To investigate the degradation mechanism of misfolded membrane proteins from the cell surface, we used mutant cystic fibrosis transmembrane conductance regulators (CFTRs) exhibiting conformational defects in post-Golgi compartments. Here, we show that the folding state of CFTR determines the post-endocytic trafficking of the channel. Although native CFTR recycled from early endosomes back to the cell surface, misfolding prevented recycling and facilitated lysosomal targeting by promoting the ubiquitination of the channel. Rescuing the folding defect or down-regulating the E1 ubiquitin (Ub)-activating enzyme stabilized the mutant CFTR without interfering with its internalization. These observations with the preferential association of mutant CFTRs with Hrs, STAM-2, TSG101, hVps25, and hVps32, components of the Ub-dependent endosomal sorting machinery, establish a functional link between Ub modification and lysosomal degradation of misfolded CFTR from the cell surface. Our data provide evidence for a novel cellular mechanism of CF pathogenesis and suggest a paradigm for the quality control of plasma membrane proteins involving the coordinated function of ubiquitination and the Ub-dependent endosomal sorting machinery.

Deletion of phenylalanine at position 508 (⌬F508) is the most common cystic fibrosis (CF)-associated mutation in the CF transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The consensus notion is that ⌬F508 imposes a temperature-sensitive folding defect and targets newly synthesized CFTR for degradation at endoplasmic reticulum (ER). A limited amount of CFTR activity, however, appears at the cell surface in the epithelia of homozygous ⌬F508 CFTR mice and patients, suggesting that the ER retention is not absolute in native tissues. To further elucidate the reasons behind the inability of ⌬F508 CFTR to accumulate at the plasma membrane, its stability was determined subsequent to escape from the ER, induced by reduced temperature and glycerol. Biochemical and functional measurements show that rescued ⌬F508 CFTR has a temperature-sensitive stability defect in post-ER compartments, including the cell surface. The more than 4 -20-fold accelerated degradation rate between 37 and 40°C is, most likely, due to decreased conformational stability of the rescued ⌬F508 CFTR, demonstrated by in situ protease susceptibility and SDS-resistant thermoaggregation assays. We propose that the decreased stability of the spontaneously or pharmacologically rescued mutant may contribute to its inability to accumulate at the cell surface. Thus, therapeutic efforts to correct the folding defect should be combined with stabilization of the native ⌬F508 CFTR.

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5-to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.

The existence of organ-specific angiogenic factor has recently received confirmation when EG-VEGF was identified. Here we characterized its angiogenic processes in endothelial cells (ECs), compared its effects in micro- and macrovascular ECs, and differentiated the effects mediated by its two G-protein–coupled receptors within the same cell type.