CFTR Protein Interactions

CFTR 蛋白质相互作用

• 批准号: 7475756
• 负责人: Monroe JACKSON Stutts
• 金额: $ 31.78万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7475756
• 关键词: Actin-Binding Protein; Actins; Address; Affinity; Anabolism; Apical; Binding; Biochemical; Biological; Biological Assay; C-terminal; Cell Line; Cell membrane; Cell surface; Cells; Childhood; Code; Complex; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeleton; Data; Disease; Endosomes; Epithelial; Epithelial Cells; Family; Genes; Half-Life; Human; Ion Channel; Localized; Lung; Mammalian Cell; Mediating; Membrane; Mutation; N-terminal; Null Lymphocytes; PDZ protein; Phosphatidylinositol Phosphates; Process; Protein Binding; Protein Family; Proteins; RNA Interference; Recycling; Regulation; Role; Sorting - Cell Movement; Surface; System; Testing; Work; apical membrane; disease-causing mutation; drug development; filamin; member; mutant; nexin; novel; numb protein; phosphatidylinositol phosphate; polarized cell; receptor; research study; sorting nexins; trafficking

DESCRIPTION (provided by applicant): Cystic fibrosis (CF) is caused by mutation in the single gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane Cl- channel. Despite extensive study, there are significant gaps in our understanding of how CFTR is synthesized and processed and how CFTR is regulated and functions at the apical membrane. CFTR associates with a number of proteins that facilitate its trafficking or function, but our understanding of these interactions and how they are altered in CF is relatively poor. We find that the actin binding proteins filamin A (FLN-A) and FLN-B associate directly with residues 1-25 of human CFTR. This interaction enhances receptor-stimulated activation of CFTR and a known disease-causing mutation in the N-terminus of CFTR (S13F) decreases the affinity of the interaction. In addition, the half-life of S13F CFTR is significantly decreased when compared to CFTR proteins that can bind FLN. We also find that a novel sorting nexin, SNX27, associates with CFTR. SNX27 accumulates on subapical endosomes and RNA interference-mediated depletion of SNX27 significantly decreased the levels of cell surface-associated CFTR. Since FLNs and SNX27 associate with CFTR at the cell surface or in endosomal compartments, we hypothesize that these novel CFTR-interacting proteins modulate aspects of CFTR internalization and recycling in polarized cells. Therefore, we propose to fully characterize the biological significance of the CFTR-FLN and CFTR-SNX27 interactions using biochemical, cellular, and functional assays in airway epithelial cells. Our data suggest that CFTR is tethered to the actin cytoskeleton via two distinct linkages - an N-terminal interaction with FLN and C-terminal interaction with actin-associated PDZ proteins. Therefore we will test the hypothesis that these two cytoskeletal anchors work in concert to stabilize CFTR at the apical membrane of polarized cells. The characterization of protein interactions that modulate CFTR trafficking, stability and/or function provide one prospect for new therapies for CF and will increase our understanding of the trafficking and regulation of this complex epithelial ion channel. Lay Summary: Mutations in CFTR cause the lethal childhood disease, cystic fibrosis; our experiments address the question of how other proteins that bind CFTR alter its function in the human lung.

描述（由申请人提供）：囊性纤维化（CF）是由编码囊性纤维化跨膜电导调节器（CFTR）（一种顶膜氯离子通道）的单基因突变引起的。尽管进行了广泛的研究，但我们对 CFTR 如何合成和加工以及 CFTR 如何在顶膜上进行调节和发挥作用的理解仍存在重大差距。 CFTR 与许多促进其运输或功能的蛋白质相关，但我们对这些相互作用以及它们在 CF 中如何改变的了解相对较少。我们发现肌动蛋白结合蛋白 filamin A (FLN-A) 和 FLN-B 直接与人 CFTR 的残基 1-25 结合。这种相互作用增强了 CFTR 受体刺激的激活，而 CFTR N 末端的已知致病突变 (S13F) 降低了相互作用的亲和力。此外，与可结合 FLN 的 CFTR 蛋白相比，S13F CFTR 的半衰期显着缩短。我们还发现一种新颖的分选连接蛋白 SNX27 与 CFTR 相关。 SNX27 在根尖下内体上积累，RNA 干扰介导的 SNX27 消耗显着降低了细胞表面相关 CFTR 的水平。由于 FLN 和 SNX27 在细胞表面或内体区室中与 CFTR 相关，因此我们假设这些新型 CFTR 相互作用蛋白在极化细胞中调节 CFTR 内化和再循环。因此，我们建议使用气道上皮细胞中的生化、细胞和功能测定来充分表征 CFTR-FLN 和 CFTR-SNX27 相互作用的生物学意义。我们的数据表明，CFTR 通过两个不同的连接与肌动蛋白细胞骨架相连——N 端与 FLN 相互作用，C 端与肌动蛋白相关 PDZ 蛋白相互作用。因此，我们将测试这两个细胞骨架锚协同工作以稳定极化细胞顶膜处的 CFTR 的假设。调节 CFTR 运输、稳定性和/或功能的蛋白质相互作用的特征为 CF 新疗法提供了前景，并将增加我们对这种复杂上皮离子通道的运输和调节的理解。外行总结：CFTR 突变会导致致命的儿童疾病——囊性纤维化；我们的实验解决了结合 CFTR 的其他蛋白质如何改变其在人肺中的功能的问题。