title: Purinergic Receptor Transactivation by theβ2-Adrenergic Receptor Increases Intracellular Ca2+in Nonexcitable Cells.
abstract: Theβ2adrenergic receptor (β2AR) increases intracellular Ca2+in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which theβ2AR promotes Ca2+mobilization (pEC50= 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca2+followingβ2AR stimulation. The increase in cytoplasmic Ca2+was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of theβ2AR activation. Consistent with this mechanism,β2AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited theβ2AR-promoted Ca2+mobilization. Together, these data support a model whereby theβ2AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP3-dependent Ca2+mobilization from intracellular stores. Given thatβ2AR and P2Y receptors are coexpressed in various tissues, this novel signaling 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 signaling.
authors.affiliation: 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 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.).
authors.forename: Wayne
authors.initials: W
authors.lastname: Stallaert
authors.affiliation: 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 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.).
authors.forename: Emma T
authors.initials: ET
authors.lastname: van der Westhuizen
authors.affiliation: 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 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.).
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Schönegge
authors.affiliation: 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 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.).
authors.forename: Bianca
authors.initials: B
authors.lastname: Plouffe
authors.affiliation: 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 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.).
authors.forename: Mireille
authors.initials: M
authors.lastname: Hogue
authors.affiliation: 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 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.).
authors.forename: Viktoria
authors.initials: V
authors.lastname: Lukashova
authors.affiliation: 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 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.).
authors.forename: Asuka
authors.initials: A
authors.lastname: Inoue
authors.affiliation: 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 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.).
authors.forename: Satoru
authors.initials: S
authors.lastname: Ishida
authors.affiliation: 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 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.).
authors.forename: Junken
authors.initials: J
authors.lastname: Aoki
authors.affiliation: 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 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.).
authors.forename: Christian
authors.initials: C
authors.lastname: Le Gouill
authors.affiliation: 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 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.) michel.bouvier@umontreal.ca.
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019204:GTP-Binding Proteins / Q000378:metabolism; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058470:Receptors, Purinergic P2Y / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D015533:Transcriptional Activation
chemical_list: C109734:P2RY11 protein, human; D018048:Receptors, Purinergic P2; D058470:Receptors, Purinergic P2Y; D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D019204:GTP-Binding Proteins; D002118:Calcium