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Synergy of cAMP and calcium signaling pathways in CFTR regulation.

ABSTRACT: Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We characterize an interaction between the regulatory region of CFTR and calmodulin, the major calcium signaling molecule, and report protein kinase A (PKA)-independent CFTR activation by calmodulin. We describe the competition between calmodulin binding and PKA phosphorylation and the differential effects of this competition for wild-type CFTR and the major F508del mutant, hinting at potential therapeutic strategies. Evidence of CFTR binding to isolated calmodulin domains/lobes suggests a mechanism for the role of CFTR as a molecular hub. Together, these data provide insights into how loss of active CFTR at the membrane can have additional consequences besides impaired chloride transport.

SUBMITTER: Bozoky Z

PROVIDER: S-EPMC5358358 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Synergy of cAMP and calcium signaling pathways in CFTR regulation.

Bozoky Zoltan Z Ahmadi Saumel S Milman Tal T Kim Tae Hun TH Du Kai K Di Paola Michelle M Pasyk Stan S Pekhletski Roman R Keller Jacob P JP Bear Christine E CE Forman-Kay Julie D JD

Proceedings of the National Academy of Sciences of the United States of America 20170227 11

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, leading to defective apical chloride transport. Patients also experience overactivation of inflammatory processes, including increased calcium signaling. Many investigations have described indirect effects of calcium signaling on CFTR or other calcium-activated chloride channels; here, we investigate the direct response of CFTR to calmodulin-mediated calcium signaling. We ch ...[more]

PMID: 28242698

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Project description:BACKGROUND & AIMS:The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. METHODS:We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses. RESULTS:Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. CONCLUSIONS:Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome.

Project description:The budding yeast, Saccharomyces cerevisiae, responds to various environmental cues by invoking specific adaptive mechanisms for their survival. Under nitrogen limitation, S. cerevisiae undergoes a dimorphic filamentous transition called pseudohyphae, which helps the cell to forage for nutrients and reach an environment conducive for growth. This transition is governed by a complex network of signaling pathways, namely cAMP-PKA, MAPK and TOR, which controls the transcriptional activation of FLO11, a flocculin gene that encodes a cell wall protein. However, little is known about how these pathways co-ordinate to govern the conversion of nutritional availability into gene expression. Here, we have analyzed an integrative network comprised of cAMP-PKA, MAPK and TOR pathways with respect to the availability of nitrogen source using experimental and steady state modeling approach. Our experiments demonstrate that the steady state expression of FLO11 was bistable over a range of inducing ammonium sulphate concentration based on the preculturing condition. We also show that yeast switched from FLO11 expression to accumulation of trehalose, a STRE response controlled by a transcriptional activator Msn2/4, with decrease in the inducing concentration to complete starvation. Steady state analysis of the integrative network revealed the relationship between the environment, signaling cascades and the expression of FLO11. We demonstrate that the double negative feedback loop in TOR pathway can elicit a bistable response, to differentiate between vegetative growth, filamentous growth and STRE response. Negative feedback on TOR pathway function to restrict the expression of FLO11 under nitrogen starved condition and also with re-addition of nitrogen to starved cells. In general, we show that these global signaling pathways respond with specific sensitivity to regulate the expression of FLO11 under nitrogen limitation. The holistic steady state modeling approach of the integrative network revealed how the global signaling pathways could differentiate between multiple phenotypes.

Dataset Information

Synergy of cAMP and calcium signaling pathways in CFTR regulation.

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Synergy of cAMP and calcium signaling pathways in CFTR regulation.

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