A cytoplasmic tyrosine is essential for the basolateral localization of mutants of the human nerve growth factor receptor in Madin-Darby canine kidney cells.
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.
Monlauzeur
L
L
Laboratoire de Génétique et Physiologie du Développement, Unité Mixte de Recherche 9943, Faculté des Sciences de Luminy, Marseille, France.
Rajasekaran
A
A
Chao
M
M
Rodriguez-Boulan
E
E
Le Bivic
A
A
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002460:Cell Line; D016764:Cell Polarity; D018076:DNA, Complementary / Q000235:genetics; D004285:Dogs; D004705:Endocytosis; D004848:Epithelium; D006801:Humans; D007668:Kidney; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection; D014443:Tyrosine / Q000502:physiology*
D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D000469:Alkaline Phosphatase