Yuanyuan Duan

Mechanosensitive gating of CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion and intracellular ligand-gated channel associated with cystic fibrosis, a lethal genetic disorder common among Caucasians. Here we show that CFTR is robustly activated by membrane stretch induced by negative pressures as small as 5 mmHg at the single-channel, cellular and tissue levels. Stretch increased the product of the number of channels present and probability of being open (NPo), and also increased the unitary conductance of CFTR in cell-attached membrane patches. CFTR stretch-mediated activation appears to be an intrinsic property independent of cytosolic factors and kinase signalling. CFTR stretch-mediated activation resulted in chloride transport in Calu-3 human airway epithelial cells and mouse intestinal tissues. Our study has revealed an unexpected function of CFTR in mechanosensing, in addition to its roles as a ligand-gated anion channel and a regulator of other membrane transporters, demonstrating for the first time a mechanosensitive anion channel with a clearly defined molecular identity. Given that CFTR is often found in mechanically dynamic environments, its mechanosensitivity has important physiological implications in epithelial ion transport and cell volume regulation in vivo.

A novel mechanism of control of NFκB activation and inflammation involving A2B adenosine receptors

Summary The nuclear factor kappa B (NF&kgr;B) pathway controls a variety of processes, including inflammation, and thus, the regulation of NF&kgr;B has been a continued focus of study. Here, we report a newly identified regulation of this pathway, involving direct binding of the transcription factor NF&kgr;B1 (the p105 subunit of NF&kgr;B) to the C-terminus of the A2B adenosine receptor (A2BAR), independent of ligand activation. Intriguingly, binding of A2BAR to specific sites on p105 prevents polyubiquitylation and degradation of p105 protein. Ectopic expression of the A2BAR increases p105 levels and inhibits NF&kgr;B activation, whereas p105 protein levels are reduced in cells from A2BAR-knockout mice. In accordance with the known regulation of expression of anti- and pro-inflammatory cytokines by p105, A2BAR-null mice generate less interleukin (IL)-10, and more IL-12 and tumor necrosis factor (TNF-&agr;). Taken together, our results show that the A2BAR inhibits NF&kgr;B activation by physically interacting with p105, thereby blocking its polyubiquitylation and degradation. Our findings unveil a surprising function for the A2BAR, and provide a novel mechanistic insight into the control of the NF&kgr;B pathway and inflammation.

Keratin K18 Increases Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Surface Expression by Binding to Its C-terminal Hydrophobic Patch

Background: CFTR function is tightly regulated by many interacting proteins. Results: Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR. Conclusion: K18 controls the function of CFTR. Significance: These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved “hydrophobic patch” (1413FLVI1416) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18−/− mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.

Functional coupling of Gs and CFTR is independent of their association with lipid rafts in epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) has been found to be colocalized with G-protein-coupled receptors (GPCRs) and the downstream signaling molecules; however, the mechanisms of the colocalization remain largely elusive. The present work has investigated the role of lipid rafts in the localized signaling from GPCRs to CFTR. Using commonly used sucrose gradient centrifugation, we found that CFTR along with

Regulation of CFTR channels by HCO3−-sensitive soluble adenylyl cyclase in human airway epithelial cells

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TRIP-Br1 is a novel adaptor protein for ubiquitinating and degrading multiple adenylyl cyclase isofroms by XIAP E3 ligase

was associated with lipid rafts, and the association was disrupted by cholesterol depletion with methyl-β-cyclodextrin (MCD) treatment in Calu-3 human airway epithelial cells. Using short-circuit current (Isc) as a readout of CFTR in Calu-3 cells or T84 human colonic epithelial cells, we showed that MCD, while increasing basal membrane permeability, had no effect on the Isc induced by several GPCR agonists. Similar results were also obtained with a cholesterol biosynthesis inhibitor lovastatin and a cholesterol-binding agent filipin in Calu-3 cells. Furthermore, cholesterol depletion did not impair cyclic AMP production elicited by the GPCR agonists in Calu-3 cells. Our data suggest that GPCR-mediated signaling maintain their integrity after lipid raft disruption in Calu-3 and T84 epithelial cells and cast doubts on the role of lipid rafts as signaling platforms in GPCR-mediated signaling.

Ligand-independent control of the NFkappaB pathway and inflammation by A2B adenosine receptors

Adenylyl cyclases (ACs) generate cAMP, a second messenger of utmost importance that regulates a vast array of biological processes in all kingdoms of life. However, almost nothing is known about how AC activity is regulated through protein degradation mediated by ubiquitination or other mechanisms. Here, we show that transcriptional regulator interacting with the PHD-bromodomain 1 (TRIP-Br1, Sertad1), a newly identified protein with poorly characterized functions, acts as an adaptor that bridges the interaction of multiple AC isoforms with X-linked inhibitor of apoptosis protein (XIAP), a RING-domain E3 ubiquitin ligase. XIAP ubiquitinates a highly conserved Lys residue in AC isoforms and thereby accelerates the endocytosis and degradation of multiple AC isoforms in human cell lines and mice. XIAP/TRIP-Br1-mediated degradation of ACs forms part of a negative-feedback loop that controls the homeostasis of cAMP signaling in mice. Our findings reveal a previously unrecognized mechanism for degrading multiple AC isoforms and modulating the homeostasis of cAMP signaling. DOI: http://dx.doi.org/10.7554/eLife.28021.001