Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2017/03/09/mol.116.106419.DC1 1521-0111/91/5/533–544$25.00 https://doi.org/10.1124/mol.116.106419 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:533–544, May 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Purinergic Receptor Transactivation by the b2-Adrenergic Receptor Increases Intracellular Ca21 in Nonexcitable Cells s Wayne Stallaert,1,2 Emma T. van der Westhuizen,1,3 Anne-Marie Schönegge, Bianca Plouffe, Mireille Hogue, Viktoria Lukashova, Asuka Inoue, Satoru Ishida, Junken Aoki, Christian Le Gouill, and Michel Bouvier Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Downloaded from Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.) Received August 14, 2016; accepted March 6, 2017 molpharm.aspetjournals.org ABSTRACT 21 The b2 adrenergic receptor (b2AR) increases intracellular Ca in receptor antagonist, suggesting a role for this Gq-coupled a variety of cell types. By combining pharmacological and receptor family downstream of the b2AR activation. Consistent genetic manipulations, we reveal a novel mechanism through with this mechanism, b2AR stimulation promoted the extracel- 21 which the b2AR promotes Ca mobilization (pEC50 5 7.32 6 lular release of ATP, and pretreatment with apyrase inhibited the 21 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. b2AR-promoted Ca mobilization. Together, these data sup- Downregulation of Gs with sustained cholera toxin pretreatment port a model whereby the b2AR stimulates a Gs-dependent and the use of Gs-null HEK293 (ΔGs-HEK293) cells generated release of ATP, which transactivates Gq-coupled P2Y receptors using the clustered regularly interspaced short palindromic through an inside-out mechanism, leading to a Gq- and IP3- 21 repeat-associated protein-9 nuclease (CRISPR/Cas9) system, dependent Ca mobilization from intracellular stores. Given that at ASPET Journals on September 25, 2021 combined with pharmacological modulation of cAMP formation, b2AR and P2Y receptors are coexpressed in various tissues, this revealed a Gs-dependent but cAMP-independent increase in novel signaling paradigm could be physiologically important and 21 intracellular Ca following b2AR stimulation. The increase in have therapeutic implications. In addition, this study reports the cytoplasmic Ca21 was inhibited by P2Y purinergic receptor generation and validation of HEK293 cells deleted of Gs using antagonists as well as a dominant-negative mutant form of Gq, a the CRISPR/Cas9 genome editing technology that will undoubt- Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) edly be powerful tools to study Gs-dependent signaling. This work was supported, in part, by Canadian Institutes for Health Introduction Research (CIHR) [Grant MOP 11215 to M.B.] and grants from Precursory Research for Embryonic Science and Technology, Japan Science and The b2 adrenergic receptor (b2AR) has been shown to Technology Agency [to A.I.], and Japan Agency for Medical Research and regulate a vast signaling network, leading to the activation of Development, Core Research for Evolutional Science and Technology [to J.A.]. W.S. was supported by the Vanier Canada Graduate Scholarship from CIHR. key cellular effectors such as adenylyl cyclase (AC), extracellu- E.T.v.d.W. was supported by postdoctoral research fellowships from CIHR, lar signal-regulated kinase 1/2, and Akt (De Blasi, 1990; Daaka Canadian Hypertension Society, Fonds de la Recherche en Santé du Quebec (FRSQ), and National Health and Medical Research Council Australia [Grant et al., 1998; Bommakanti et al., 2000) to control a variety of GNT-1013819]. A.-M.S. was supported by postdoctoral research fellowships physiologic processes, including the regulation of cardiac func- from FRSQ, and B.P. was supported by postdoctoral research fellowships from CIHR. M.B. holds the Canada Research Chair in Signal Transduction and tion, smooth muscle tone, immunologic responses, fat metabo- Molecular Pharmacology. lism, as well as both central and peripheral nervous system 1W.S. and E.T.v.d.W. contributed equally to this work and should be considered co-first authors. activity (Guimarães and Moura, 2001; Collins et al., 2004; 2Current affiliation: Department of Systemic Cell Biology, Max Planck Sitkauskiene and Sakalauskas, 2005; Pérez-Schindler et al., Institute of Molecular Physiology, Dortmund, Germany. 3Current affiliation: Monash Institute for Pharmaceutical Sciences, Monash 2013). Although much is known about the signaling repertoire University, Parkville, Victoria, Australia. of the b2AR, new insights into its full signaling capabilities and https://doi.org/10.1124/mol.116.106419. s This article has supplemental material available at molpharm. its role in cellular biology continue to be discovered (Stallaert aspetjournals.org. et al., 2012; van der Westhuizen et al., 2014). ABBREVIATIONS: AC, adenylyl cylase; b2AR, b2 adrenergic receptor; BRET, bioluminescent resonance energy transfer; Cch, carbamylcholine chloride; CRISPR/Cas9, clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease; CTX, cholera toxin; FBS, fetal bovine serum; FRET, fluorescent resonance energy transfer; GPCR, G protein-coupled receptor; HA, hemagglutinin; HEK, human embryonic kidney; IP3, inositol 1,4,5-trisphosphate; ISO, isoproterenol; PCR, polymerase chain reaction; PEI, polyethylenimine. 533 534 Stallaert et al. In addition to the abovementioned signaling pathways, the [imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]] 21 9 b2AR can also stimulate an increase in intracellular Ca . bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt], NF340 [4,4 - This signaling response is well established in excitable cells, (Carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis 9 such as cardiomyocytes, via a mechanism involving cAMP- (naphthalene-2,6-disulfonic acid) tetrasodium salt], NF279 [8,8 - mediated regulation of plasma membrane L-type Ca21 chan- [Carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)] bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt], A-804598 nels (Zhang et al., 2001; Christ et al., 2009; Benitah et al., 99 9 21 [N-Cyano-N -[(1S)-1-phenylethyl]-N -5-quinolinyl-guanidine], 5-BDBD 2010). However, increases in intracellular Ca levels have [5-(3-Bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin- also been observed in nonexcitable cells, such as alveolar 2-one], CGS 15943 [9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c] epithelial cells (Keller et al., 2014), and in human embryonic quinazolin-5-amine] and NF449 [4,49,499,4999-[Carbonylbis(imino- kidney (HEK)293 cells endogenously or stably overexpressing 5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedisulfonic the b2AR (Schmidt et al., 2001; Stallaert et al., 2012; van der acid octasodium salt] were obtained from R&D Systems (Bio-Techne, Westhuizenetal.,2014).Previous observations from our labora- Minneapolis, MN). Coelenterazine 400a and coelenterazine h were purchased from Nanolight Technologies (Pinetop, AZ), and coelenter- tory indicated that such b2AR-mediated increases in intra- cellular Ca21 in HEK293 cells are inhibited by an inositol azine cp was purchased from Biotium (Hayward, CA). Arginine-vasopressin and A23187 [5-(Methylamino)-2-[[2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8- 1,4,5-trisphosphate (IP3) receptor antagonist, 2-APB (Stallaert 1 [(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2- et al., 2012), suggesting the release of Ca2 from intracellular yl]methyl]-4-benzoxazolecarboxylic acid] were from Tocris (Bio-Techne). Downloaded from stores, which points to a mechanism distinct from the plasma 21 Cell culture reagents were from Wisent (Montreal, QC, Canada). The Gq membrane Ca channel activation described in excitable cells. selective inhibitor, FR900359 (Schrage et al., 2015), was obtained from E. Several studies have reported that activation of the b2AR Kostenis and S. Kehraus from the University of Bonn (Bonn, Germany). can also stimulate the release of extracellular mediators as part The Gq and Gq(Q209L/D277N) constructs were purchased from Missouri of its signaling repertoire. Activation of the b2AR in mouse S&T cDNA Resource Center (Rolla, MO). HapII, PvuII, and HaeII skeletal muscle, vascular smooth muscle cells, or cardiac restriction enzymes were from Takara Bio (Kusatsu, Shiga, Japan). All fibroblasts promotes the extracellular release of cAMP, which other reagents were obtained from Sigma-Aldrich, unless otherwise stated. molpharm.aspetjournals.org is converted to AMP by ecto-phosphodiesterases and then to Cell Culture and Transfections. Parental HEK293S cells or adenosine by ecto-59-nucleotidase on the extracellular surface HEK293S cells stably expressing an amino-terminal tagged human b AR [3.17 6 0.32 pmol/mg protein; hemagglutinin (HA)-b AR- of the cells (Dubey et al., 1996, 2001; Duarte et al., 2012). In 2 2 HEK293S cells] (Galandrin and