Dataset Information

AP2 ? modulates cystic fibrosis transmembrane conductance regulator function in the human intestine.

ABSTRACT: AP2 is a clathrin-based endocytic adaptor complex comprising ?, ?2, ?2 and ?2 subunits. ?2 regulates CFTR endocytosis. The ? subunit interacts with CFTR in the intestine but its physiologic significance is unclear.CFTR short circuit current was measured in intestinal T84 cells following shRNA knock down of AP2? (AP2?KD). Clathrin-coated structures (CCS) were immunolabeled and quantified in AP2?KD intestinal Caco2BBe (C2BBe) cells. GST tagged human AP2? appendage domain was cloned and its interaction with CFTR determined by GST pull down assay.AP2?KD in T84 cells resulted in higher CFTR current (57%) compared to control, consistent with increased functional CFTR and delayed endocytosis. Depletion of AP2? reduced CCS in C2BBe cells. Pull down assays revealed an interaction between human AP2? appendage domain and CFTR.AP2 ? interacts with and modulates CFTR function in the intestine by participating in clathrin assembly and recruitment of CFTR to CCS.

SUBMITTER: Kumari V

PROVIDER: S-EPMC5502754 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

AP2 α modulates cystic fibrosis transmembrane conductance regulator function in the human intestine.

Kumari Vandana V Desai Shruti S Ameen Nadia A NA

Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 20170421 3

<h4>Background</h4>AP2 is a clathrin-based endocytic adaptor complex comprising α, β2, μ2 and σ2 subunits. μ2 regulates CFTR endocytosis. The α subunit interacts with CFTR in the intestine but its physiologic significance is unclear.<h4>Methods</h4>CFTR short circuit current was measured in intestinal T84 cells following shRNA knock down of AP2α (AP2αKD). Clathrin-coated structures (CCS) were immunolabeled and quantified in AP2αKD intestinal Caco2BBe (C2BBe) cells. GST tagged human AP2α appendag ...[more]

PMID: 28438500

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

AP2 ? modulates cystic fibrosis transmembrane conductance regulator function in the human intestine.

Publications

AP2 α modulates cystic fibrosis transmembrane conductance regulator function in the human intestine.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets