scholarly journals In vivo crystals reveal critical features of the interaction between cystic fibrosis transmembrane conductance regulator (CFTR) and the PDZ2 domain of Na+/H+ exchange cofactor NHERF1

2020 ◽  
Vol 295 (14) ◽  
pp. 4464-4476
Author(s):  
Eleanor R. Martin ◽  
Alessandro Barbieri ◽  
Robert C. Ford ◽  
Robert C. Robinson
Keyword(s):  
Cystic Fibrosis ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Binding Motif ◽  
Transmembrane Conductance Regulator ◽  
Protein Protein Interactions ◽  
Reporter System ◽  
Transmembrane Conductance ◽  
Cystic Fibrosis Transmembrane

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.

scholarly journals Functional regulation of cystic fibrosis transmembrane conductance regulator-containing macromolecular complexes: a small-molecule inhibitor approach

2011 ◽  
Vol 435 (2) ◽  
pp. 451-462 ◽  
Author(s):  
Weiqiang Zhang ◽  
Himabindu Penmatsa ◽  
Aixia Ren ◽  
Chandanamali Punchihewa ◽  
Andrew Lemoff ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Small Molecule ◽  
Protein Interactions ◽  
Pdz Domain ◽  
Transmembrane Conductance Regulator ◽  
Protein Protein Interactions ◽  
Transmembrane Conductance ◽  
Macromolecular Complexes ◽  
Channel Function ◽  
Cystic Fibrosis Transmembrane

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.

scholarly journals In-vivo crystals reveal protein interactions

2019 ◽  
Author(s):  
Eleanor R. Martin ◽  
Alessandro Barbieri ◽  
Robert C. Ford ◽  
Robert C. Robinson
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Pdz Domain ◽  
Cell Types ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
Reporter System ◽  
X Ray Crystallography

AbstractCrystallisation of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under non-physiological extremes of protein, salt, and H+ concentrations. Here, we describe the development of the robust iBox-PAK4cat system that spontaneously crystallises in several mammalian cell types. The developments described here allow the quantitation of in-vivo protein-protein interactions using a novel GFP-linked reporter system. Here, we have combined this assay with in-vitro X-ray crystallography and molecular dynamics studies characterise the molecular determinants of the interaction between NHERF1 PDZ2 and CFTR, a protein complex pertinent to the genetic disease cystic fibrosis. These studies have revealed the crystal structure of the extended PDZ domain of NHERF1, and indicated, contrary to previous reports, that residue selection at −1 and −3 PDZ-binding motif positions influence the affinity and specificity of the interaction. The results presented here demonstrate that the iBox-PAK4cat assay could easily be utilised to screen other protein-protein interactions.

Examining basal chloride transport using the nasal potential difference response in a murine model

2001 ◽  
Vol 281 (5) ◽  
pp. L1173-L1179 ◽  
Author(s):  
Kristine G. Brady ◽  
Thomas J. Kelley ◽  
Mitchell L. Drumm
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Transport ◽  
Inbred Mice ◽  
Inbred Strains ◽  
Potential Difference ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Inbred Strains Of Mice ◽  
Cystic Fibrosis Transmembrane

Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate ( Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.

Biosynthesis and Degradation of CFTR

1999 ◽  
Vol 79 (1) ◽  
pp. S167-S173 ◽  
Author(s):  
RON R. KOPITO
Keyword(s):  
Cystic Fibrosis ◽  
Secretory Pathway ◽  
Covalent Modification ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Binding Domains ◽  
The Common ◽  
Effects Of Temperature ◽  
Cystic Fibrosis Transmembrane

Kopito, Ron R. Biosynthesis and Degradation of CFTR. Physiol. Rev. 79, Suppl.: S167–S173, 1999. — Many of the mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that cause cystic fibrosis interfere with the folding and biosynthetic processing of nascent CFTR molecules in the endoplasmic reticulum. Mutations in the cytoplasmic nucleotide binding domains, including the common allele ΔF508, decrease the efficiency of CFTR folding, reduce the probability of its dissociation from molecular chaperones, and largely prevent its maturation through the secretory pathway to the plasma membrane. These mutant CFTR molecules are rapidly degraded by cytoplasmic proteasomes by a process that requires covalent modification by multiubiquitination. The effects of temperature and chemical chaperones on the intracellular processing of mutant CFTR molecules suggest that strategies aimed at increasing the folding yield of this protein in vivo may eventually lead to the development of novel therapies for cystic fibrosis.

scholarly journals PDZ Domain Interaction Controls the Endocytic Recycling of the Cystic Fibrosis Transmembrane Conductance Regulator

2002 ◽  
Vol 277 (42) ◽  
pp. 40099-40105 ◽  
Author(s):  
Agnieszka Swiatecka-Urban ◽  
Marc Duhaime ◽  
Bonita Coutermarsh ◽  
Katherine H. Karlson ◽  
James Collawn ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pdz Domain ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Domain Interaction ◽  
Endocytic Recycling ◽  
Cystic Fibrosis Transmembrane

Deactivation of CFTR-Cl conductance by endogenous phosphatases in the native sweat duct

1996 ◽  
Vol 270 (2) ◽  
pp. C474-C480 ◽  
Author(s):  
M. M. Reddy ◽  
P. M. Quinton
Keyword(s):  
Cystic Fibrosis ◽  
Okadaic Acid ◽  
Apical Membrane ◽  
Transmembrane Conductance Regulator ◽  
Sweat Duct ◽  
Transmembrane Conductance ◽  
Phosphatase Inhibitors ◽  
Cystic Fibrosis Transmembrane ◽  
Cl Conductance

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel. However, very little is known about the endogenous mechanism(s) of deactivation of CFTR-Cl conductance (CFTR-GCl) in vivo. We studied the action of endogenous phosphatases in regulation of the adenosine 3',5'-cyclic monophosphate (cAMP)- and ATP-induced CFTR-GCl in the apical membrane of microperfused preparations of basolaterally permeabilized native sweat duct. Activation of CFTR-GCl was monitored by measuring the apical Cl diffusion potentials and GCl, which spontaneously deactivated on removal of cAMP. This spontaneous loss of CFTR-GCl activity could be prevented by a cocktail of phosphatase inhibitors (fluoride, vanadate, and okadaic acid). We studied the effects of each of these phosphatase antagonists on the rate of deactivation of CFTR-GCl after cAMP washout. In contrast to vanadate or fluoride, okadaic acid virtually prevented deactivation of CFTR-GCl after cAMP washout. We conclude that either or both protein phosphatases 1 and 2A are responsible for the dephosphorylation deactivation of CFTR-GCl in vivo.

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