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Research ArticleCellular and Molecular

Adenylyl Cyclase 2 Selectively Couples to E Prostanoid Type 2 Receptors, Whereas Adenylyl Cyclase 3 Is Not Receptor-Regulated in Airway Smooth Muscle

Amy S. Bogard, Piyatilake Adris and Rennolds S. Ostrom
Journal of Pharmacology and Experimental Therapeutics August 2012, 342 (2) 586-595; DOI: https://doi.org/10.1124/jpet.112.193425
Amy S. Bogard
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Piyatilake Adris
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Rennolds S. Ostrom
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Abstract

Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because β-adrenergic receptor (βAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to βAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with β2AR but not E prostanoid type 2 receptor (EP2R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the β2AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP2R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.

Footnotes

  • This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL079166].

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    http://dx.doi.org/10.1124/jpet.112.193425.

  • ↵Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    GPCR
    G protein-coupled receptor
    AC
    adenylyl cyclase
    βAR
    β-adrenergic receptor
    mBSMC
    mouse bronchial smooth muscle cell
    HEK
    human embryonic kidney
    PGE2
    prostaglandin E2
    EP
    E prostanoid
    EP2R
    prostacyclin EP2 receptor
    RT
    reverse transcriptase
    PCR
    polymerase chain reaction
    PKA
    protein kinase A
    PDE
    phosphodiesterase
    AKAP
    A kinase anchoring protein
    IBMX
    3-isobutyl-1-methylxanthine
    PBS
    phosphate-buffered saline
    MBS
    25 mM 4-morpholineethanesulfonic acid and 150 mM NaCl, pH 6.5
    AdV
    adenovirus
    Fsk
    forskolin
    Iso
    isoproterenol
    Cav
    caveolin
    IPR
    I prostanoid receptor.

  • Received February 20, 2012.
  • Accepted May 21, 2012.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 342 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 342, Issue 2
1 Aug 2012
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Research ArticleCellular and Molecular

AC Isoform Compartments in Airway Smooth Muscle

Amy S. Bogard, Piyatilake Adris and Rennolds S. Ostrom
Journal of Pharmacology and Experimental Therapeutics August 1, 2012, 342 (2) 586-595; DOI: https://doi.org/10.1124/jpet.112.193425

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Research ArticleCellular and Molecular

AC Isoform Compartments in Airway Smooth Muscle

Amy S. Bogard, Piyatilake Adris and Rennolds S. Ostrom
Journal of Pharmacology and Experimental Therapeutics August 1, 2012, 342 (2) 586-595; DOI: https://doi.org/10.1124/jpet.112.193425
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