Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator.

Abstract

Deletion of phenylalanine at position 508 (AF608) in the f i s t nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator (CFTR)is the most common mutation in patients with cystic fibrosis.Although retaining functional C1channel activity, this mutant is recognized as abnormal by the cellular "quality control" machinery and is retained within the endoplasmic reticulum (ER).W e have used human epithelial cells and recombinant Chinese hamster ovary cells to identify molecular interactions that may contribute to this intracellular retention.Based upon coimmunoprecipitation and cosedimentation through glycerol density gradients, newly synthesized wild-type and AFS08 mutant CFTRa associated specifically with calnexin, the calcium-binding transmembrane chaperone of the E R This association was restricted to the immature (or ERassociated) forms of the CFTR proteins.Although the bulk of wild-type and AFS08 CFTRs were present initially in complexes containing calnexin, only wild-type CFTR was able to escape from this association and exit the ER.Calnexin retains misfolded or incompletely assembled proteins in the ER and thus is likely to contribute to the mislocalization of mutant CFTR.Mutations in the gene encoding CFTR' (1-3) result in the clinical symptoms of CF due to the absence or malfunction of a regulated C1-channel in the apical membrane of secretory epithelia (for recent reviews, see Refs.4-6).The demonstration that CFTR itself forms the channel (7) has fueled intensive research into methods to alleviate CF through gene therapy (e.g.Refs.8 and 9).Alternative approaches involve attempts to overcome the functional impairment of various mutant CFTRs, especially that in which phenylalanine at position 508 is deleted from the first nucleotide-binding fold (AF508 CFTR).This mutation is found in -70% of CF chromosomes and results in a severe form of the disease (3); >90% of CF patients have at