Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach.
PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.
Wolde
Michael
M
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Fellows
Abigail
A
Cheng
Jie
J
Kivenson
Aleksandr
A
Coutermarsh
Bonita
B
Talebian
Laleh
L
Karlson
Katherine
K
Piserchio
Andrea
A
Mierke
Dale F
DF
Stanton
Bruce A
BA
Guggino
William B
WB
Madden
Dean R
DR
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism; D000077614:Golgi Matrix Proteins; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000502:physiology*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C505032:CFTR protein, human; D002352:Carrier Proteins; C434484:GOPC protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D015534:Trans-Activators; D019005:Cystic Fibrosis Transmembrane Conductance Regulator