Dataset Information

Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation.

ABSTRACT: Although the cystic fibrosis transmembrane conductance regulator (CFTR) is primarily implicated in the regulation of plasma-membrane chloride permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood. To delineate the internalization pathway, both the rate and extent of CFTR accumulation in endosomes were monitored in stably transfected Chinese hamster ovary (CHO) cells. The role of clathrin-dependent endocytosis was assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-dependent endocytosis by >90%, as verified by measurements of 125I-transferrin uptake. Functional association of CFTR with newly formed endosomes was determined by an endosomal pH dissipation protocol [Lukacs, Chang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 267, 14568-14572]. As a second approach, endocytosis of CFTR was determined after cell-surface biotinylation with the cleavable sulphosuccinimidyl-2-(biotinamido)ethyl-1,3-dithio- propionate. Both the biochemical and the functional assays indicated that arresting the formation of clathrin-coated vesicles inhibited the retrieval of the CFTR from the plasma membrane to endosomes. An overall arrest of membrane traffic cannot account for the inhibition of CFTR internalization, since the fluid-phase endocytosis was not effected by the treatments used. Thus the efficient, constitutive internalization of surface CFTR (5% per min) occurs, predominantly by clathrin-dependent endocytosis. Stimulation of protein phosphorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CFTR, and contributed to a substantial diminution of the internal CFTR pool compared with that of unstimulated cells. These results suggest that the rate of CFTR internalization may participate in the determination of the CFTR channel density, and consequently, of the cAMP-stimulated chloride conductance of the plasma membrane.

SUBMITTER: Lukacs GL

PROVIDER: S-EPMC1218928 | biostudies-other | 1997 Dec

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin-proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Dataset Information

Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets