A Cytoplasmic Tyrosine Is Essential for the Basolateral Localization of Mutants of the Human Nerve Growth Factor Receptor in Madin- Darby Canine Kidney Cells (*)
- Laure Monlauzeur(1),
- Ayyapan Rajasekaran(2),
- Moses Chao(2),
- Enrique Rodriguez-Boulan(2) and
- André Le Bivic(1)(§)
- From the (1) From Laboratoire de Génétique et Physiologie du Développement, Unité Mixte de Recherche 9943, Faculté des Sciences de Luminy, 13288 Marseille, Cedex 09 France and the
- (2) Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York 10021
- §To whom correspondence and reprint requests should be addressed. Fax: 33-91-26-97-48.
Abstract
Deletion of 58 internal amino acids from the C-terminal cytoplasmic domain of p75 human nerve growth factor receptor (hNGFR) changes its localization from apical to basolateral in transfected Madin-Darby Canine Kidney (MDCK) cells (Le Bivic, A., Sambuy, Y., Patzak, A., Patil, N., Chao, M., and Rodriguez-Boulan, E. (1991) J. Cell Biol. 115, 607-618). The mutant protein, PS-NGFR, also shows a dramatic increase in its ability to endocytose NGF and to recycle through basolateral endosomes. We report here the site-directed mutagenesis analysis of PS-NGFR to localize and characterize its basolateral and endocytic sorting signals. Both signals reside in the proximal part of the PS cytoplasmic tail, between positions 306 and 314. Transferring the cytoplasmic tail (19 residues) and transmembrane domain of a truncated PS mutant to the ectodomain of the placental alkaline phosphatase, an apical glypiated ectoenzyme, redirected it to the basolateral membrane and the endocytic compartments. A tyrosine at position 308, present in this short cytoplasmic segment, was mutated into phenylalanine or alanine. The resulting mutants were expressed predominantly on the apical membrane of MDCK cells. Their ability to endocytose NGF was reduced with the alanine mutant showing the stronger diminution. The PS mutant contains a short cytoplasmic sequence necessary both for basolateral targeting and endocytosis, and the requirement for tyrosine at position 308 is crucial for basolateral targeting.
Footnotes
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↵* This work was supported by CNRS Unité Mixte de Recherche Grant 9943, Association pour la Recherche sur le Cancer Grant 6421, INSERM Grant 930203, and an Association Franaise de Lutte contre la Mucoviscidose grant (to A. L. B.), a North Atlantic Treaty Organization Collaborative Research Program, and National Institutes of Health Grant 34107 (to E. R. B.). 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.
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↵1 The abbreviations used are:
- LDLR
-
light density lipoprotein receptor
- hNGFR
-
human nerve growth factor receptor
- MDCK
-
Madin-Darby canine kidney
- PLAP
-
placental alkaline phosphatase
- NGF
-
nerve growth factor
- pIgR
-
polymeric immunoglobulin receptor
- WT
-
wild type
- LAP
-
lysosomal acid phosphatase
- PS315 Y
F -
point mutation of Tyr-308
Phe-308
- PS315 Y
A -
point mutation of Tyr-308
Ala-308.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
