Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, as 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 that these proteins are co-expressed in lipid rafts. We overexpressed AC2, AC3 or AC6 in mouse bronchial smooth muscle cells (mBSMC) and HEK-293 cells using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCR). 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 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 we tested. Forskolin-induced arborization of mBSMC 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 addition of the PDE4 inhibitor, rolipram, AC2 did accelerate forskolin-stimulated arborization. Thus, AC2 selectively couples to EP2R but signals from this complex are limited by PDE4 activity. AC3 does not appear to couple to GPCR in either mBSMC or HEK-293 cells, so likely exists in a distinct signaling domain.
- Received February 20, 2012.
- Revision received May 21, 2012.
- Accepted May 21, 2012.
- The American Society for Pharmacology and Experimental Therapeutics