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 do main of p75 human nerve growth factor receptor (hNGFR) changes its loc alization 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 a bility to endocytose NGF and to recycle through basolateral endosomes. We report here the site-directed mutagenesis analysis of PS-NGFR to l ocalize 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 ect odomain of the placental alkaline phosphatase, an apical glypiated ect oenzyme, redirected it to the basolateral membrane and the endocytic c ompartments. A tyrosine at position 308, present in this short cytopla smic segment, was mutated into phenylalanine or alanine. The resulting mutants were expressed predominantly on the apical membrane of MDCK c ells. Their ability to endocytose NGF was reduced with the alanine mut ant showing the stronger diminution. The PS mutant contains a short cy toplasmic sequence necessary both for basolateral targeting and endocy tosis, and the requirement for tyrosine at position 308 is crucial for basolateral targeting.