| Popis: |
Two signals are known that regulate the entry of the polymeric immunoglobulin receptor (pIgR) into the transcytotic pathway: binding of the ligand, dimeric IgA, and phosphorylation of Ser664 in the cytoplasmic domain of the receptor. Mutation of Ser664 to an alanine residue generates a receptor that is transcytosed slowly and exhibits increased recycling at the basolateral plasma membrane. In contrast, if Ser664 is mutated to an aspartate (which bears a constant negative charge-like a phosphate group) the receptor is transcytosed at a rate that is greater than the wild-type pIgR. We have constructed fusions of placental alkaline phosphatase (PLAP) with the transmembrane region and cytoplasmic domain of the wild-type or mutant pIgRs described above (PLAP/WT, PLAP/Ala, and PLAP/Asp) in order to study transcytotic signals present in the cytoplasmic domain of the pIgR independently of those present in the lumenal domain. Transcytosis of the fusion proteins was measured by cell surface immunoprecipitation of a single cohort of basolaterally biotinylated fusion proteins as it arrived at the apical plasma membrane. Significantly more of the PLAP/WT fusion (27%) than the PLAP/Ala fusion protein (5%) was transcytosed during a 2-h chase period. In addition, it was found that approximately 10% of a pseudoligand, monovalent 125I-labeled Fabs (derived from affinity-purified anti-PLAP antibodies), was transcyosed by the PLAP/WT fusion, 7% by the PLAP/Ala fusion, and 22% by the PLAP/Asp fusion over a 4-h period. These data are consistent with the hypothesis that the cytoplasmic domain of the pIgR contains information necessary to direct proteins into the transcytotic pathway, albeit inefficiently, and that phosphorylation of Ser664 is at least a partially autonomous signal for transcytosis. |
