Identification and Characterization of a Novel Protein (p137) Which Transcytoses Bidirectionally in Caco-2 Cells

doi:10.1074/jbc.270.35.20717

Identification and Characterization of a Novel Protein (p137) Which Transcytoses Bidirectionally in Caco-2 Cells (*)

Juliet A. Ellis and
J. Paul Luzio(§)

From the Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR, United Kingdom

^§ To whom correspondence should be addressed. Tel.: 1223-336780; Fax: 1223-330598.

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°C, followed by chasing at 37°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.

Footnotes

↵* This work was supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank]and ID HSGPI137.
↵1 The abbreviations used are:

TGN

trans-Golgi network

CRD

Cross Reacting Determinant

DTT

dithiothreitol

ECL

enhanced chemiluminescence

GPI

glycosylphosphatidylinositol

MDCK

Madin-Darby canine kidney

ORF

open reading frame

PI-PLC

phosphatidylinositol specific phospholipase C

UTR

predicted untranslated region

PAGE

polyacrylamide gel electrophoresis

bp

base pair(s).
↵² M. B. Soares, unpublished results.
↵³ M. D. Adams, unpublished results.
↵⁴ R. W. Davies, unpublished results.
- Received February 8, 1995.
- Revision received April 27, 1995.