Mutations in the Amino Terminus of the Cystic Fibrosis Transmembrane Conductance Regulator Enhance Endocytosis

James F. Collawn,John P. Clancy,Eric J. Sorscher,Zsuzsa Bebok,Yao Li,Karoly Varga,Krzysztof Nowotarski,Kevin L. Kirk,Asta Jurkuvenaite

doi:10.1074/jbc.m508131200

Abstract

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.

Highlights

IntroductionThis sequence conforms to a consensus internalization signal that consists of YXX⌽, where ⌽ is a large hydrophobic residue and X is any residue [13]
Class II mutations that affect protein maturation are the most prevalent, and the prototype of this class, ⌬F508, is the most common disease-causing mutation. This mutation confers a temperature-sensitive folding defect [18]; when cell lines expressing this protein are cultured at 27 °C for 2 days, ⌬F508 CFTR is released to the cell surface [18], where it reduces single channel activity [20]
Our results indicate that both R31C and R31L have compromised biogenesis and enhanced endocytosis compared with wild-type CFTR

Summary

Introduction

This sequence conforms to a consensus internalization signal that consists of YXX⌽, where ⌽ is a large hydrophobic residue and X is any residue [13]. Class II mutations that affect protein maturation are the most prevalent, and the prototype of this class, ⌬F508, is the most common disease-causing mutation This mutation confers a temperature-sensitive folding defect [18]; when cell lines expressing this protein are cultured at 27 °C for 2 days, ⌬F508 CFTR is released to the cell surface [18], where it reduces single channel activity [20]. Our results indicate that both R31C and R31L have compromised biogenesis and enhanced endocytosis compared with wild-type CFTR These two additive defects contribute to the low surface expression of the mutants. Patch clamp studies confirmed the biochemical data and showed a dramatic decrease in the functional surface pool of both Arg-31 CFTR mutants

Methods

Results

Conclusion