Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

Cathleen D Valentine,Gergely L Lukacs,Alan S Verkman,Peter M Haggie

doi:10.1074/jbc.m112.421172

Abstract

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing.

Highlights

Phenylalanine 508 deletion from CFTR (⌬F508CFTR) is the most common cause of cystic fibrosis (CF)
Increased Cell Surface Diffusion of r⌬F508CFTR Compared with wtCFTR—Initial studies were done using HeLa cells expressing CFTR constructs containing triplet hemagglutinin tags (CFTR3HA) engineered into the fourth external loop region; these cells were used previously to characterize and identify cellular components involved in r⌬F508CFTR processing [15]
single particle tracking (SPT) analysis of the dynamics and interactions of rescued ⌬F508CFTR revealed novel properties relative to wtCFTR that were related to the peripheral quality control mechanism and, potentially, ⌬F508CFTR internalization dynamics

Summary

Background

Phenylalanine 508 deletion from CFTR (⌬F508CFTR) is the most common cause of CF. Results: Diffusional mobility of surface ⌬F508CFTR is greater than that of wild type CFTR, irrespective of ⌬F508CFTR rescue mechanism. Deletion of phenylalanine 508 (⌬F508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). To investigate the role of scaffold protein interactions in rescued ⌬F508CFTR surface instability, the plasma membrane mobility of ⌬F508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ⌬F508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Cell membrane chloride conductance can be partially restored by maneuvers that correct (or rescue) ⌬F508CFTR biosynthetic processing, promoting its exit from the ER and targeting to the cell surface. We found increased cell surface mobility of r⌬F508CFTR compared with that of wtCFTR, which were found to involve reduced PDZ-interactions and elements of the peripheral quality control machinery. Disruption of CFTRPDZ interactions enhanced endocytosis of wild type and ⌬F508CFTR, suggesting a possible role of reduced PDZ interactions in the accelerated internalization of ⌬F508CFTR

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION