Interplay between ER Exit Code and Domain Conformation in CFTR Misprocessing and Rescue

Abstract

Impaired conformation underlies CFTR misprocessing in cystic fibrosis. Defective conformation interferes with ER export and post-ER stability. In ΔF508 CFTR, low temperature or R555K improves both through a global conformational reversion. The interplay between the ER exit code and domain conformation governs CFTR misprocessing and rescue.

Human Heat Shock Protein 105/110 kDa (Hsp105/110) Regulates Biogenesis and Quality Control of Misfolded Cystic Fibrosis Transmembrane Conductance Regulator at Multiple Levels

“…5). This is consistent with the findings that ⌬F508 CFTR associates more extensively with Hsc70 and Hsp70 (34,46) and Hdj-2 (24) than wild-type CFTR. The preferential association of Hsp105 with the misfolded ⌬F508 CFTR in the ER is consistent with the preferential stabilizing effect of Hsp105 overexpression on ⌬F508 CFTR (Fig.…”

“…The CFTR expression plasmids pcDNA3.1(ϩ)-CFTR-WT, pcDNA3.1(ϩ)-CFTR-⌬F508 and pcDNA3.1(ϩ)-CFTR-DAA (34), and the pcDNA3.1(Ϫ)-GST-EGFP plasmid (35) have been described previously. The pcDNA3.1(Ϫ)-Myc-Rab3A plasmid was provided by Dr. William Balch (The Scripps Research Institute, La Jolla, CA) and was used as the control plasmid for Hsp105 overexpression experiments.…”

“…The HEK cells stably expressing ⌬F508 (HEK-⌬F) or wild-type (HEK-WT) CFTR (34,36) were maintained in the above medium supplemented with 150 g/ml hygromycin B (EMD Chemicals, Gibbstown, NJ) as described previously (34). CF airway epithelial IB3-1 cells (37) were maintained in LHC-8 medium without gentamicin (Invitrogen), 5% fetal bovine serum, and 100 units/ml each of penicillin and streptomycin.…”

“…We treated the CFTRexpressing HEK cells with brefeldin A to block the ER-to-Golgi trafficking so that the vast majority of the CFTR molecules were retained in the ER. Although ⌬F508 mutation is known to cause global misfolding in CFTR (34,(42)(43)(44), an ER exit code mutation (DAA) (45) was recently shown to impair CFTR export with minimal impact on CFTR conformation (34). Therefore, we included this DAA mutant as an additional control.…”

Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2

“…Likewise, whereas the mutation of the di-acidic ER export motif causes enhanced protein degradation of CFTR (63), mutating the COOH-terminal valine, another ER exit code, leads, in contrast, to a stabilization of Pmel17 protein (64). Whether our NKCC2 mutants are mainly conformation mutants, like the cystic fibrosis ⌬F508 mutants (63), as opposed to relatively "pure" sorting mutants, similar to DAA for CFTR (63) 1049 , are highly conserved in the COOH-terminal tails of all members of the cation chloride co-transporter family. Indeed, all three motifs are present in all members of the CCC transporters and are evolutionarily conserved from insect to human homologs.…”