Biochemical characterization of the cystic fibrosis transmembrane conductance regulator in normal and cystic fibrosis epithelial cells.
Affinity-purified polyclonal antibodies, raised against two synthetic peptides corresponding to the R domain and the C terminus of the human cystic fibrosis transmembrane conductance regulator (CFTR), were used to characterize and localize the protein in human epithelial cells. Employing an immunoblotting technique that ensures efficient detection of large hydrophobic proteins, both antibodies recognized and approximately 180-kDa protein in cell lysates and isolated membranes of airway epithelial cells from normal and cystic fibrosis (CF) patients and of T84 colon carcinoma cells. Reactivity with the anti-C terminus antibody, but not with the anti-R domain antibody, was eliminated by limited carboxypeptidase Y digestion. When normal CFTR cDNA was overexpressed via a retroviral vector in CF or normal airway epithelial cells or in mouse fibroblasts, the protein produced had an apparent molecular mass of about 180 kDa. The CFTR expressed in insect (Sf9) cells by a baculovirus vector had a molecular mass of about 140 kDa, probably representing a nonglycosylated form. The CFTR in epithelial cells appears to exist in several forms. N-glycosidase treatment of T84 cell membranes reduces the apparent molecular mass of the major CFTR band from 180 kDa to 140 kDa, but a fraction of the T84 cell CFTR could not be deglycosylated, and the CFTR in airway epithelial cell membranes could not be deglycosylated either. Moreover, wheat germ agglutinin absorbs the majority of the CFTR from detergent-solubilized T84 cell membranes but not from airway cell membranes. The CFTR in all epithelial cell types was found to be an integral membrane protein not solubilized by high salt or lithium diiodosalicylate treatment. Sucrose density gradient fractionation of crude membranes prepared from the airway epithelial cells, previously surface-labeled by enzymatic galactosidation, showed a plasma membrane localization for both the normal CFTR and the CFTR carrying the Phe508 deletion (delta F 508). The CFTR in all cases co-localized with the Na+, K(+)-ATPase and the plasma membrane calcium ATPase, while the endoplasmic reticulum calcium ATPase and mitochondrial membrane markers were enriched at higher sucrose densities. Thus, the CFTR appears to be localized in the plasma membrane both in normal and delta F 508 CF epithelial cells.
Sarkadi
B
B
Department of Medicine, University of North Carolina, Chapel Hill 27599.
Bauzon
D
D
Huckle
W R
WR
Earp
H S
HS
Berry
A
A
Suchindran
H
H
Price
E M
EM
Olson
J C
JC
Boucher
R C
RC
Scarborough
G A
GA
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001980:Bronchi / Q000737:chemistry; D002268:Carboxypeptidases; D002460:Cell Line; D002461:Cell Line, Transformed; D003110:Colonic Neoplasms; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004848:Epithelium / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G; D007313:Insecta; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009297:Nasal Mucosa / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D012016:Reference Values; D014132:Trachea / Q000737:chemistry; D014162:Transfection
D000906:Antibodies; D000911:Antibodies, Monoclonal; C505032:CFTR protein, human; D007074:Immunoglobulin G; D008565:Membrane Proteins; D010455:Peptides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D002268:Carboxypeptidases; C022633:serine carboxypeptidase