CFTR (cystic fibrosis transmembrane conductance regulator) has been shown to form multiple protein macromolecular complexes with its interacting partners at discrete subcellular microdomains to modulate trafficking, transport and signalling in cells. Targeting protein–protein interactions within these macromolecular complexes would affect the expression or function of the CFTR channel. We specifically targeted the PDZ domain-based LPA2 (type 2 lysophosphatidic acid receptor)–NHERF2 (Na+/H+ exchanger regulatory factor-2) interaction within the CFTR–NHERF2–LPA2-containing macromolecular complexes in airway epithelia and tested its regulatory role on CFTR channel function. We identified a cell-permeable small-molecule compound that preferentially inhibits the LPA2–NHERF2 interaction. We show that this compound can disrupt the LPA2–NHERF2 interaction in cells and thus compromises the integrity of macromolecular complexes. Functionally, it elevates cAMP levels in proximity to CFTR and upregulates its channel activity. The results of the present study demonstrate that CFTR Cl− channel function can be finely tuned by modulating PDZ domain-based protein–protein interactions within the CFTR-containing macromolecular complexes. The present study might help to identify novel therapeutic targets to treat diseases associated with dysfunctional CFTR Cl− channels.
- cystic fibrosis transmembrane conductance regulator (CFTR)
- type 2 lysophosphatidic acid receptor (LPA2)
- Na+/H+ exchanger regulatory factor-2 (NHEFR2)
- PDZ domain
- protein–protein interaction
- small-molecule inhibitor
CFTR [CF (cystic fibrosis) transmembrane conductance regulator] is the product of the gene mutated in patients with CF, a lethal autosomal recessive genetic disease most common among Caucasians . CFTR is a cAMP-regulated chloride (Cl−) channel primarily localized at the apical surfaces of epithelial cells which line the airway, gut, exocrine glands etc., where it is responsible for transepithelial salt and water transport [2,3]. CFTR function is also critical in maintaining fluid homoeostasis, airway fluid clearance and airway submucosal glands secretion in healthy and disease phenotypes [4,5].
NHERF2 (Na+/H+ exchanger regulatory factor-2) is a PDZ domain-containing protein that is primarily localized at the apical surfaces of epithelial cells. It contains two tandem PDZ domains and a C-terminal ERM (ezrin/radixin/moesin) domain, which mediates the association of NHERF2 with MERM proteins (merlin/ERM) and links NHERF2 to the cytoskeleton . NHERF2 has been shown to cluster signalling molecules into supramolecular complexes [7–11].
LPAs (lysophosphatidic acids) are growth-factor-like phospholipids present in biological fluids and foods. LPAs mediate diverse cellular responses, such as cell proliferation, differentiation, migration, survival, angiogenesis, inflammation and platelet aggregation [12,13]. At least eight G-protein-coupled LPA receptors have been identified which couple to Gs, Gi/o, Gq and/or G12/13 protein to activate various signalling pathways [7,13]. Among these LPA receptors, LPA2 (type 2 LPA receptor) belongs to the EDG (endothelial differentiation gene) family and is structurally unique at the C-terminal tail, in which it contains a di-leucine motif and several putative palmitoylated cysteine residues in the proximal region that are responsible for binding to several zinc-finger proteins such as TRIP6 (thyroid-hormone-receptor-interacting protein 6). The last four amino acids of LPA2 (DSTL; a class I PDZ domain-binding motif) mediate its interaction with several PDZ proteins, including NHERF2 [7,13,14]. Through the interaction with LPA2, NHERF2 regulates the LPA-mediated PLC-β3 (phospholipase C-β3) signalling pathway and the activation of ERKs (extracellular signal-regulated kinases)  and Akt . It has also been reported that LPA induces the formation of a ternary complex containing LPA2, TRIP6 and NHERF2 at microdomains on the plasma membrane and regulates the anti-apoptotic signalling of LPA2 .
A growing number of studies suggest that CFTR interacts directly or indirectly with other ion channels, transporters, scaffolding proteins, protein kinases, effectors and cytoskeletal elements to form macromolecular complexes at specialized subcellular domains. These dynamic protein–protein interactions influence CFTR channel function as well as its localization and processing within cells [8,10,11,17–20]. Previously, we found that CFTR, LPA2 and NHERF2 (along with other signalling molecules) form macromolecular complexes at the plasma membrane of gut epithelia, which functionally couple LPA2 signalling to CFTR-mediated Cl− transport . We demonstrated that LPA inhibits CFTR-mediated Cl− transport through the LPA2-mediated Gi pathway in a compartmentalized manner in cells, and that LPA inhibits CFTR-dependent cholera toxin-induced mouse intestinal fluid secretion in vivo . The formation of CFTR–NHERF2–LPA2-containing macromolecular complexes and their importance in compartmentalized cAMP signalling are further supported by the observation that disruption of the integrity of the macromolecular complexes by using a cell-permeable LPA2-specific peptide reversed LPA2-mediated inhibition . Recently, Singh et al.  investigated the roles of NHERF1/2/3 in regulating CFTR-dependent murine duodenal HCO3− secretion in mice. They demonstrated that the absence of each NHERF protein resulted in distinct alteration in the regulation of HCO3− secretion. NHERF1 ablation strongly reduced basal as well as FSK (forskolin)-stimulated HCO3− secretory rates and blocked β2AR (β2-adrenergic receptor) stimulation. PDZK1 (NHERF3) ablation reduced basal, but not FSK-stimulated, secretion. As for NHERF2, the authors showed that FSK-stimulated HCO3− secretion was significantly increased in Nherf2−/− mice and that NHERF2 is absolutely required for LPA2-mediated inhibition of HCO3− secretion , which is consistent with our previous findings . These findings imply that targeting individual NHERF proteins might provide new approaches for therapeutic interventions of CFTR-associated diseases [8,21].
To study the formation of CFTR–NHERF2–LPA2-containing macromolecular complexes in airway epithelia and their importance in regulating CFTR Cl− channel function, and to explore the possibility of identifying new therapeutic targets (by perturbing PDZ domain-based protein–protein interactions within the CFTR-containing macromolecular complexes) for treating diseases associated with a dysfunctional CFTR Cl− channel, we identified a cell-permeable small-molecule compound that preferentially inhibits the biochemical LPA2–NHERF2 interaction. We demonstrate that this compound indeed disrupts the LPA2–NHERF2 interaction in cells and thus compromises the integrity of the CFTR–NHERF2–LPA2-containing macromolecular complexes. Functionally, it abolishes the inhibitory effect of LPA2-dependent events on the CFTR Cl− channel (mediated by NHERF2), and thus augments CFTR Cl− channel activity in Calu-3 cells and also in fluid secretion from pig tracheal submucosal glands.
Cell culture and transfection
The Calu-3 cell line was purchased from A.T.C.C. and cultured in MEM (minimal essential medium) (Invitrogen) containing 15% (v/v) serum, 1% (w/v) penicillin/streptomycin 1 mM sodium pyruvate, and 1×non-essential amino acids. HEK (human embryonic kidney)-293 cells overexpressing FLAG–LPA2 and HA (haemagglutinin)–NHERF2 were cultured in DMEM-F12 (Dulbecco's modified Eagle's medium-F12) (Invitrogen) containing 10% (v/v) serum and 1% (w/v) penicillin/streptomycin. The cells were maintained in a 5% CO2 incubator at 37 °C. The FLAG (M2) tag was introduced into LPA2 (FLAG tag on N-terminal tail on the outer loop of the protein) by using a two-step QuikChange® Mutagenesis kit (Invitrogen). The sequence was confirmed, and the FLAG–LPA2 was cloned into the pcDNA3 vector (Invitrogen). Lipofectamine™ 2000 (Invitrogen) was used to transfect plasmids containing FLAG–LPA2, HA–NHERF2, FLAG–CFTR or FLAG–PLC-β3 in HEK-293 cells. Stable cell lines were generated by selection using 2 μg/ml puromycin in the medium. Lipofectamine™ was also used to transfect plasmids containing CFP (cyan fluorescent protein)–EPAC (exchange protein activated by cAMP)–YFP (yellow fluorescent protein) into Calu-3 cells. Pig tracheas were harvested less than 1 h postmortem from piglets that had been killed for projects unrelated to the present study. The experimental procedures using pigs were carried out in accordance with the guidelines provided by the Institutional Animal Care and Use Committee of the University of Tennesee Health Science Center (Memphis, TN, U.S.A.).
Screening for potent inhibitors that disrupt the LPA2–NHERF2 interaction by using the AlphaScreen™ assay
Biotin–LPA2 peptide (10 μM final concentration) and GST (glutathione transferase)–NHERF2 (100 nM final concentration; see the Supplementary Experimental section for more details at http://www.BiochemJ.org/bj/435/bj4350451add.htm) were mixed in the assay buffer [25 mM Hepes, 100 mM NaCl, 0.1% BSA and 0.05% Tween 20, pH 7.4], into which the compounds were added, serially diluted (final concentration: 1 mM–10 nM), and incubated at room temperature (22 °C) for 30 min. Each sample solution (15 μl) was transferred to a 384-well white opaque OptiPlate™ (PerkinElmer) in triplicate and anti-GST acceptor beads (5 μl, 20 μg/ml final concentration) were added and incubated for 30 min. Streptavidin donor beads (5 μl, 20 μg/ml final concentration) were then added and incubated for 1 h at room temperature. The plates were read on an EnVison plate reader (PerkinElmer). The binding curve and IC50 value were generated using GraphPad Prism software.
Co-immunoprecipitation and immunoblotting
HEK-293 cells stably expressing FLAG–LPA2 and HA–NHERF2 (HEK-293-FLAG–LPA2-HA–NHERF2 cells) were treated with compound CO-068 (50 μM) or an equal volume of DMSO for 1 h at 37 °C. The cells were washed with PBS (1×) and then lysed in lysis buffer [1×PBS containing 0.2% Triton X-100 and protease inhibitors: PMSF (1 mM), pepstatin A (1 μg/ml), leupeptin (1 μg/ml) and aprotinin (1 μg/ml)]. The lysate was centrifuged at 16000 g for 10 min at 4 °C. The protein concentration of the clear supernatant was determined by using the bicinchoninic acid assay (Pierce). The clear supernatant was subjected to immunoprecipitation by using anti-FLAG beads (Sigma). The immunoprecipitated beads were washed three times with lysis buffer, and the proteins were eluted from the beads using Laemmli sample buffer [5×; containing 2.5% (v/v) 2-mercaptoethanol]. The eluted proteins were denatured, subjected to SDS/PAGE on 4–15% gels (Bio-Rad), transferred on to PVDF membrane, and immunoblotted for NHERF2 and LPA2 with an anti-HA monoclonal antibody (Sigma) and an anti-LPA2 monoclonal antibody (rabbit-2143, against the last 11 amino acids) respectively. The immunoreactive bands were visualized by ECL (Amersham Biosciences). HEK-293 cells expressing HA–NHERF2 (HEK-293-HA–NHERF2 cells) were also used as a negative control.
Co-immunoprecipitation of NHERF2 and CFTR in the presence of 50 μM compound CO-068 (or an equal volume of DMSO) was performed in HEK-293 cells stably expressing FLAG–CFTR (HEK-293-FLAG–CFTR cells) using the method described above. Specific antibodies were used to detect CFTR (R1104 monoclonal mouse antibody) and NHERF2 (affinity purified rabbit polyclonal antibody) levels.
Co-immunoprecipitation of NHERF2 and PLC-β3 in the presence of 50 μM compound CO-068 (or an equal volume of DMSO) was performed in HEK-293 cells expressing FLAG–PLC-β3 (HEK-293-FLAG–PLC-β3 cells) using the method described above. An anti-FLAG monoclonal antibody was used to detect PLC-β3. Affinity-purified rabbit polyclonal antibody (rabbit-2346; see the Supplementary Experimental section) was used to detect NHERF2 levels.
Short-circuit currents (Isc) measurements (Ussing chamber experiments)
Polarized Calu-3 cell monolayers were grown to confluency on Costar Transwell permeable supports (Cambridge; filter area=0.33 cm2) and then mounted in a modified Ussing chamber. Short-circuit currents mediated by the CFTR Cl− channel were measured as described previously [19,20]. The cells were bathed in Ringer's solution (basolateral: 140 mM NaCl, 5 mM KCl, 0.36 mM K2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 0.5 mM MgCl2, 4.2 mM NaHCO3, 10 mM Hepes and 10 mM glucose, pH 7.2, [Cl−]=149 mM), and low Cl− Ringer's solution (apical: 133.3 mM sodium gluconate, 5 mM potassium gluconate, 2.5 mM NaCl, 0.36 mM K2HPO4, 0.44 mM KH2PO4, 5.7 mM CaCl2, 0.5 mM MgCl2, 4.2 mM NaHCO3, 10 mM Hepes and 10 mM mannitol, pH 7.2, [Cl−]=14.8) at 37 °C and gassed with 95% O2 and 5% CO2. All reagents were added to the apical side of the cell monolayers. At the end of the experiments, a specific CFTR channel inhibitor, CFTRinh-172 (20 μM), was added to the apical sides of both chambers to inhibit the Cl− currents to verify that the Isc responses observed were CFTR-dependent.
Pig tracheal submucosal gland fluid secretion
Fluid secretion from pig tracheal submucosal glands was monitored as described by Wine and co-workers . Pig tracheas were collected within 1 h of killing, placed in ice-cold KRB buffer (Krebs–Ringer's bicarbonate buffer; 120 mM NaCl, 25 mM NaHCO3, 3.3 mM KH2PO4, 0.8 mM K2HPO4, 1.2 mM MgCl2, 1.2 mM CaCl2, 10 mM D-glucose and 1 μM indomethacin) and bubbled with 95% O2 and 5% CO2 gas. The tracheal ring was cut open along the dorsal fold in ice-cold KRB buffer. The mucosa with underlying glands was carefully dissected from the cartilage, and a 1 cm piece was mounted in a chamber with the mucosal side up. The mucosal side was blotted clean, further dried with a gentle stream of 95% O2 and 5% CO2 gas, and then partly covered with a thin layer of water-saturated mineral oil. To establish a baseline, KRB buffer was added to the serosal side, maintained at 37 °C and superfused with 95% O2 and 5% CO2. All pharmacological reagents were diluted to the final concentration in 37 °C appropriately gassed KRB buffer, and added to the serosal side after monitoring basal secretion. Carbachol (10 μM) was added at the end of the experiments to check for the viability of the submucosal glands. The tissues covered with water-saturated oil were obliquely illuminated to visualize the spherical droplets of secreted mucus within the oil. Digital images were collected at 1 min intervals with a digital camera attached to a stereoscopic microscope (National Optical) and controlled by Motic Images 2.0 ML software. ImageJ software (NIH) was used to analyse the fluid secretion. Briefly, a 1 mm×1 mm grid was placed on the tissue in the last image to set the scales, which were used to measure the area of secreted bubbles. The volumes of the secreted bubbles were calculated from Area using the formula V=4/3Πr3, where r is the radius. The fluid secretion rates were calculated as slopes of Volume against Time plots using linear regression (R2>0.8). For basal secretion, the fluid secretion rates were calculated from data over a 10 min period; for secretion induced by FSK (10 μM), FSK (10 μM) plus CFTRinh-172 (50 μM), compound CO-068 (50 μM), compound CO-068 (50 μM) plus CFTRinh-172 (50 μM), FSK (10 μM) plus compound CO-068 (50 μM), or FSK (10 μM) plus compound CO-068 (50 μM) plus CFTRinh-172 (50 μM), the secretion rates were calculated from results over a 25 min period; for carbachol (10 μM), or carbachol (50 μM) plus compound CO-068 (50 μM) induced secretion, the secretion rates were calculated from results over a 5 min period.
Ratiometric FRET (fluorescence resonance energy transfer) microscopy and data analysis
Calu-3 cells expressing a cAMP sensor, CFP–EPAC–YFP, were grown on 35 mm glass-bottom dishes (MatTek), washed twice with HBSS (Hanks balanced salt solution), added in 1 ml of HBSS, and mounted on an inverted Olympus microscope (IX51; the microscope is described in detail in the Supplementary Experimental section). Cells were maintained in HBSS in the dark at room temperature. After establishing the baseline, compound CO-068 (50 μM) or FSK (10 μM) was added to the buffer, and ratiometric FRET imaging was performed as described previously [19,20]. Briefly, time-lapse images were captured with 100–300 ms exposure time and 1 min interval with a cooled EM-CCD camera (Hamamatsu) controlled by Slidebook 4.2 software (Intelligent Imaging Innovations). Following background subtraction, multiple regions of interest (10–20) were selected (three to five cells) for data analysis with the ratio module of Slidebook 4.2 software. The emission ratio (CFP/FRET) was obtained from CFP and FRET emission of background-subtracted cells.
Data are represented as means±S.E.M. unless otherwise indicated. Student's t test (two-tailed) was used to compare the data of different groups. **P<0.01 or *P<0.05 was considered significant.
Screening for small-molecule inhibitors that disrupt the LPA2–NHERF2 interaction
Development of small-molecule inhibitors for protein–protein interactions is of great importance. The compounds identified can be used as tools to study the cell physiology associated with the protein–protein interactions and may also have a therapeutic potential [23–25]. Generally, two sequential approaches are adopted to identify inhibitors for protein–protein interactions. One can produce chemical libraries of derivatives of a chemical scaffold that is rationally designed, and use high-throughput screening to identify the hit compounds. For the present study, we used the AlphaScreen™ assay (Amplified Luminescent Proximity Homogeneous Assay) to screen a library of compounds that was previously designed to inhibit PDZ domain-based protein–protein interactions [26–29]. We first developed a direct binding assay between the purified full-length GST–NHERF2 protein and a biotin–LPA2 peptide (biotin–NGHPLMDSTL-COOH, which is derived from the C-terminal sequence of LPA2 containing the PDZ motif DSTL; the schematic representation of this assay is shown in Supplementary Figure S1A at http://www.BiochemJ.org/bj/435/bj4350451add.htm). As shown in Figure 1(A), the biotin–LPA2 peptide binds to GST–NHERF2 in a dose- and pH-dependent manner. At pH 6 and 8, the binding signals were weak, whereas at physiological pH (pH 7.4) the binding signals were strong. The strongest signal was observed when the concentration of biotin–LPA2 peptide reached 10 μM. Further increase of the biotin–LPA2 peptide concentration led to decreased signals (the typical ‘Hook effect’ usually observed in AlphaScreen™ assays). To verify whether the observed signals were specific to the LPA2–NHERF2 interaction, we performed competition assays in which a non-biotinylated LPA2 peptide was used to compete against biotin–LPA2 peptide for binding to GST–NHERF2 (the schematic representation of this assay is shown in Supplementary Figure S1B). A dose-dependent inhibitory effect was observed which confirmed the specificity of the binding signals (Figure 1B). The IC50 was determined to be 12 μM.
The optimized assay conditions were then used to screen for small-molecule inhibitors. Among 80 compounds screened so far, one compound (named compound CO-068 in the present paper; the structure is shown in Figure 1C) showed the best inhibitory effect (IC50=63 μM; Figure 1D). To check the selectivity of its inhibitory effect, we developed other AlphaScreen™ assays for protein–protein interactions that have been reported to get involved in CFTR-containing macromolecular complexes [such as CFTR–NHERF2, MRP4 (multidrug-resistance protein 4)–PDZK1 and CFTR–NHERF1] [10,11], and then tested the inhibitory effects of compound CO-068 in these systems. Our results show that compound CO-068 did not preferentially perturb these PDZ domain-based protein–protein interactions (Table 1). For a proof-of-concept study, we used this compound to study the CFTR–NHERF2–LPA2-containing macromolecular complexes in airway epithelia and their importance in regulating CFTR Cl− channel function.
Synthesis of compound CO-068
Compound CO-068 inhibits the LPA2–NHERF2 interaction in cells
To test whether compound CO-068 inhibits the LPA2–NHERF2 interaction in cells, we co-transfected HEK-293 cells with FLAG–LPA2 and HA–NHERF2 constructs and generated a stable cell line (HEK-293-FLAG–LPA2-HA–NHERF2 cells). Since the potency of compound CO-068 for inhibiting the LPA2–NHERF2 interaction is ~5-fold weaker than that of LPA2–peptide, we used this compound at a concentration of 50 μM throughout the present study. It is to be noted that this concentration (50 μM) is below its IC50 for the LPA2–NHERF2 interaction and well below its IC50 for the CFTR–NHERF2 interaction, which would minimize the possibility of its disruption on the CFTR–NHERF2 interaction. We treated the cells with 50 μM compound CO-068 for 1 h at 37 °C and then lysed the cells. The protein complex was immunoprecipitated from clear supernatant by using anti-FLAG beads. The proteins were eluted from the beads, subjected to SDS/PAGE, and immunoblotted for LPA2 and NHERF2 by using specific anti-LPA2 or anti-HA antibodies. Cells pretreated with an equal volume of DMSO (solvent used to solvate compound CO-068) were used as a control. HEK-293 cells expressing HA–NHERF2 (HEK-293-HA–NHERF2 cells) were also used as a negative control. As shown in Figure 2, in immunoprecipitated protein complex, the FLAG–LPA2 levels remained the same for cells treated with compound CO-068 or DMSO. However, the NHERF2 level decreased substantially (42%; as analysed by quantifying the blots with Scion Image software) for cells treated with compound CO-068 compared with that from cells treated with DMSO, indicating that compound CO-068 disrupts the LPA2–NHERF2 interaction in these cells.
To further test whether compound CO-068 disrupts the CFTR–NHERF2 interaction in cells at a concentration of 50 μM, we treated HEK-293 cells stably expressing FLAG–CFTR (HEK-293-FLAG–CFTR cells) with compound CO-068 (or DMSO) for 1 h at 37 °C, lysed the cells, and immunoprecipitated the protein complex from clear supernatant by using α-FLAG beads. The proteins were eluted from the beads, subjected to SDS/PAGE, and immunoblotted for CFTR and NHERF2 by using specific anti-CFTR or anti-NHERF2 antibodies. HEK-293 parental cells were also used as a control. As shown in Figure 3(A), in the immunoprecipitated protein complex, both FLAG–CFTR levels and NHERF2 levels remained the same for cells treated with compound CO-068 or DMSO, indicating that at a concentration of 50 μM, compound CO-068 does not disrupt the CFTR–NHERF2 interaction in cells.
We also used a similar method to study whether compound CO-068 disrupts the NHERF2–PLC-β3 interaction in cells. HEK-293 cells overexpressing FLAG–PLC-β3 (HEK-293-FLAG–PLCβ3 cells) were treated with compound CO-068 (or DMSO) under the same conditions as described above. As shown in Figure 3(B), in immunoprecipitated protein complex, both FLAG–PLC-β3 levels and NHERF2 levels remained the same for cells treated with compound CO-068 or DMSO, suggesting that, at a concentration of 50 μM, compound CO-068 does not disrupt the PLC-β3–NHERF2 interaction in cells.
For these co-immunoprecipitation experiments, compound CO-068 was added externally at a concentration of 50 μM. The cell permeability of compound CO-068 was tested using HEK-293 cells. The cell numbers were counted and the cells were incubated with compound CO-068 (50 μM) for 1 h at 37 °C. The culture medium was removed by centrifugation and the cells were pelleted. The cell pellets were washed twice with RIPA buffer (without SDS; see the Supplementary Experimental section for the composition), and then lysed in the RIPA buffer (without SDS). Cells treated with an equal amount of DMSO were used as controls. LC MS/MS (liquid chromatography tandem MS) was used to measure the concentrations of compound CO-068 in these cell lysates (see Supplementary Table S1 at http://www.BiochemJ.org/bj/435/bj4350451add.htm). As expected, samples from cells treated with DMSO did not show a compound CO-068 peak. For samples from cells treated with compound CO-068, a mean concentration of 28.74 μM in cell lysates was detected (n=5, S.D.=1.74; the volume for cell lysates is 63.3 μl). Considering the volume of cell pellets is approximately 40 μl, the mean intracellular concentration of compound CO-068 was determined as ~46 μM, indicating that it is quite cell permeable.
In conclusion, at a concentration of 50 μM, compound CO-068 seems to specifically disrupt LPA2–NHERF2 interaction in cells.
Compound CO-068 augments CFTR Cl− channel function in lung epithelial cells (Calu-3 cells)
Given the facts that: (i) LPA2 has an inhibitory effect on AC (adenylate cyclase) [8,13] and AC generates cAMP which regulates CFTR Cl− channel function; (ii) CFTR, LPA2 and NHERF2 form macromolecular complexes at the plasma membrane of gut and lung epithelial cells (HT29-CL19A cells and Calu-3 cells), which forms the molecular basis for functional coupling between LPA2-mediated signalling events and CFTR-mediated Cl− transport ; and (iii) compound CO-068 disrupts the LPA2–NHERF2 interaction and thus would disrupt the macromolecular complexes, we envisioned that disruption of LPA2 from CFTR–NHERF2–LPA2-containing complexes would increase CFTR Cl− channel function. To test this hypothesis, we measured CFTR-mediated short-circuit currents (Isc) in polarized Calu-3 monolayers mounted in an Ussing chamber with treatment of compound CO-068. DMSO was used as a control. Another compound, FJL-3-18 (whose structure is shown in Supplementary Figure S2A at http://www.BiochemJ.org/bj/435/bj4350451add.htm) which has very weak potency to inhibit the LPA2–NHERF2 interaction (IC50=850 μM; AlphaScreen™ result), was also used as a control. Calu-3 cells are airway serous gland epithelial cells that endogenously express CFTR, LPA2 and NHERF2 at the apical surfaces when polarized and have been used as a model system to study CFTR channel function [8,30,31]. When polarized Calu-3 cells were treated with compound CO-068 (50 μM), a significant increase in CFTR-dependent Isc was detected (Figures 4A and 4B), whereas DMSO or compound FJL-3-18 did not induce significant Isc responses (Figures 4A and 4B and Supplementary Figures S2B and S2C). A specific CFTR channel inhibitor, CFTRinh-172, was added towards the end of the experiments to verify that the observed Isc responses were indeed CFTR-dependent. These results demonstrate that disruption of the LPA2–NHERF2 interaction by using compound CO-068 increased basal CFTR Cl− channel function.
We further tested whether compound CO-068 could potentiate agonist-stimulated CFTR Cl− channel function by using FSK. As shown in Figures 4(C) and 4(D), compound CO-068 indeed further potentiated FSK-induced CFTR Cl− channel function. It is interesting to note that in the presence of compound CO-068, the FSK-activation rate was faster; possibly suggesting that compound CO-068 changed the three-dimensional arrangement of signalling components within the CFTR–NHERF2–LPA2-containing macromolecular complexes by disrupting the LPA2–NHERF2 interaction, which accounted for the changes in magnitude and kinetics of CFTR Cl− channel activation.
In summary, these observations support our hypothesis that CFTR, NHERF2 and LPA2 form macromolecular complexes at the plasma membrane of Calu-3 cells and disruption of LPA2 from the macromolecular complexes augments CFTR Cl− channel function. The data also imply that: (i) targeting PDZ domain-based protein–protein interactions within the CFTR–NHERF2–LPA2-containing macromolecular complexes can locally regulate CFTR Cl− channel function, which might provide potential therapeutic targets for treating CFTR-related diseases; and (ii) compound CO-068 could be a seed compound for developing improved leads to augment CFTR function in CF patients who have CFTR mutants with impaired channel function, such as G551D or R117H.
Compound CO-068 augments CFTR-dependent fluid secretion from pig tracheal submucosal glands
Submucosal gland secretion plays important roles in maintaining airway and lung health. It can be stimulated by cholinergic agonists or agonists that elevate cAMP or Ca2+ levels. CFTR is present in the apical membrane of gland serous cells and mediates at least part of the fluid secretion. Loss of CFTR function reduces the capacity of glands to secrete fluid and has been suggested to link to the airway pathology of CF [4,31,32]. In the present study, we used the pig tracheal submucosal glands secretion model to investigate whether compound CO-068 could potentiate CFTR-dependent fluid secretion from submucosal glands. Pig is considered a closer model to human CF, and a CF pig model is available for studying CFTR function . Because it has been reported that FSK can induce fluid secretion from pig tracheal submucosal glands , we first used FSK to validate the method. Our results showed that FSK induced a 5-fold increase in mean fluid secretion rate compared with basal secretion rate (Figure 5B). CFTRinh-172 markedly inhibited FSK-induced secretion, indicating that the observed secretion was CFTR-dependent. When compound CO-068 (50 μM) was added in combination with FSK, we observed a 2.5-fold increase in mean secretion rate compared with FSK-induced secretion (Figure 5B), suggesting that compound CO-068 potentiated FSK-induced fluid secretion. This potentiation effect was inhibited by CFTRinh-172, indicating that it was CFTR-dependent. These results are consistent with the results from Isc measurement (Figures 4C and 4D). We then tested the effect of compound CO-068 on basal CFTR-dependent fluid secretion. As shown in Figures 5(A) and 5(B), compound CO-068 induced a 4-fold increase in mean fluid secretion rate compared with basal secretion, and the effect was reversed by CFTRinh-172. These findings are also consistent with the results from Isc measurements (Figures 4A and 4B).
To further verify that the increased fluid secretion by using compound CO-068 is specific to CFTR and not through another mechanism, we investigated whether compound CO-068 could augment carbachol-induced fluid secretion. Our results demonstrate that, when added in combination with carbachol, compound CO-068 did not increase the mean fluid secretion rate (see Supplementary Figure S3 at http://www.BiochemJ.org/bj/435/bj4350451add.htm).
In summary, compound CO-068 increases both basal and FSK-induced CFTR-dependent submucosal glands fluid secretion in pig, a finding that could be potentially useful to restore the impaired mucociliary clearance process in diseased airways due to a dysfunctional CFTR Cl− channel such as the G551D-CFTR mutant.
Compound CO-068 augments CFTR Cl− channel function by means of elevating cAMP levels in cells
The results described above show that compound CO-068 disrupts the LPA2–NHERF2 interaction and augments CFTR Cl− channel function in Calu-3 cells and in pig tracheal submucosal glands. To gain direct evidence for whether compound CO-068 acts through the cAMP pathway, we transfected a FRET-based cAMP sensor, CFP–EPAC–YFP, into Calu-3 cells and then performed ratiometric FRET measurements to directly visualize cAMP dynamics in live cells. This highly sensitive cAMP sensor can be used to monitor cAMP dynamics in intact cells with very high temporal and spatial resolution [19,20,35]. After establishing the baseline, compound CO-068 (50 μM) was added into the buffer and ratiometric FRET signals were monitored. As shown in Figures 6(A)–6(C), cAMP levels (represented by CFP/FRET emission ratio) increased 1.2-fold upon treatment of the cells with compound CO-068. FSK (10 μM) was added at the end of the experiments as a positive control which elicited a further increase in cAMP levels. When FSK was added after basal levels were established, it induced a 1.5-fold increase in cAMP levels. Addition of compound CO-068 further increased the intracellular cAMP levels (Figure 6D). The results provide direct evidence that compound CO-068 indeed elevates cAMP levels and consequently augments CFTR Cl− channel function, a finding that supports our hypothesis that compound CO-068 disrupts LPA2 from the CFTR–NHERF2–LPA2-containing macromolecular complexes and abolishes the inhibitory effect of LPA2 on AC, and consequently increases cAMP levels.
Formation of multiple protein complexes at discrete subcellular microdomains increases the specificity and efficiency of signalling [e.g. cAMP–PKA (protein kinase A) signalling] in cells [19,36,37]. For polarized epithelial cells (e.g. Calu-3 cells and HT-29 cells), it has been observed that the signals originating at cell surfaces do not always induce detectable changes for specific intracellular second messengers (e.g. cAMP, cGMP or Ca2+). However, the cellular response transduced by these specific second messengers is specifically and efficiently accomplished. These observations suggest that receptors, effectors, ion channels, transporters and signalling intermediates form macromolecular complexes and compartmentalize into discrete subcellular microdomains that, at the molecular level, ensure that the right signalling components are localized at the right place (spatially) and at the right time (temporally), thus increasing the velocity of response and specificity of signalling .
PDZ domains are conserved protein–protein interaction modules of ~90 amino acids in length that fold to form a peptide-binding groove that binds to the specific short peptide motif (PDZ motif) found in the C-terminus or internal region of a variety of target proteins . PDZ domain-containing proteins (PDZ proteins) often contain multiple PDZ domains and can interact simultaneously with multiple binding partners (e.g. receptors, ion channels or transporters) to assemble larger protein complexes at specific subcellular compartments involved in signalling, trafficking or subcellular transport in a variety of tissues [10,11,40,41]. Many PDZ domain proteins can interact with disease-associated proteins, and the regulation of disease-associated proteins by PDZ domain proteins gives them provisional roles in many disease states. The discrete properties of PDZ domain-based protein–protein interactions make them promising candidates for modulation to understand cell physiology and to develop novel therapeutic agents against diseases [24,25,42]. Developing small-molecule inhibitors to compete against PDZ targets for binding to PDZ protein is a very attractive approach in formulating pharmaceutical agents [25–29,43,44].
CFTR has been shown to interact directly or indirectly with a wide variety of proteins and to form distinct multiprotein macromolecular complexes at different subcellular microdomains and tissues [10,11,18]. Previously, we reported the multiprotein macromolecular complex formation between CFTR, NHERF2 and LPA2 (along with other signalling molecules) at the apical plasma membranes of gut epithelia, and their importance in compartmentalized cAMP signalling and in local regulation of CFTR Cl− channel function . To further study the regulatory roles of PDZ domain-based protein–protein interactions within the macromolecular complexes on regulating CFTR Cl− channel function, and to explore the potential therapeutic value of using such an approach to treat diseases associated with dysfunctional CFTR protein (e.g. G551D-CFTR and R117H-CFTR), we screened a specially designed chemical library and identified a compound (compound CO-068) that preferentially disrupts the LPA2–NHERF2 interaction. Our results from the present study demonstrate that this compound does inhibit the LPA2–NHERF2 interaction in cells and consequently disrupts the integrity of the CFTR–NHERF2–LPA2-containing macromolecular complexes, which leads to increased cAMP levels and augments CFTR Cl− channel function.
On the basis of our findings, we propose a model to depict the formation of multiple protein macromolecular complexes at airway epithelia and the regulatory role of the LPA2–NHERF2 interaction on CFTR channel function (Figure 7). Since LPA2 binds to only the PDZ2 domain of NHERF2 whereas CFTR can bind to both PDZ domains, one possible way to form the macromolecular complexes is for NHERF2 to self-associate through PDZ domains  and thereby bridge LPA2 and CFTR. Other signalling intermediates such as ezrin and PKA should also be present in the macromolecular complexes . Under basal conditions, LPA2 exerts inhibitory effects on AC through the Gi pathway, which results in reduced cAMP levels in proximity to CFTR and thus down-regulates its channel function (Figure 7A). However, perturbing the LPA2–NHERF2 interaction within the macromolecular complexes would scatter LPA2 from its binding partners, which would abolish the inhibitory effects of LPA2 on AC, leading to cAMP generation and consequently augmenting CFTR Cl− channel function (Figure 7B).
Our observations support in vivo studies, which showed that, upon deletion of NHERF2 in mice, the basal CFTR-dependent murine duodenal HCO3− secretion was slightly (but not significantly) higher than that in wild-type mice . Deletion of NHERF2 would completely disrupt the macromolecular complex formation between CFTR, NHERF2 and LPA2, and lead to the complete loss of compartmentalized cAMP signalling. In the present study, we sought to disrupt the NHERF2–LPA2 interaction and leave the CFTR–NHERF2 interaction intact, which would possibly contribute to the substantial increase in CFTR channel function at both the basal and agonist-induced levels.
The molecular assembly of CFTR with its interacting proteins is of great interest and importance because: (i) in addition to serving as a channel to transport Cl− and HCO3−, CFTR also regulates a wide variety of other channels, transporters and processes [10,11,17]; (ii) several human diseases are attributed to altered regulation of CFTR, among which CF and secretory diarrhoea are two major disorders [1,17]. CF is caused by the loss or dysfunction of CFTR Cl− channel activity resulting from mutations that decrease either the biosynthesis or the ion channel function of the protein . Secretory diarrhoea is caused by excessive activation of the CFTR Cl− channel in the gut . It is therefore reasonable to propose that any reagent (or approach) that can specifically enhance CFTR Cl− channel activity would be potentially beneficial in treating diseases such as CF. Conversely, any reagent (or approach) that can specifically decrease CFTR Cl− channel activity would be potentially beneficial in treating CFTR-mediated secretory diarrhoea. CFTR itself has been targeted to develop inhibitors for therapy of secretory diarrhoeas, and activators for therapy in CF [49,50]. By using high-throughput screening, Verkman et al.  have identified some small-molecule inhibitors and activators that show promising potential in the treatment of CF and CFTR-mediated secretory diarrhoea. In the present study, we targeted the PDZ domain-based LPA2–NHERF2 interaction within the CFTR–NHERF2–LPA2-containing macromolecular complexes in airway epithelia and demonstrated that a synthetic cell-permeable inhibitor (compound CO-068) could specifically increase CFTR Cl− channel activity at both basal and agonist-induced states. To our knowledge, this is the first study that specifically targeted one type of PDZ domain-based protein–protein interaction within the CFTR-containing macromolecular complexes and demonstrated that a small-molecule inhibitor could potentiate CFTR Cl− channel function in cells and tissues. The present study implies that these macromolecular complexes could potentially be a new therapeutic target for treating CFTR-associated diseases. Moreover, the present study suggests that by targeting different PDZ domain-based protein–protein interactions within the macromolecular complexes, we can modulate CFTR channel function on a use-dependent mode for treating different diseases, that is, targeting the LPA2–NHERF2 interaction to potentiate CFTR Cl− channel function for drug development to treat CF (especially those due to the presence of G551D- or R117H-CFTR); and targeting the CFTR–NHERF2 interaction to down-regulate CFTR Cl− channel function for drug development to treat CFTR-mediated secretory diarrhoea.
It is to be noted that when cells were treated with compound CO-068 (50 μM), no side effects on cell viability or morphology were observed. Currently, development of more potent inhibitors for the LPA2–NHERF2 interaction, as well as more potent inhibitors for the CFTR–NHERF2 interaction, is under way.
The manuscript was written by Weiqiang Zhang and supervised by Anjaparavanda Naren and Naoaki Fujii. The project was designed and supervised by Anjaparavanda Naren. Naoaki Fujii provided the chemical library and supervised chemical synthesis; Weiqiang Zhang performed AlphaScreen™ assays, chemical synthesis, and pig tracheal submucosal glands secretion experiments. Himabindu Penmatsa conducted ratiometric FRET measurements; Aixia Ren performed short-circuit current measurements; and Anjaparavanda Naren conducted co-immunoprecipitation experiments. Chandanamali Punchihewa assisted in developing AlphaScreen™ assays. Andrew Lemoff and Bing Yan performed LC MS/MS analysis. All authors discussed the results and commented on the manuscript.
We thank The American Lebanese Syrian Associated Charities (ALSAC) and St Jude Children's Research Hospital for support. This work was supported by US National Institutes of Health (NIH) [grant number DK080834 (to A.P.N.)].
We thank Dr P.G. Suh (Pohang University of Science and Technology, Republic of Korea) for the FLAG–PLC-β3 construct (pCMV2-FLAG–PLC-β3); Dr D. Armbruster (UTHSC, Memphis, TN, U.S.A.) for editing the manuscript prior to submission; Dr R.K. Buddington (University of Memphis, Memphis, TN, U.S.A.) for supplying pig trachea; and Dr A. Mayasundari and Dr N. Mahindroo (St Jude Children's Research Hospital, Memphis, TN) for support in chemical synthesis of compound CO-068.
Abbreviations: AC, adenylate cyclase; CF, cystic fibrosis; CFP, cyan fluorescent protein; CFTR, cystic fibrosis transmembrane conductance regulator; EPAC, exchange protein activated by cAMP; ERM, ezrin/radixin/moesin; FRET, fluorescence resonance energy transfer; FSK, forskolin; GST, glutathione transferase; HA, haemagglutinin; HBSS, Hanks balanced salt solution; HEK cell, human embryonic kidney cell; Isc, short-circuit currents; KRB buffer, Krebs–Ringer's bicarbonate buffer; LPA, lysophosphatidic acid; LPA2, type 2 LPA receptor; MRP4, multidrug-resistance protein 4; NHERF, Na+/H+ exchanger regulatory factor; PLC-β3, phospholipase C-β3; PKA, protein kinase A; TRIP6, thyroid-hormone-receptor-interacting protein 6; YFP, yellow fluorescent protein
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