Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression

Krisztina Peter,Karoly Varga,Zsuzsa Bebok,Carmel M Mcnicholas-Bevensee,Lisa Schwiebert,Eric J Sorscher,Erik M Schwiebert,James F Collawn

doi:10.1074/jbc.m209275200

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999) J. Biol. Chem. 274, 3602-3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr(1424)-X-X-Ile(1427) where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.

Highlights

The cystic fibrosis transmembrane conductance regulator (CFTR)1 is a cAMP-activated chloride channel that resides at the apical surface of epithelial cells
Because the chloride channel activity and relative surface expression of Y1424A and I1427A CFTR are elevated to a similar extent, we propose that these substitutions affect protein trafficking but not CFTR chloride channel function
Mutations in the Carboxyl-terminal Tail of CFTR Increase Surface Expression—Our hypothesis in these experiments is that if both tyrosine 1424 and isoleucine 1427 are important for CFTR internalization, complete disruption of these residues should increase CFTR surface expression

Summary

Introduction

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that resides at the apical surface of epithelial cells. Because tyrosine-based signals have been proposed to consist of the motif YXX⌽ where ⌽ is a large hydrophobic residue and X is any residue [10], we tested the hypotheses that the isoleucine residue at position 1427 is important for CFTR endocytosis and that ablation of this putative signal YXXI would increase the steady-state surface expression of CFTR. To this end, we performed an integrated series of biochemical and electrophysiological assays designed to study maturation efficiency, trafficking, and ClϪ channel function of the wild-type and two COOH-terminal mutant CFTR proteins. This is the first CFTR mutant that has enhanced rather than diminished activity at the cell surface because of attenuation of internalization

Methods

Results

Conclusion