Ablation of internalization signals in the carboxyl-terminal tail of the cystic fibrosis transmembrane conductance regulator enhances cell surface expression.
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.
Peter
Krisztina
K
Department of Cell Biology, Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294-0005, USA.
Varga
Karoly
K
Bebok
Zsuzsa
Z
McNicholas-Bevensee
Carmel M
CM
Schwiebert
Lisa
L
Sorscher
Eric J
EJ
Schwiebert
Erik M
EM
Collawn
James F
JF
D000818:Animals; D019921:Biotinylation; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D018118:Chloride Channels / Q000378:metabolism; D002713:Chlorine / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis* / Q000737:chemistry*; D004705:Endocytosis; D007532:Isoleucine / Q000737:chemistry; D009154:Mutation; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D018118:Chloride Channels; D007532:Isoleucine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D014443:Tyrosine; D002713:Chlorine