Rab11b regulates the apical recycling of the cystic fibrosis transmembrane conductance regulator in polarized intestinal epithelial cells.
The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, DeltaF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.
Silvis
Mark R
MR
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, PA 15261, USA.
Bertrand
Carol A
CA
Ameen
Nadia
N
Golin-Bisello
Franca
F
Butterworth
Michael B
MB
Frizzell
Raymond A
RA
Bradbury
Neil A
NA
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D016764:Cell Polarity; D002712:Chlorides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D005453:Fluorescence; D005799:Genes, Dominant; D006801:Humans; D018189:Immunomagnetic Separation; D007422:Intestines / Q000166:cytology*; D015640:Ion Channel Gating; D050505:Mutant Proteins / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D022142:Secretory Vesicles / Q000648:ultrastructure; D020691:rab GTP-Binding Proteins / Q000378:metabolism*; D000099193:rab11 GTP-Binding Proteins
D002712:Chlorides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D050505:Mutant Proteins; D034741:RNA, Small Interfering; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins