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Quantitative phosphoproteomic analysis reveals unique cAMP signaling pools emanating from AC2 and AC6 in human airway
Isabella Cattani-Cavalieri1, Yue Li2, Jordyn Margolis1
1Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, CA, United States.
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.
Insights
This study reveals distinct signaling networks activated by different adenylyl cyclase (AC) isoforms in human airway smooth muscle cells. Understanding these pathways is key for developing targeted asthma and COPD therapies.
Area of Science:
- Cellular Biology
- Molecular Signaling
Background:
- Human airway smooth muscle (HASM) cells utilize cyclic adenosine monophosphate (cAMP) signaling for bronchodilation, primarily mediated by beta-adrenergic receptor (ßAR) agonists.
- Different G-protein coupled receptors (GPCRs) can activate distinct adenylyl cyclase (AC) isoforms, leading to compartmentalized cAMP production and varied cellular responses.
- The downstream signaling networks regulated by specific AC isoforms in HASM cells remain largely uncharacterized.
Purpose of the Study:
- To delineate the distinct downstream signaling networks activated by adenylyl cyclase 2 (AC2) and adenylyl cyclase 6 (AC6) in human airway smooth muscle cells.
- To compare the phosphoproteomic profiles resulting from cAMP generated by localized AC isoforms.
Main Methods:
- Quantitative phosphoproteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed.
- Human airway smooth muscle cells with differential overexpression of AC2 (non-raft membranes) or AC6 (lipid raft membranes) were stimulated with forskolin.
- Differentially phosphorylated proteins (DPPs) were identified and analyzed for protein-protein interactions using STRING.
Main Results:
- AC2 activity was associated with 14 DPPs, primarily impacting RNA/DNA binding and microtubule/spindle body proteins.
- AC6 activity was associated with 34 DPPs, regulating pathways like autophagy, calcium-calmodulin signaling, Rho GTPases, and cytoskeletal regulation.
- The protein OFD1 exhibited opposing phosphorylation changes in response to AC2 and AC6 activity.
Conclusions:
- Quantitative phosphoproteomics effectively deciphers complex signaling networks arising from compartmentalized cAMP production.
- Distinct cAMP pools generated by AC2 and AC6 orchestrate different cellular responses in HASM.
- These findings highlight the importance of AC isoform localization in determining downstream signaling outcomes and cellular functions.
