Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle

Abstract

Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1; also known as ezrin-radixin-moesin–binding phosphoprotein 50) is a PSD-95, disc large, zona occludens-1 adapter that acts as a scaffold for signaling complexes and cytoskeletal-plasma membrane interactions. NHERF1 is crucial to β-2-adrenoceptor (β₂AR)-mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells, and NHERF1 has been proposed to mediate the recycling of internalized β₂AR back to the cell membrane. In the current study, we assessed the role of NHERF1 in regulating cAMP-mediated signaling and immunomodulatory functions in airway smooth muscle (ASM). NHERF1 knockdown attenuated the induction of (protein kinase A) phospho–vasodilator-stimulated phosphoprotein (p-VASP) by isoproterenol (ISO), prostaglandin E₂ (PGE₂), or forskolin (FSK) as well as the induction of p–heat shock protein 20 after 4 h of stimulation with ISO and FSK. NHERF1 knockdown fully abrogated the ISO-, PGE₂-, and FSK-induced IL-6 gene expression and cytokine production without affecting cAMP-mediated phosphodiesterase 4D (PDE4D) gene expression, phospho–cAMP response element–binding protein (p-CREB), and cAMP response element (CRE)–Luc, or PDGF-induced cyclin D1 expression. Interestingly, NHERF1 knockdown prevented ISO-induced chromatin-binding of the transcription factor CCAAT-enhancer–binding protein-β (c/EBPβ). c/EBPβ knockdown almost completely abrogated the cAMP-mediated IL-6 but not PDE4D gene expression. The differential regulation of cAMP-induced signaling and gene expression in our study indicates a role for NHERF1 in the compartmentalization of cAMP signaling in ASM.—Pera, T., Tompkins, E., Katz, M., Wang, B., Deshpande, D. A., Weinman, E. J., Penn, R. B. Specificity of NHERF1 regulation of GPCR signaling and function in human airway smooth muscle.

GPCR signaling is a complex cascade of events subject to regulation at multiple loci inside the cell. Following ligand binding, a conformational change occurs in the 3-dimensional structure of the GPCR, initiating a series of events resulting in the generation of second messenger molecules and activation of downstream signaling effectors that regulate cellular functions. For many GPCRs, ligand binding will also initiate the desensitization and down-regulation of receptors, which may limit their signaling abilities. During the process of receptor down-regulation GPCRs are internalized via clathrin-coated pits and are subsequently recycled back to the cell membrane, or they can be sorted into endosomes, which destines them for lysosomal degradation.

Na⁺/H⁺ exchanger (NHE) regulatory factor 1 [NHERF1; also known as ezrin-radixin-moesin (ERM)-binding phosphoprotein 50] contains postsynaptic density protein 95 (PSD-95), disc large, zona occludens-1 (PDZ) domains, which enable protein-protein interactions with molecules containing PDZ-binding motifs. In addition, its ERM domain renders it capable of binding to the actin cytoskeleton. NHERF1 was initially identified as a cofactor required for the cAMP-dependent protein kinase (PKA)-mediated inhibition of the NHE in kidney brush border membranes (1).

Hall et al. (2) was the first to demonstrate a direct interaction of NHERF1 with GPCRs, in which NHERF1 was shown to interact with the PDZ-binding motif (D-S/T-x-L) in the C terminus of the β-2-adrenoceptor (β₂AR). These initial studies described the potential of NHERF1 to function as a signaling molecule that transduces β₂AR signaling independently of PKA to regulate NHE. Subsequent work by the von Zastrow lab also revealed that NHERF1 is required for the efficient recycling of internalized β₂AR (3). Impaired NHERF1 binding to β₂AR, imposed either by truncation of NHERF1 PDZ domains or mutations in the β₂AR C terminus (PDZ-binding motifs), leads to diminished recycling of internalized β₂AR back to the cell membrane, instead diverting receptors to lysosomes for degradation. The ERM domain of NHERF1, which allows interaction of NHERF1 with the actin cytoskeleton, was similarly crucial for efficient recycling of β₂AR. Since these initial studies, multiple GPCRs, including parathyroid hormone receptor, κ opioid receptor, P2Y purinoceptor 1, C-C chemokine receptor 5, calcitonin receptor–like receptor, and thromboxane A2 receptor, have been shown to bind NHERF1 to modulate their down-regulation and recycling dynamics (4). In addition to its role in receptor trafficking, NHERF1 has been shown to form complexes to either promote C-X-C motif chemokine receptor 2 (CXCR2) – phospholipase C-3β (PLC3β) (5) or inhibit platelet-derived growth factor receptor (PDGFR) - phosphatase and tensin homolog (PTEN), frizzled class receptor 4 (Fzd4) – disheveled (Dvl) (6, 7) signaling.

Moreover, NHERF1 has been shown to bind the A-kinase anchoring protein ezrin to form a signaling complex with PKA to promote immunomodulatory actions of cAMP in T cells (8, 9) or to promote the stability and cAMP-mediated activation of cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells (10–13). The ability of NHERF1 to regulate GPCR desensitization or recycling, to direct GPCR signaling, and to engage in formation of signaling complexes makes it very well positioned to affect signaling and functional outcomes in cells. Although numerous studies by our group and others have examined the regulation and functional significance of cAMP/PKA signaling in airway smooth muscle (ASM) cells (14–25), no studies to date have examined the role of NHERF1 in ASM. Herein, we delineate the regulatory role of NHERF1 in Gs-coupled GPCR signaling in human ASM cells.

MATERIALS AND METHODS

Human ASM cell isolation and cell culture

Human ASM cultures were established as previously described (26) from human airways obtained from lung transplant donors under procedures approved by the University of Maryland, and the Thomas Jefferson University Institutional Review Board. Characterization of these cells regarding immunofluorescence of smooth muscle actin and agonist-induced changes in cytosolic calcium has been previously reported (27). Third to sixth passage cells were plated at a density of 10⁴ cells/cm² and maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum. Cells were growth arrested 24 h prior to stimulation by washing once in PBS and refeeding with serum-free Ham’s F-12 medium.

Small interfering RNA–mediated knockdown of NHERF1 in ASM

Small interfering RNA (siRNA) On-Targetplus Smartpool oligos (Dharmacon, Lafayette, CO, USA) directed against NHERF1 or CCAAT-enhancer–binding protein-β (c/EBPβ) or scrambled (SCR; control) siRNA oligos were annealed at 37°C for 1 h; 5 μg of the annealed oligo mixture was used to transfect human ASM cells using Dharmafect 1 (Dharmacon) per the manufacturer’s instruction. Twenty-four hours after transfection, the cells were replated for subsequent assessment of protein expression, cAMP accumulation, or gene expression.

cAMP accumulation in cultured ASM cells

Cells transfected with SCR or NHERF1 siRNA were plated into 24-well plates and analyzed for agonist-induced cAMP accumulation as previously described (27). Briefly, cells were growth arrested, washed, and stimulated with PBS containing 300 µM ascorbic acid, 1 mM 3-isobutyl-1-methylxanthine, and either vehicle, isoproterenol (ISO; 50 nM or 1 µM), prostaglandin E₂ (PGE₂; 1 µM), or forskolin (FSK; 100 µM) for 10 min at 37°C. Reactions were quenched by buffer aspiration and addition of 400 µl cold 100% ethanol. Isolated cAMP was subsequently quantified by radioimmunoassay (27). Data are expressed as picomoles cAMP/well.

Immunoblot analysis

Cells were growth arrested for 24 h, refed with serum-free medium, and stimulated for the indicated times. Cells were then washed with ice-cold PBS and solubilized in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitor cocktail (Biotool, Houston, TX, USA). For cell fractionation, a subcellular protein fractionation kit for cultured cells (Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer’s instructions. Lysates were buffered with Laemmli solution and loaded and separated on 10% or 4–20% gradient Tris-glycine polyacrylamide gels (Criterion; Bio-Rad, Hercules, CA, USA). Blots were probed with specific antibodies directed against NHERF1 and phosphorylated (p)–heat shock protein 20 (HSP20; Abcam, Cambridge, MA, USA), vasodilator-stimulated phosphoprotein (VASP; BD Biosciences, San Jose, CA, USA), p–cAMP response element–binding protein (CREB), c/EBPβ (Cell Signaling Technology, Danvers, MA, USA), and β-actin (MilliporeSigma, Burlington, MA, USA).

Quantitative PCR

Total RNA was isolated by standard procedures using Trizol (Thermo Fisher Scientific) and Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA) converted to cDNA, and mRNA abundance was assessed by quantitative PCR as previously described (28). Primers used are GAPDH forward: 5′-CCCTTCATTGACCTCAACTACATGGT-3′, and reverse: 5′-TGATGACAAGCTTCCCGTTCTCAG-3′; IL-6 forward: 5′-AAGTCCTGATCCAGTTCCTG-3′, and reverse: 5′-GCGCAGAATGAGATGAGTTG-3′; and phosphodiesterase 4D (PDE4D) forward: 5′-ATCCATGCTGCAGATGTTGTCC-3′, and reverse: 5′-AAAGCCCACAGCCAAATGATGG-3′. All primers were designed to span an intron.

Luciferase reporter assay

For luciferase assays, stable expression of a reporter construct for cAMP response element (CRE)-Luc was established in human telomerase reverse transcriptase ASM cells using lentivirus (Cignal Lenti luciferase reporter viral particles; SA Biosciences, Frederick, MD, USA) as per the manufacturer’s recommendations. Stable lines were selected and maintained in complete medium containing puromycin. Cells were plated into 24-well plates post–NHERF1 knockdown, allowed to attach overnight, and then serum–deprived for 24 h. Cells were subsequently stimulated for 6 h and then harvested in passive lysis buffer according to the manufacturer’s instructions and stored at −20°C. After thawing, 20 µl of cell lysate was loaded per well of a white 96-well plate, and firefly luciferase substrate reagent (100 µl; Luciferase Assay System; Promega, Madison, WI, USA) was added to each well. Luminescence was measured using a microplate luminometer (Synergy 2; BioTek, Winooski, VT, USA).

Statistical analysis

Data analysis was performed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA), and data are expressed as means ± sem. Group comparisons were performed using 2-way or 1-way ANOVA followed by Bonferroni’s multiple comparison test. A value of P < 0.05 was considered sufficient to reject the null hypothesis.

RESULTS

NHERF1 regulates cAMP signaling in human ASM cells

In primary human ASM cells, expression of NHERF1 was confirmed by Western blotting and quantitative PCR (unpublished results). Transfection with siRNA directed against NHERF1 consistently resulted in >80% knockdown of NHERF1 protein levels (Fig. 1A) as determined by Western blotting. Knockdown of NHEFR1 did not affect acute (10 min stimulation) ISO-, PGE₂-, or FSK-induced cAMP production in human ASM cells (Fig. 1B). However, a time-course of ISO-stimulated phosphorylation of the PKA substrate VASP revealed that knockdown of NHERF1 resulted in decreased VASP phosphorylation after 180 and 240 min of stimulation (Fig. 2A). In addition, ISO-induced HSP20 phosphorylation after 4 h of stimulation was lower under NHERF1 knockdown conditions compared with SCR controls (Fig. 2B). Similarly, PGE₂- and FSK-induced VASP phosphorylation was reduced after 180 and 240 min of stimulation (Fig. 3). Interestingly, at 30 min of stimulation, PGE₂- and FSK-induced HSP20 phosphorylation was increased under NHERF1 knockdown condition, whereas at 180 and 240 min poststimulation, FSK-induced p-HSP20 was lower under NHERF1 knockdown vs. SCR controls (Fig. 3). PGE₂-induced pHSP20 at 180 and 240 min was not different between NHERF1 and SCR conditions.

Figure 1.

NHERF1 knockdown does not affect cAMP accumulation in human ASM cells. A) NHERF1 knockdown was confirmed by immunoblotting; a representative blot is shown. B) After SCR or NHERF1 siRNA transfection, cells were stimulated with cAMP-elevating agents for 10 min in the presence of IBMX. Data are means ± sem; n = 3.

Figure 2.

NHERF1 knockdown decreases ISO-induced VASP (A) and HSP20 (B) phosphorylation in human ASM cells. Cells were stimulated with ISO (0–240 min) post–siRNA transfection and cell lysates collected. Representative blot is shown. Data are means ± sem; n = 5. Two-way ANOVA followed by Bonferroni multiple comparison test. *P < 0.05 vs. own SCR control.

Figure 3.

Role of NHERF1 in PGE₂- and FSK-induced VASP and HSP20 phosphorylation in human ASM cells. Cells were stimulated with PGE₂ (A) or FSK (B) (30–240 min) post–siRNA transfection and cell lysates collected. Representative blots are shown. Data are means ± sem; n = 4–7 (PGE₂) or n = 3–5 (FSK). Two-way ANOVA followed by Bonferroni multiple comparison test. *P < 0.05 vs. own SCR control.

NHERF1 differentially regulates cAMP-driven gene expression and cyclin D1 expression in human ASM cells

Among the important functions of ASM are its immunodulatory and synthetic functions, including the production of cytokines that cooperate in the regulation of allergic airway inflammation (29, 30). Stimulation of human ASM cells with ISO, PGE₂, or FSK increased IL-6 gene expression by ∼6-fold as determined by quantitative PCR (Fig. 4A). NHERF1 knockdown resulted in a 90–100% inhibition of IL-6 gene expression for all 3 cAMP-elevating agents. NHERF1 knockdown did not have an effect on TNF-α–induced IL-6 gene expression (SCR: 26.7 ± 2.8; NHERF1: 22.8 ± 8.3 fold basal). Similar results were obtained for IL-6 cytokine protein measured by ELISA (Fig. 4B). Conversely, PDE4D mRNA expression induced by ISO and PGE₂ was not affected by NHERF1 knockdown; FSK-induced PDE4D gene expression was modestly affected but not statistically different (Fig. 4C). Combined average Ct values from all experiments for the SCR vehicle condition were GAPDH, 16.7 ± 0.2; IL-6, 25.7 ± 0.6; and PDE4D, 24.5 ± 0.6.

Figure 4.

NHERF1 differentially regulates cAMP-driven gene expression and IL-6 production in human ASM cells. Cells were stimulated with ISO (1 µM), PGE₂ (1 µM), or FSK (10 µM) post–siRNA transfection for either 4 h (mRNA) or 24 h (ELISA). A, B) IL-6 mRNA (A) was quantified by quantitative PCR and normalized to SCR basal; IL-6 concentration (B) in cell supernatants was determined by ELISA. C) PDE4D mRNA was quantified by quantitative PCR and normalized to SCR basal. Data are means ± sem; n = 5 (mRNA) or n = 7 (ELISA); 2-way ANOVA followed by Bonferroni multiple comparison test. *P < 0.05 vs. SCR control.

Immunoblot analysis of ISO-induced (1 µM; 30–240 min) CREB phosphorylation suggested that NHERF1 knockdown did not affect ISO-induced p-CREB (Fig. 5A). Furthermore, ISO-, PGE₂-, and FSK-induced CRE-Luc activity was not affected by NHERF1 knockdown (Fig. 5B). Similarly, the ability of cAMP-elevating agents to inhibit PDGF-induced cyclin D1 expression was not limited by NHERF1 knockdown (Fig. 5C).

Figure 5.

NHERF1 knockdown does not limit cAMP-driven CREB signaling in human ASM cells. A) Cells were stimulated with ISO (1 µM; 30–240 min), and p-CREB was determined in whole cell lysates. B) Human telomerase reverse transcriptase–expressing human ASM cells stably transfected with CRE-Luc reporter construct were stimulated with ISO (1 µM), PGE₂ (1 µM), or FSK (10 µM) for 6 h, lysates were collected, and luciferase activity was determined. C) For cyclin D1 expression analysis, cells were stimulated with PDGF (10 ng/ml) with or without ISO (1 µM), PGE₂ (1 µM), or FSK (10 µM) post–siRNA transfection and cell lysates collected. Representative blots are shown. Data are means ± sem; n = 3. Two-way ANOVA followed by Bonferroni multiple comparison test. *P < 0.05 vs. own SCR control.

NHERF1 mediates cAMP-driven gene expression via c/EBPβ

In order to evaluate the role of NHERF1 in ISO-mediated activation of the transcription factor c/EBPβ, we performed cell fractionation experiments and evaluated the expression of c/EBPβ in cytosolic, nuclear, and nuclear chromatin–bound fractions. c/EBPβ was detected in the nuclear but not the cytosolic fractions. Stimulation with ISO (1 µM; 30 min) increased the c/EBPβ chromatin-bound nuclear fraction, indicating increased c/EBPβ binding to DNA. This ISO-induced increase in c/EBPβ binding to DNA was fully abrogated by NHERF1 knockdown (Fig. 6A, B). cAMP-driven IL-6, but not PDE4D, gene expression (Fig. 6C, D) was eliminated by c/EBPβ knockdown (60 ± 5%; Fig. 6E). Collectively, these data demonstrate an important role for NHERF1 in directing cAMP signaling toward c/EBPβ, driving IL-6 production, but not toward CREB pathways.

Figure 6.

c/EBPβ knockdown eliminates cAMP-mediated IL-6 gene expression in human ASM cells. A) Human ASM cells were stimulated with ISO (1 µM) for 30 min post–siRNA transfection, and cell fractionation was performed. c/EBPβ, lamin A/C, β-actin, and histone [³H] expression in the cytosolic, nuclear, and chromatin-bound nuclear fraction was determined by immunoblotting. Representative blots are shown. B) Quantification of c/EBPβ expression in the chromatin-bound nuclear fraction; histone [³H] was used as a loading control. C, D) IL-6 (C) and PDE4D (D) mRNA was quantified by quantitative PCR and normalized to SCR basal. Cells were stimulated with ISO (1 µM), PGE₂ (1 µM), or FSK (10 µM) post–siRNA transfection for 4 h. E) c/EBP knockdown was confirmed by immunoblotting; a representative blot is shown. Data are means ± sem; n = 3. Two-way ANOVA (gene expression) or 1-way ANOVA (c/EBPβ chromatin binding), followed by Bonferroni multiple comparison test. *P < 0.05 vs. own SCR control.

DISCUSSION

In the current study, we demonstrate that NHERF1 regulates various aspects of cAMP signaling and cAMP-mediated functions in ASM cells. Regulation of signaling at the receptor locus (i.e., receptor desensitization, internalization, and recycling) has been studied extensively using heterologous and overexpression systems. However, the impact of these regulatory mechanisms on cell functions in physiologic model systems still needs to be determined for many processes.

Compartmentalized signaling involves signaling inputs being transduced via multiple independent signaling cascades to engage different effectors and modulate distinct cell functions; it appears to be dependent, in large part, on the ability of scaffolding proteins to form signaling complexes that route signaling. In recent years, there has been a growing appreciation of how signaling is compartmentalized in various pathways. cAMP pathways are compartmentalized by formation of various signaling complexes facilitated by A-kinase anchoring proteins (AKAP) of which now 30 have been identified (reviewed in ref. 31). In addition, cAMP signaling may be directed by the coupling of Gs subunits to distinct adenylyl cyclase isoforms (32) and by the involvement of distinct PDE isoforms (33, 34). In its ability to form signaling complexes with PKA and to bind ezrin, NHERF1 resembles AKAPs.

Although initially discovered as a regulator of NHE transporter in kidney epithelial cells, NHERF1 has since emerged as a regulator of a surprisingly wide range of signaling pathways affecting multiple cell functions. NHERF1 interactions with GPCRs have been characterized best for the parathyroid hormone (PTH) receptor. The Friedman Lab has demonstrated an important role for NHERF1 in PTH-receptor endocytosis and recycling dynamics (35–38). NHERF1 has also been identified as a binding partner for CFTR, promoting cAMP-mediated activation of CFTR as well as CFTR shuttling from the Golgi to the plasma membrane (10, 12, 39), which may be highly relevant to cystic fibrosis given that both activity and transport of mutant CFTR to the plasma membrane are impaired in this disease. Regulation of Gs-coupled receptors in ASM, including the β₂AR, is highly relevant to obstructive lung diseases. Of note, with respect to Gq signaling, we determined that NHERF1 knockdown does not affect muscarinic M3 acetylcholine receptor–mediated calcium mobilization in M3-expressing ASM cells (unpublished results).

In T cells, NHERF1 forms a macromolecular complex with ezrin and PKA, which allows cAMP-mediated inhibition of T-cell receptor signaling (40). In this particular setting, NHERF1 appears to act in the capacity of a scaffold required to form the signaling complex as well as to enable the localizing of the complex to lipid rafts (41). This indicates that the two broadly defined roles of NHERF1, complex formation and molecular shuttling and localization, are not mutually exclusive and may even act concomitantly to regulate signaling.

The phosphorylation status of NHERF1 is subject to regulation by multiple kinases (including GRK6A, Akt, PKC, cdc2, and RSK1) and phosphatases (PP1 and PP2A). At least 11 serine and threonine phosphorylation sites on NHERF1 have been identified thus far (reviewed in ref. 31). In addition, NHERF1 protein expression may also be subject to regulation (42, 43). Increased expression of NHERF1 has been observed in hepatic (44), biliary (45), and colorectal (46) cancer cells.

In the current study of ASM cells, NHERF1 knockdown did not affect acute cAMP production and early cAMP signaling (30 min–2 h), but it did impair signaling (VASP and HSP20 phosphorylation) at 3–4 h of stimulation. The delayed nature of this effect suggests that deficient receptor recycling is the underlying mechanism. This is consistent with observations from the von Zastrow group (3), showing the absence of NHERF1 results in increased lysosomal degradation of β₂AR after 4 h of stimulation. However, given that we observed a trend toward reduced p-VASP and p-HSP20 induction with FSK stimulation, receptor internalization may not be the only mechanism contributing to the decrease in cAMP signaling with NHERF1 knockdown.

Our data show no discernable role for NHERF1 in regulating CREB pathways. NHERF1 knockdown did not affect cAMP-driven CREB phosphorylation, CRE-Luc activity, or PDE4D mRNA expression. CRE binding sites in the PDE4D promotor region have been identified as crucial for cAMP-induced up-regulation of PDE4D mRNA expression (47). Another study has shown that in murine NHERF1-null mesenchymal stem cells, the phosphorylation status of CREB is not changed compared with wild-type cells (48).

c/EBPβ is a member of the Basic Leucine Zipper (bZIP) family of transcriptional activators that was initially named NF-IL6 when it was identified as a c/EBP homolog that binds to CCAAT consensus sequences in the IL-6 gene (49). c/EBPβ promoter binding is often supportive of, but not required for, NF-κB– and AP1-mediated transcription, and c/EBPβ promotes transcription of a wide range of proinflammatory cytokines (reviewed in ref. 50). c/EBPβ was shown to promote increased IL-8 release from cultured asthmatic human ASM cells (51). Of note, this study also suggested that nuclear translocation of c/EBPβ is not required for transcriptional activity in ASM, and that despite similar levels of nuclear c/EBPβ expression between cells from healthy and asthmatic tissues, c/EBPβ DNA binding is increased in asthmatic ASM cells. This is consistent with observations by us and others (52), which indicate that, at least in some cell types, c/EBPβ is localized in the nucleus, and changes in DNA binding, rather than nuclear translocation, govern transcriptional activity. cAMP-mediated regulation of c/EBP-mediated transcription has long been established (53, 54), but only one previous study linked NHERF1 to transcriptional activation at the CCAAT promoter sequences, showing a requirement for NHERF1 expression for the type IIa sodium phosphate cotransporter gene transcription (55). Though additional regulation of cAMP production at later time points has not been excluded, our current study identifies NHERF1 as a critical adapter in cAMP-driven c/EBPβ DNA binding, leading to IL-6 gene expression and cytokine production.

Collectively, these data suggest a role for NHERF1 in maintaining cAMP and PKA signaling as well as in compartmentalizing cAMP signaling, with a crucial role for NHERF1 in promoting c/EBPβ-dependent IL-6 production but not in CREB- or CRE-driven processes.

Glossary

ASM

airway smooth muscle

β₂AR

β-2-adrenoceptor

c/EBPβ

CCAAT-enhancer–binding protein β

CFTR

cystic fibrosis transmembrane conductance regulator

CRE

cAMP response element

CREB

cAMP response element–binding protein

EBP50

ezrin-radixin-moesin–binding phosphoprotein 50

ERM

ezrin radixin moesin

FSK

forskolin

HSP20

heat shock protein 20

ISO

isoproterenol

NHE

Na⁺/H⁺ exchanger

NHERF1

NHE regulatory factor

PDE4D

phosphodiesterase 4D

PDZ

PSD-95, disc large, zona occludens-1

PGE₂

prostaglandin E₂

PKA

cAMP-dependent protein kinase

SCR

scrambled

siRNA

small interfering RNA

VASP

vasodilator-stimulated phosphoprotein

AUTHOR CONTRIBUTIONS

T. Pera designed and performed research, analyzed data, and wrote the manuscript; E. Tompkins, M. Katz, B. Wang, and D. A. Deshpande performed experiments and analyzed data; E. J. Weinman designed research; and R. B. Penn designed the research and wrote the manuscript.