Basolateral Sorting Signal Research Articles

W(h)ither default? sorting and polarization in epithelial cells

The discovery of basolateral sorting signals in the past year may leave the default pathway with nowhere to go. With new results suggesting that even more GTP-binding proteins and coatamers might be involved in transport and targeting, it is clear that the age of mapmaking in polarization research is nearly over.

The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal.

The C-terminal cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) contains two tyrosine residues, Tyr668 and Tyr734. Previous work identifying Tyr734 as a critical residue in the endocytosis of the pIgR in Madin-Darby canine kidney (MDCK) cells also suggested that a second functional internalization signal was present (Breitfeld, P. P., Casanova, J. E., McKinnon, W. C., and Mostov, K. E. (1990) J. Biol. Chem. 265, 13750-13757). To test this hypothesis, Tyr668 and Tyr734 were mutated singly or together by oligonucleotide-directed mutagenesis of pIgR cDNA, and the mutants were expressed in MDCK cells. The amount of ligand internalized within 5 min from the basolateral membrane by the pIgR in which cytoplasmic tyrosines were mutated separately to Cys668 or Ser734 or together to Cys668, Ser734 was 58, 39, and 20%, respectively, of the internalized by the wild-type pIgR. The cytoplasmic and transmembrane domains of the pIgR, when joined to the external domain of the influenza virus hemagglutinin, retained the capacity to mediate rapid internalization. As with the full-length pIgR, mutation of either tyrosine in the chimera resulted in impairment of endocytosis, with mutation of Tyr734 having a significantly greater effect than mutation on Tyr668 on the initial rate of endocytosis (3 and 44% of control values, respectively). However, unlike the full-length pIgR, mutation of both tyrosines together in the chimera did not reduce internalization further. The two tyrosines in the cytoplasmic sequence of the pIgR, although widely separated in the linear amino acid sequence, both contribute to internalization of the protein, suggesting that both can function as internalization signals. In addition, the correlation between endocytosis and basolateral targeting of the pIgR in MDCK cells was investigated. Neither tyrosine of the cytoplasmic domain was necessary for basolateral targeting of the pIgR.