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.
↵ 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
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|