Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Feb 28:14:1149063.
doi: 10.3389/fphys.2023.1149063. eCollection 2023.

Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway smooth muscle cells

Isabella Cattani-Cavalieri  1 , Yue Li  2 , Jordyn Margolis  1 , Amy Bogard  3 , Moom R Roosan  2 , Rennolds S Ostrom  1
Affiliations

Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway smooth muscle cells

Isabella Cattani-Cavalieri et al. Front Physiol. .

Abstract

Human airway smooth muscle (HASM) is the primary target of ßAR agonists used to control airway hypercontractility in asthma and chronic obstructive pulmonary disease (COPD). ßAR agonists induce the production of cAMP by adenylyl cyclases (ACs), activate PKA and cause bronchodilation. Several other G-protein coupled receptors (GPCR) expressed in human airway smooth muscle cells transduce extracellular signals through cAMP but these receptors elicit different cellular responses. Some G-protein coupled receptors couple to distinct adenylyl cyclases isoforms with different localization, partly explaining this compartmentation, but little is known about the downstream networks that result. We used quantitative phosphoproteomics to define the downstream signaling networks emanating from cAMP produced by two adenylyl cyclases isoforms with contrasting localization in uman airway smooth muscle. After a short stimulus of adenylyl cyclases activity using forskolin, phosphopeptides were analyzed by LC-MS/MS and differences between cells overexpressing AC2 (localized in non-raft membranes) or AC6 (localized in lipid raft membranes) were compared to control human airway smooth muscle. The degree of AC2 and AC6 overexpression was titrated to generate roughly equal forskolin-stimulated cAMP production. 14 Differentially phosphorylated proteins (DPPs) resulted from AC2 activity and 34 differentially phosphorylated proteins resulted from AC6 activity. Analysis of these hits with the STRING protein interaction tool showed that AC2 signaling is more associated with modifications in RNA/DNA binding proteins and microtubule/spindle body proteins while AC6 signaling is associated with proteins regulating autophagy, calcium-calmodulin (Ca2+/CaM) signaling, Rho GTPases and cytoskeletal regulation. One protein, OFD1, was regulated in opposite directions, with serine 899 phosphorylation increased in the AC6 condition 1.5-fold but decreased to 0.46-fold by AC2. In conclusion, quantitative phosphoproteomics is a powerful tool for deciphering the complex signaling networks resulting from discreet signaling events that occur in cAMP compartments. Our data show key differences in the cAMP pools generated from AC2 and AC6 activity and imply that distinct cellular responses are regulated by these two compartments.

Keywords: adenylyl cyclase; cAMP; g-protein coupled receptors; human airway smooth muscle; phosphoproteomics.

PubMed Disclaimer

Conflict of interest statement

AB was employed by AB Research LLC. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
cAMP responses measured using the cADDis biosensor in HASM with AC2 or AC6 overexpression. Cells were incubated for 24 h with the indicated recombinant adenovirus and the cADDis baclovirus for 24 h. Fluorescent decay curves were measured for 20 min following addition of either vehicle (not shown) or various concentrations of forskolin. The response to each forskolin concentration was fit to a one-site decay model and the K (decay rate) and plateau for each concentration were multiplied together and plotted on a log scale. The resultant concentration-response curves were fit with non-linear regression. The data represent the mean ± SEM of three different HASM cell lines.
FIGURE 2
FIGURE 2
Venn diagram showing the overlap of up and downregulated differentially phosphorylated proteins in AC2 and AC6 samples. (A) 25 and nine upregulated proteins were detected compared to control samples whereas (B) nine and five downregulated proteins were detected in AC6 and AC2 samples, respectively. Only four and three proteins were common between the two groups in up and downregulated proteins. The rest of the detected proteins were unique to each treatment condition. Overall, twice as many differentially phosphorylated proteins were identified in AC6 samples compared to AC2 samples.
FIGURE 3
FIGURE 3
Schematic diagram of cAMP signaling via transmembrane ACs. Once activated by forskolin or a GPCR agonist acting through Gs, an AC isoform catalyzes the reaction of ATP to cAMP. The cAMP signal is terminated though the activity of one or more PDEs that degrade cAMP. The cAMP signal is advanced though the activation of PKA, Epac, and POPDC. When PKA is activated by cAMP, AKAP-directed targeting directs phosphorylation of specific proteins to generate signals through distinct pathways. POPDC and EPAC also initiate signaling and, in addition to the PKA-initiated signals, generate complex signaling networks via crosstalk between pathways. The complex signaling events can lead to increased or decreased phosphorylation of various proteins. For example, lipid raft AC6 led to increased phosphorylation of OFD1 (green arrow) potentially producing physiological effects such as mitosis, cilia assembly and hedgehog signaling. However, non-raft localized AC2 led to decreased phosphorylation of OFD1 (red arrow) potentially causing different effects such as microtubule association and epithelial cilium movement. A few of the other phosphoproteins that were increased or decreased by a specific AC signaling pool in our studies are shown along with some of the physiological effects associated with those proteins.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Agarwal S. R., Yang P. C., Rice M., Singer C. A., Nikolaev V. O., Lohse M. J., et al. (2014). Role of membrane microdomains in compartmentation of cAMP signaling. PLoS One 9, e95835. 10.1371/journal.pone.0095835 - DOI - PMC - PubMed
    1. Agarwal S. R., Miyashiro K., Latt H., Ostrom R. S., Harvey R. D. (2017). Compartmentalized cAMP responses to prostaglandin EP2 receptor activation in human airway smooth muscle cells. Br. J. Pharmacol. 174, 2784–2796. 10.1111/bph.13904 - DOI - PMC - PubMed
    1. Agarwal S. R., Gratwohl J., Cozad M., Yang P. C., Clancy C. E., Harvey R. D. (2018). Compartmentalized cAMP signaling associated with lipid raft and non-raft membrane domains in adult ventricular myocytes. Front. Pharmacol. 9, 332. 10.3389/fphar.2018.00332 - DOI - PMC - PubMed
    1. Agarwal S. R., Fiore C., Miyashiro K., Ostrom R. S., Harvey R. D. (2019). Effect of adenylyl cyclase type 6 on localized production of cAMP by beta-2 adrenoceptors in human airway smooth-muscle cells. J. Pharmacol. Exp. Ther. 370, 104–110. 10.1124/jpet.119.256594 - DOI - PMC - PubMed
    1. Baillie G. S. (2009). Compartmentalized signalling: Spatial regulation of cAMP by the action of compartmentalized phosphodiesterases. FEBS J. 276, 1790–1799. 10.1111/j.1742-4658.2009.06926.x - DOI - PubMed