EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1

Lobo, Miguel J; Amaral, Margarida D.; Zaccolo, Manuela; Farinha, Carlos M.

doi:10.1242/jcs.185629

Cited by 58 publications

(54 citation statements)

References 58 publications

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“…Nevertheless, a substantial amount of rescued CFTR remained stable, being present up to 8 h after cycloheximide administration. Perhaps our results obtained by co-administering C4 and C18 may be enhanced by including a "stabilizer" compound, such as those that promote the interaction between rescued CFTR mutant and Na + /H + exchanger regulatory factor 1 [43, 44]. However, co-administration of C4 and C18 did not rectify the trafficking, function, or stability of CFTR bearing G85E, leaving a continuing need for an effective treatment to rescue this mutant.…”

Section: Discussionmentioning

confidence: 99%

Combination of Correctors Rescues CFTR Transmembrane-Domain Mutants by Mitigating their Interactions with Proteostasis

Lopes‐Pacheco¹,

Boinot²,

Sabirzhanova³

et al. 2017

Cell Physiol Biochem

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Background/Aims: Premature degradation of mutated cystic fibrosis transmembrane conductance regulator (CFTR) protein causes cystic fibrosis (CF), the commonest Mendelian disease in Caucasians. Despite recent advances in precision medicines for CF patients, many CFTR mutants have not been characterized and the effects of these new therapeutic approaches are still unclear for those mutants. Methods: Cells transfected or stably expressing four CFTR transmembrane-domain mutants (G85E, E92K, L1077P, and M1101K) were used to: 1) characterize the mutants according to their protein expression, thermal sensitivity, and degradation pathways; 2) evaluate the effects of correctors in rescuing them; and 3) explore the effects of correctors on CFTR interactions with proteostasis components. Results: All four mutants exhibited lower protein expression than did wild type-CFTR, and they were degraded by proteasomes and aggresomes. At low temperature, only cells expressing the mutants L1077P and M1101K exhibited increased CFTR maturation. Co-administration of C4 and C18 showed the greatest effect, restoring functional expression and partial stability of CFTR bearing E92K, L1077P, or M1101K at the cell surface. However, this treatment was inefficient in rectifying the defect of CFTR bearing G85E. Correctors rescued CFTR mutants by reducing their interactions with proteostasis components associated with protein retention in the endoplasmic reticulum and ubiquitination. Conclusion: Co-administration of C4 and C18 rescued CFTR transmembrane-domain mutants by remodeling the CFTR interactome.

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Section: Discussionmentioning

confidence: 99%

Combination of Correctors Rescues CFTR Transmembrane-Domain Mutants by Mitigating their Interactions with Proteostasis

Lopes‐Pacheco¹,

Boinot²,

Sabirzhanova³

et al. 2017

Cell Physiol Biochem

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“…Arora et al have shown that F508del can be stabilized at the cell surface with the use of VX-809 (a corrector molecule) by potentiation of its interaction with NHERF1 (3). More recently, it has been demonstrated that exchange protein directly activated by cAMP (EPAC1) interacts with NHERF1 through CFTR, and its activation is additive to F508del CFTR rescue with VX-809 (41). Taken together, the characterization of the 1417 EEN-KVR 1422 motif provides a new potential mechanism to manipulate the interaction between CFTR and NHERF1 to amplify the effect of small-molecule treatments.…”

Section: Discussionmentioning

confidence: 99%

A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction

Sharma

LaRusch

Sosnay

et al. 2016

American Journal of Physiology-Lung Cellular and Molecular Phys

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The development of cystic fibrosis transmembrane conductance regulator (CFTR) targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins, such as sodium/hydrogen exchange regulatory factor-1 (NHERF1). In this study, we explored whether COOH-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, K = 1,382 ± 61.1 nM) at concentrations well above physiological levels, estimated at 240 nM from RNA-sequencing and 260 nM by liquid chromatography tandem mass spectrometry in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in coimmunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six-amino acid sequence EENKVR and the terminal TRL (PDZ-binding motif) in the COOH-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in Madin-Darby canine kidney epithelial cells fostered NHERF1 oligomerization that was substantially reduced (∼5-fold) on alanine substitution of EEN, KVR, or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that COOH-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR, a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.

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“…Previously, we uncovered a prominent long-range allosteric regulatory behavior in NHERF1: Upon Ezrin binding to NHERF1, the binding affinities of the PDZ domains of NHERF1 for target membrane proteins are dramatically increased, even though the PDZ domains are located more than 120 Å away from the Ezrin-binding site (6,20,21*). Thus, Ezrin allosterically modulates NHERF1 to assemble membrane protein complexes and to tether the assembled complexes to the cytoskeletal actin network, which modulates the cell surface targeting and intracellular trafficking of the assembled signaling complexes (22-24). …”

Section: Nherf1: a Paradigm Of Long-range Allosterymentioning

confidence: 99%

Visualizing the nanoscale: protein internal dynamics and neutron spin echo spectroscopy

Callaway

2017

Current Opinion in Structural Biology

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The most complex molecular machines are proteins found within cells. Protein dynamics, in particular dynamics on nanoscales, presents us with a novel paradigm for cell signaling: the idea that proteins and protein complexes can communicate directly within themselves to effect long-range information transfer, via coupled domains and correlated residue clusters. This idea has been little explored, in large part because of a paucity of experimental techniques that can address the necessary questions. Here we review recent progress in developing a promising new approach, neutron spin echo spectroscopy.

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EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1

Cited by 58 publications

References 58 publications

Combination of Correctors Rescues CFTR Transmembrane-Domain Mutants by Mitigating their Interactions with Proteostasis

Combination of Correctors Rescues CFTR Transmembrane-Domain Mutants by Mitigating their Interactions with Proteostasis

A sequence upstream of canonical PDZ-binding motif within CFTR COOH-terminus enhances NHERF1 interaction

Visualizing the nanoscale: protein internal dynamics and neutron spin echo spectroscopy

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