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

Abstract

Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because ␤-adrenergic receptor (␤AR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to ␤AR 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 proteincoupled 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 ␤ 2 AR but not E prostanoid type 2 receptor (EP 2 R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the ␤ 2 AR-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 EP 2 R, 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.

Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor

“…AC2 is also stimulated in a PKC-independent manner by Ga s in response to activation of G s -coupled receptors and directly via the small molecule AC activator forskolin. PGE 2 is known to bind and activate the G s -coupled EP2/4 prostanoid receptors that are endogenously expressed in HEK293 cells (Willoughby et al, 2007;Bogard et al, 2012). As expected, PGE 2 treatment resulted in a concentration-dependent increase in cAMP accumulation in HEK-hAC2 cells (EC 50 : 160 6 78 nM, n 5 3).…”

Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

“…Toll-like receptor 4 is associated with AC6 in lipid rafts in a murine macrophage cell line (24), while stomatin-related olfactory protein and AC3 are found in lipid rafts in olfactory cilia (22). By contrast, AC2 and E prostanoid type 2 receptors are found in non-lipid rafts in mouse bronchial smooth muscle cells (21). …”

Prostaglandin E Receptor EP1 Forms a Complex with Dopamine D1 Receptor and Directs D1-Induced cAMP Production to Adenylyl Cyclase 7 through Mobilizing GβγSubunits in Human Embryonic Kidney 293T Cells

“…It has been shown that several GPCRs use a specific AC isoform for cAMP production. For example, in mouse smooth muscle cells and HEK-293 cells, AC2 and AC6 mediate cAMP increase induced by EP2 and ADRB2, respectively (Bogard et al, 2012). Consistent with specific GPCR-AC coupling as such, subcellular fractionation showed that ADRB2 and AC6 are localized in lipid rafts, from which EP2, AC2, and AC7 are excluded (Crossthwaite et al, 2005;Bogard et al, 2012).…”

“…For example, in mouse smooth muscle cells and HEK-293 cells, AC2 and AC6 mediate cAMP increase induced by EP2 and ADRB2, respectively (Bogard et al, 2012). Consistent with specific GPCR-AC coupling as such, subcellular fractionation showed that ADRB2 and AC6 are localized in lipid rafts, from which EP2, AC2, and AC7 are excluded (Crossthwaite et al, 2005;Bogard et al, 2012). Likewise, D1 receptors that are coupled to AC6 without EP1 and those that are coupled to AC7 with EP1 could be localized in different membrane domains, lipid raft domains, and nonlipid raft domains, respectively.…”