulon 4 receptor-like 1 3.65 0.221128 LIG4 ligase IV, DNA, ATP-dependent -2.66 0.221500 CDH23 cadherin-related 23 2.50 0.222620 GPM6A glycoprotein M6A -2.63 0.223775 ADGRE5 adhesion G protein-coupled receptor E5 -2.35 0.223775 LMX1B LIM homeobox transcription factor 1, beta 2.45 0.224040 TMEM130 transmembrane protein 130 -2.50 0.224040 TNF tumor necrosis factor -5.37 0.224040 KIAA1549L KIAA1549-like 2.39 0.225116 EPHA4 EPH receptor A4 2.27 0.225245 CCSER1 coiled-coil serine rich protein 1 2.31 0.225443 ZNF503 zinc finger protein 503 2.23 0.225601 STMN3 stathmin-like 3 2.76 0.226887 LRRC8C leucine rich repeat containing 8 family, member C -2.04 0.227782 PGR progesterone receptor -4.12 0.227782 MMP13 matrix metallopeptidase 13 -8.58 0.227782 ZNF32 zinc finger protein 32 2.02 0.227782 GABBR2 gamma-aminobutyric acid (GABA) B receptor, 2 2.43 0.227782 TRIM55 tripartite motif containing 55 -2.06 0.227782 ANKRD18B ankyrin repeat domain 18B 2.03 0.227782 FRK fyn-related Src family tyrosine kinase -2.25 0.227782 ZDHHC22 zinc finger, DHHC-type containing 22 2.22 0.227782 RASA4 RAS p21 protein activator 4 -2.13 0.227974 COLCA2 colorectal cancer associated 2 2.06 0.228844 ARRDC3 arrestin domain containing 3 2.13 0.228864 PKIG protein kinase (cAMP-dependent, catalytic) inhibitor gamma -2.03 0.228864 ZFP36 ZFP36 ring finger protein -2.04 0.229162 KMT5C lysine (K)-specific methyltransferase 5C 2.04 0.229550 VWA5A von Willebrand factor A domain containing 5A -2.67 0.229568 SCAI; GOLGA1 suppressor of cancer cell invasion; golgin A1 2.28 0.229568 XPNPEP3 X-prolyl aminopeptidase 3, mitochondrial 2.54 0.229568 SYT14 synaptotagmin XIV -3.78 0.230916 FAM216A family with sequence similarity 216, member A 2.37 0.231097 CEACAM1 carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) -2.04 0.231097 AR androgen receptor 2.21 0.231097 PCBP3 poly(rC) binding protein 3 -2.21 0.232701 SPRY2 sprouty RTK signaling antagonist 2 2.38 0.232874 RUNX2 runt-related transcription factor 2 -2.50 0.233182 MIR99AHG mir-99a-let-7c cluster host gene -2.26 0.233279 GJB7 gap junction protein beta 7 2.45 0.233571 FPGS folylpolyglutamate synthase 2.10 0.233936 SERF1B; SERF1A small EDRK-rich factor 1B (centromeric); small EDRK-rich factor 1A (telomeric) 2.03 0.234194 SERF1A small EDRK-rich factor 1A (telomeric) 2.03 0.234194 IL31RA interleukin 31 receptor A -5.13 0.235161 Supplemental Table 2: continue
ALDH1L2 aldehyde dehydrogenase 1 family, member L2 2.36 0.235237 TLR1 toll-like receptor 1 -2.28 0.235469 ARMCX2 armadillo repeat containing, X-linked 2 -2.51 0.235892 C8orf4 chromosome 8 open reading frame 4 -2.25 0.235892 KRTAP21-2 keratin associated protein 21-2 -3.44 0.235919 ZBTB20; MIR568 zinc finger and BTB domain containing 20; microRNA 568 -3.82 0.236947 RFPL4A ret finger protein-like 4A -2.75 0.237185 SDR16C5 short chain dehydrogenase/reductase family 16C, member 5 -2.19 0.237315 FAM20C family with sequence similarity 20, member C 2.05 0.240017 DDIT3 DNA-damage-inducible transcript 3 -2.56 0.240083 PLIN2 perilipin 2 2.42 0.240226 ARL15 ADP-ribosylation factor like GTPase 15 2.17 0.240227 SYNPO synaptopodin -2.29 0.240227 ALPK3 alpha kinase 3 -2.23 0.240474 SOX11 SRY box 11 -2.41 0.241008 COL9A3 collagen, type IX, alpha 3 2.64 0.241459 SAMD9 sterile alpha motif domain containing 9 -3.80 0.241735 F2RL2 coagulation factor II (thrombin) receptor-like 2 -3.64 0.241735 ARRDC4 arrestin domain containing 4 -2.12 0.241735 CAMK1G calcium/calmodulin-dependent protein kinase IG -2.65 0.241735 LHPP phospholysine phosphohistidine inorganic pyrophosphate phosphatase -2.15 0.241735 DES desmin -2.05 0.241735 FOSL1 FOS-like antigen 1 -2.79 0.241735 RARB retinoic acid receptor, beta 3.94 0.242651 REXO1L10P REX1, RNA exonuclease 1 homolog-like 10, pseudogene -3.05 0.243730 LMNA lamin A/C -2.01 0.244792 SLITRK5 SLIT and NTRK-like family, member 5 -2.26 0.245036 THBS1 thrombospondin 1 -2.74 0.245486 DGKA diacylglycerol kinase alpha -3.09 0.246302 FZD4 frizzled class receptor 4 2.18 0.246357 ANKRD19P ankyrin repeat domain 19, pseudogene 2.32 0.246769 SLC45A3 solute carrier family 45, member 3 -2.06 0.246971 PRR5-ARHGAP8 PRR5-ARHGAP8 readthrough 2.32 0.247324 IFIH1 interferon induced, with helicase C domain 1 -2.47 0.247324 CYP3A5 cytochrome P450, family 3, subfamily A, polypeptide 5 -2.38 0.248155 LHX2 LIM homeobox 2 2.40 0.248155 HLA-DOB major histocompatibility complex, class II, DO beta -2.27 0.248159 CCL20 chemokine (C-C motif) ligand 20 -2.52 0.252810 WFDC2 WAP four-disulfide core domain 2 -2.25 0.252810 USP17L1 ubiquitin specific peptidase 17-like family member 1 -2.46 0.253684 BEGAIN brain-enriched guanylate kinase-associated -2.46 0.254223 DBF4 Transcript Identified by AceView, Entrez Gene ID(s) 10926 2.09 0.254235 MYOM3 myomesin 3 -2.33 0.254235 PCDH10 protocadherin 10 -2.18 0.254758 C1S complement component 1, s subcomponent -2.27 0.257500 HOXA5 homeobox A5 2.62 0.258495 CSF1 colony stimulating factor 1 (macrophage) -2.09 0.258596 CXCL8 chemokine (C-X-C motif) ligand 8 -3.45 0.258596 ENDOG endonuclease G 3.89 0.262148 PLA2G7 phospholipase A2, group VII (platelet-activating factor acetylhydrolase, plasma) 2.83 0.263764 RFPL4AL1 ret finger protein-like 4A-like 1 -2.74 0.264409 RENBP renin binding protein -2.80 0.265119 Supplemental Table 3: Expression levels of the different G protein subunits (α, β and γ) in the parental and ∆Gs cells. The statistical analysis (unpaired one-way ANOVA between-samples; p < 0.05 being considered statistically significant) was done using the “transcriptome analysis console” from Affymetrix. Using a threshold of a minimum of 2 fold change (<-2 or >2), none of the G-protein encoding genes was found to be differentially expressed. The false discovery rate (FDR) adjustment has been calculated for each gene and is indicated.
parental Bi-weight ∆Gs Bi-weight Fold Change ANOVA FDR Gene Description Avg Signal Avg Signal (linear) p-value p-value Symbol (log2) (log2) (parental vs ∆Gs) (parental vs ∆Gs) (parental vs ∆Gs) GNA11 guanine nucleotide binding protein (G protein), alpha 11 12.79 12.85 -1.04 0.135977 0.394487 GNA12 guanine nucleotide binding protein (G protein) alpha 12 11.64 11.22 1.34 0.126440 0.383426 GNA13 guanine nucleotide binding protein (G protein), alpha 13 11.84 12.22 -1.30 0.023307 0.211094 GNA14 guanine nucleotide binding protein (G protein), alpha 14 5.35 5.38 -1.03 0.490967 0.723730 GNA15 guanine nucleotide binding protein (G protein), alpha 15 7.75 7.64 1.08 0.630218 0.815803 GNAI1 guanine nucleotide binding protein (G protein), alpha i1 12.77 13.38 -1.52 0.034347 0.236947 GNAI2 guanine nucleotide binding protein (G protein), alpha i2 12.92 13.29 -1.30 0.000435 0.112203 GNAI3 guanine nucleotide binding protein (G protein), alpha i3 13.29 12.97 1.25 0.106507 0.355931 GNAO1 guanine nucleotide binding protein (G protein), alpha o 8.66 7.80 1.82 0.179868 0.447122 GNAQ guanine nucleotide binding protein (G protein), alpha q 10.67 9.73 1.92 0.014903 0.187846 GNAT1 guanine nucleotide binding protein (G protein), alpha t-rod 6.45 6.49 -1.03 0.823536 0.920852 GNAT2 guanine nucleotide binding protein (G protein), alpha t-cone 7.20 7.21 -1.01 0.920552 0.966923 GNAT3 guanine nucleotide binding protein (G protein), alpha t-gus 4.29 4.73 -1.35 0.312938 0.583998 GNAZ guanine nucleotide binding protein (G protein), alpha z 10.50 9.83 1.59 0.001963 0.129017 GNB1 guanine nucleotide binding protein (G protein), beta 1 15.07 15.24 -1.12 0.128356 0.386013 GNB2 guanine nucleotide binding protein (G protein), beta 2 12.73 13.33 -1.52 0.011808 0.179296 GNB3 guanine nucleotide binding protein (G protein), beta 3 7.91 7.67 1.19 0.198847 0.469394 GNB4 guanine nucleotide binding protein (G protein), beta 4 11.51 11.39 1.09 0.370855 0.631538 GNB5 guanine nucleotide binding protein (G protein), beta 5 7.53 7.98 -1.37 0.039106 0.244321 GNGT1 guanine nucleotide binding protein (G protein), gamma 1 4.98 5.45 -1.39 0.240187 0.512642 GNG2 guanine nucleotide binding protein (G protein), gamma 2 7.33 7.48 -1.11 0.511698 0.739180 GNG3 guanine nucleotide binding protein (G protein), gamma 3 4.16 4.28 -1.09 0.750803 0.884181 GNG4 guanine nucleotide binding protein (G protein), gamma 4 11.38 12.32 -1.91 0.002438 0.133236 GNG5 guanine nucleotide binding protein (G protein), gamma 5 13.84 13.82 1.02 0.503797 0.733463 GNG7 guanine nucleotide binding protein (G protein), gamma 7 9.57 9.31 1.20 0.074743 0.308821 GNG8 guanine nucleotide binding protein (G protein), gamma 8 5.77 5.83 -1.04 0.729230 0.872955 GNGT2 guanine nucleotide binding protein (G protein), gamma 9 6.14 6.69 -1.46 0.332215 0.600536 GNG10 guanine nucleotide binding protein (G protein), gamma 10 10.74 10.19 1.46 0.047841 0.262132 GNG11 guanine nucleotide binding protein (G protein), gamma 11 7.36 8.22 -1.81 0.006633 0.157845 GNG12 guanine nucleotide binding protein (G protein), gamma 12 11.50 12.22 -1.65 0.019686 0.202782 GNG13 guanine nucleotide binding protein (G protein), gamma 13 6.21 5.98 1.17 0.380751 0.638756 Supplemental Figure 1. Genomic sequences of GNAS- and GNAL-double mutant HEK293 cells. sgRNA-target sequences of the three mutant clones (A, CL1; B, CL2; C, CL3) were determined by a TA-cloning method. sgRNA target sequences are boxed and PAM sequences (NGG) are underlined. Arrows indicate a putative double-stranded break site. Restriction enzyme sites (Hap II (GNAS and GNAL #2), Pvu II (GNAL #3) and Hae III (GNAL #1)) are highlighted in red.
10 Supplemental Figure 2. Genotyping of Gs-mutant clones identified by a restriction enzyme- digested fragment method. sgRNA targets including one site (A) in the GNAS gene site and the three sites (sgRNA #1, #2 and #3, shown in B, C and D, respectively) in the GNAL gene were PCR- amplified and treated with the corresponding restriction enzymes (RE; Hap II (A and C), Hae III (B) or Pvu II (D)). The digested samples were subjected to a capillary electrograph analysis (MultiNA, Shimadzu) and pseudo-gel images of electropherogram were visualized by an accessory software with DNA markers of Hae III-digested phaiX174. Filled (grey) and open arrowheads indicate undigested and RE-digested, respectively, PCR fragments of the targeted sites in the parental HEK293 cells. An open arrowhead with an asterisk (CL2 in A) denotes a heteroduplex between the small and large single-stranded DNA. Note that there are four Hae III sites in a parental PCR fragment (B).
11 A An alignment of the Gs amino acids
GNAs-targeting sgRNA #2
B An alignment of the Golf amino acids
GNAL-targeting sgRNA #3
GNAL-targeting sgRNA #1
GNAL-targeting sgRNA #2
Supplemental Figure 3. An alignment of deduced amino acid sequences of the GNAS-(Gαs, A) and GNAL- (Gαolf, B) mutant alleles from the ∆Gs-HEK293 clones. Genomic sequences of the GNAS (A) and GNAL (B) gene from the three mutant clones (CL1, CL2 and CL3) were determined with a TA cloning method. A) Note that all alleles were introduced with a frame-shift mutation. Parent Gαs sequence refers to the isoform GNASL (long isoform). B) Note that except for one allele (CL3 allele #1, which has 28-amino acid insertion), all alleles were introduced with a frame-shift mutation. Parent Gαolf sequence refers to the isoform 2. Arrows indicate the GNAS-andGNAL-(#1, #2 and #3 constructs were used to generate the CL1, CL2 and CL3, respectively) targeting sgRNA site. Aligned regions with 100% identity are shaded in black and those above 50% identity are shaded grey.
12 epnefloigete aorsi AP 0n)o osoi Fk 10 (Fsk, Forskolin or 100nM) ∆ (AVP, vasopressin either following response ∆ epnefloigete spoeeo IO 10 (ISO, isoproterenol either following response the of of Characterization clones – 4 Figure Supplemental A esrdfloigete S rinpoeA38 1 M ciain h ubra h o fteISO the of top the Ca at number the The of activation. mM) % (10 the A23187 represent ionophore or columns ISO either following measured ...o needn xeiet,ec efre ntilct.Clm rpswr nlzdb two- by analyzed were graphs Column comparison triplicate. multiple in Bonferroni’s a performed by each followed ANOVA experiments, way independent 3 of S.E.M. C B sHK9Tclswr rninl rnfce with transfected transiently were cells Gs-HEK293T with transfected transiently cells Gs-HEK293T cAMP response (BRET2) cAMP response (BRET2) Ca2+ response (AUC x 105) 0 2 4 6 8 sHK9Tclstasetytasetdwt h PCBE isno eetse o cAMP for tested were biosensor EPAC-BRET the with transfected transiently cells ∆Gs-HEK293T HEK293 HEK293 HEK293 Overexpressed Overexpressed V2R L L CL3 CL2 CL1 Endogenous ∆Gs-HEK293 L L CL3 CL2 CL1 L L CL3 CL2 CL1 ∆Gs-HEK293 ∆Gs-HEK293 β 2AR 2+ β 2 epneotie ihA28.Dt r xrse stema ± mean the as expressed are Data A32187. with obtained response AR )o osoi Fk 10 (Fsk, Forskolin or M) A23187 ISO 2 n PCBE isno eetse o cAMP for tested were biosensor EPAC-BRET and V2R 13 sHK9 cells. ∆Gs-HEK293 β A n blnboesr n Ca and biosensor, obelin and 2AR post-hoc test. A aetlHK9Tadthree and HEK293T Parental (A) )atvto.()HK9Tor HEK293T (B) activation. M) )atvto.()HK9Tor HEK293T (C) activation. M) +2 epnewas response Supplemental Figure 5 – Scatter plot of the microarray comparing gene expression between parental and ∆Gs-HEK293 cells. Gene Level Differential Expression Analysis of the parental vs ∆Gs (clone 1) cells was performed on 21,448 genes using the Clarion S Human array type (Affymetrix). Among these, 491 genes were found to be either transcriptionally up-regulated (198, green) or down-regulated (293, red), including GNAL and GNAS that were knockdown using the CRISPR/Cas9 system. The list of 489 up- or down-regulated genes (with a minimum threshold of 2 fold) are presented in Supplemental Table 2. A Purinergic (P2X) Microarray signal (a.u.)
B Adenosine (P1Y) Purinergic (P2Y) Microarray signal (a.u.)
Supplemental Figure 6 – mRNA expression of P2X and P2Y purinergic receptor subtypes in HA-β2AR- HEK293S cells. Total mRNA expression in HA-β2AR-HEK293S cells was analyzed by gene expression array. Data were normalized using a quantile normalization procedure (see Methods). For comparison purposes, the expression levels of the housekeeping genes β-actin and α-tubulin are shown, since these genes are highly expressed in HA-β2AR-HEK293S cells. Since HEK293 cells have previously been found to not express Gαo to detectable amounts by Western blotting (Law et al., 1993), the normalized value for this probe is included on the graph to represent a baseline level of fluorescence of the microarray probes. The expression of the endogenous β2AR (not the overexpressed HA-β2AR, due to the location of the β2AR probe in the 3’- untralnslated region) is shown for comparison, as well as the expression of all of the purinergic receptor subtypes in HA-β2AR cells.
15