Insulin-Like Growth Factor 1 (IGF-1) Enhances the Protein Expression of CFTR

Ha Won Lee,Mark Donowitz,William B Guggino,Olga Kovbasnjuk,Jie Cheng,Effie C Tsilibary

doi:10.1371/journal.pone.0059992

Abstract

Low levels of insulin-like growth factor 1 (IGF-1) have been observed in the serum of cystic fibrosis (CF) patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR), whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL)- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.

Highlights

Cystic fibrosis is a genetic disease caused by mutations in CFTR [1]
CFTR-associated ligand (CAL) Binds to CFTR at the Golgi To find out how CAL regulates CFTR trafficking, we investigated the interaction between CAL and CFTR by using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay
The FRET results (Fig. 1B-E) confirmed the binding of YFP-CFTR and CAL-CFP, showing that the FRET efficiency of CAL-CFP and YFP-wildtype CFTR was significantly higher than that of the negative controls (CAL-CFP alone or with YFP-DTRL CFTR). These results demonstrate that CAL binds CFTR, but not DTRL CFTR, in vivo and that the detected FRET is specific for the binding of CAL to CFTR

Summary

Introduction

Cystic fibrosis is a genetic disease caused by mutations in CFTR [1]. CFTR’s primary function is to move chloride ions across the plasma membrane of epithelial cells; this is a key function in the normal operation of several organs, including the airways, the intestinal tract, the pancreas, the epididymis, and the sweat duct (see [2] for a review). It is well known that CFTR trafficking to the cell surface is regulated by PDZ proteins (the Golgi reassembly stacking protein [GRASP], CFTR-associated ligand [CAL], Na+/H+ exchanger regulatory factor [NHERF1/2], and CFTR-associated protein 70 [CAP70]), which bind to CFTR [7,8,9,10] These proteins assemble CFTR into protein complexes in the ER, Golgi, or plasma membrane in polarized epithelial cells [8,10] and regulate CFTR localization at the apical membrane by allowing CFTR to reach the plasma membrane, sequestering it within the cell, or targeting it for degradation (see [11] for review). NHERF and CAP70 stabilize CFTR and allow CFTR to form an efficient functional complex [8,14,15]

Methods

Results

Conclusion