Abstract
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.
Highlights
THE JOURNAL OF BIOLOGICAL CHEMISTRYVol 270, No 35, Issue of September 1, pp. 20717-20723, 1995 Printed in U.S.A. Identification and Characterization of a Novel Protein (pI37) Which Transcytoses Bidirectionally in Caco-2 Cells*
Transcytosis of selected proteins to the apical domain [2]
Sequence motifs in the cytoplasmic tail of the polymeric immunoglobulin receptor (pIgR) required for transcytosis have been defined [5, 9, 10] as well as the endocytic compartments through which it travels [8] and the proteolytic events occurring when it arrives at the apical cell surface and is released [11]
Summary
Vol 270, No 35, Issue of September 1, pp. 20717-20723, 1995 Printed in U.S.A. Identification and Characterization of a Novel Protein (pI37) Which Transcytoses Bidirectionally in Caco-2 Cells*. Antisera raised against detergent-extracted memo brane fractions from the human intestinal epithelial cell line Caco-2 were used to screen a human colon eDNA library in a bacteriophage expression vector This led to the identification, molecular cloning, and sequencing of a novel plasma membrane protein (pI37) which was present in approximately equal amounts on the basolateral and apical surfaces of the cell. We have previously shown that some antisera raised against plasma membrane fractions from Caco-2 cells react with subsets of proteins common to the apical and basolateral domains [17] Such proteins are candidates for apical to basolateral transcytosis, since ifinternalized from the apical surface they would not necessarily carry signals allowing sorting back to the apical domain. In the present study we'have identified and cloned a novel membrane protein present in both the apical and basolateral plasma membrane domains of Caco-2 cells, and show that it is transcytosed in both directions
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