An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells.
A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.
Le Bivic
A
A
Department of Cell Biology and Anatomy, Cornell University Medical College, New York, NY 10021.
Sambuy
Y
Y
Patzak
A
A
Patil
N
N
Chao
M
M
Rodriguez-Boulan
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002872:Chromosome Deletion; D004285:Dogs; D005455:Fluorescent Antibody Technique; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor; D014162:Transfection
D000911:Antibodies, Monoclonal; D009414:Nerve Growth Factors; D011956:Receptors, Cell Surface; D017475:Receptors, Nerve Growth Factor