Identification and characterization of a novel protein (p137) which transcytoses bidirectionally in Caco-2 cells.
Antisera raised against detergent-extracted membrane fractions from the human intestinal epithelial cell line Caco-2 were used to screen a human colon cDNA library in a bacteriophage expression vector. This led to the identification, molecular cloning, and sequencing of a novel plasma membrane protein (p137) which was present in approximately equal amounts on the basolateral and apical surfaces of the cell. The pattern of extraction of p137 from membranes by Triton X-114 and its release from membranes after incubation with phosphatidylinositol-specific phospholipase C were consistent with it being a glycosylphosphatidylinositol-anchored membrane protein. Using antibodies raised against bacterial fusion proteins, it was shown that p137 was present on the cell surface as a reducible homodimer of 137 kDa subunits. There was constitutive release of p137 into the culture medium as a non-reducible 280-kDa entity. Pulse-chase experiments showed that newly synthesized p137 appeared at the basolateral side of a Caco-2 cell layer before appearing at the apical domain. Domain-specific surface biotinylation of Caco-2 cells at 4 degrees C, followed by chasing at 37 degrees C, demonstrated that p137 is capable of transcytosing in both directions across Caco-2 cells. The unusual plasma membrane domain distribution of this glycosylphosphatidylinositol-linked protein and its transcytosis characteristics demonstrate the existence of a previously uncharacterized apical to basolateral transcytotic pathway in Caco-2 cells.
Ellis
J A
JA
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, United Kingdom.
Luzio
J P
JP
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D003110:Colonic Neoplasms; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007422:Intestines; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D003902:Detergents; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40