Putative O-Glycosylation Sites and a Membrane Anchor Are Necessary for Apical Delivery of the Human Neurotrophin Receptor in Caco-2 Cells*
- From the Laboratoire de Génétique et Physiologie du Développement, UMR6545, IBDM, Faculté des Sciences de Luminy, Case 907, Université de la Méditerranée, 13288 Marseille Cedex 09, France
Abstract
We have expressed the human neurotrophin receptor p75 (p75NTR) in the intestinal epithelial cell line Caco-2 as a model to study intracellular transport and subcellular sorting signals in intestinal cells. p75NTR was localized at the apical membrane of Caco-2 cells and reached this membrane mainly via an indirect pathway. Apical localization, intracellular routing, and basolateral to apical transcytosis were not affected by truncation of the cytoplasmic domain or replacement of the transmembrane domain by a glycosyl phosphatidylinositol anchor. Removal of membrane anchoring resulted in basolateral secretion of the ectodomain of p75NTR in Caco-2 cells but in apical secretion in Madin-Darby canine kidney (MDCK) cells. Substitution of potentialO-glycosylation sites present in the stalk of p75NTR led to intracellular cleavage and secretion of the ectodomain into the basolateral medium both in Caco-2 and MDCK cells. These results suggest that the stalk of p75NTR carries an apical sorting information that is recognized efficiently by Caco-2 cells only when attached to the membrane. This apical sorting information is linked to the presence of predictedO-glycosylation sites in that region. These putative O-glycosylation sites also play a role in the regulation of p75NTR transport to the cell surface and in the prevention of rapid degradation by cleavage of the stalk domain.
Footnotes
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↵* This work was supported by CNRS UMR6545, Université de la Méditerranée, Institut de Biologie du Développement de Marseille, Arc 9297, AFLM, and INSERM 930203.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed. Tel.: 33 04 91 26 97 44; Fax: 33 04 91 26 97 48; E-mail: lebivic{at}ibdm.univ-mrs.fr.
- Abbreviations:
- MDCK
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Madin-Darby canine kidney
- WT
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wild type
- PCR
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polymerase chain reaction
- PAGE
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polyacrylamide gel electrophoesis
- TGN
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trans-Golgi Network
- GPI
-
glycosylphosphatidyl inositol.
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- Received April 6, 1998.
- Revision received August 7, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
