Abstract. Cross-linking of surface receptors results in altered receptor trafficking in the endocytic system. To better understand the cellular and molecular mechanisms by which receptor cross-linking affects the intracellular trafficking of both ligand and receptor, we studied the intracellular trafficking of the transferrin receptor (TfR) bound to multivalent-transferrin (Tfl0) which was prepared by chemical cross-linking of transferrin (Tf). Tfl0 was internalized about two times slower than Tf and was retained four times longer than Tf, without being degraded in CHO cells. The intracellular localization of Tfl0 was investigated using fluorescence and electron microscopy. Tfl0 was not delivered to the lysosomal pathway followed by low density lipoprotein but remained accessible to Tf in the pericentriolar endocytic recycling compartment for at least 60 min. The retained Tfl0 was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tfl0. The presence of Tfx0 within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tfl0 within the recycling compartment did not require the cytoplasmic domain of the TfR since Tfl0 exited cells with the same rate when bound to the wild-type TfR or a mutated receptor with only four amino acids in the cytoplasmic tail. Thus, cross-linking of surface receptors by a multivalent ligand acts as a lumenal retention signal within the recycling compartment. The data presented here show that the recycling compartment labeled by Tfl0 is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.
SummaryHelper T cell recognition of antigen requires that it be processed within antigen-presenting cells (APC) to peptide fragments that subsequently bind to major histocompatibility complex (MHC) class II molecules and are displayed on the APC surface. Heretofore, processed antigen-MHC class II complexes have been detected by functional assays, measuring the activation of specific T cells. We now report direct, biochemical evidence for the assembly of processed antigen-MHC class II complexes within splenic B ceils as APC. The I-E k MHC class II molecules were immunoprecipitated from B cells that had processed the model protein antigen cytochrome c radiolabeled across its entire length by reductive methylation of lysine residues and covalently coupled to Ig-specific antibodies, allowing internalization after binding to surface Ig. Our previous studies showed that I-E ~ immunoaffinity purified from B cells that had processed cytochrome c contains functional processed antigen-MHC class II complexes and that approximately 0.2% of the I-E k molecules are specifically associated with one of two predominant processed antigenic fragments. Here we show that these complexes are rapidly assembled, within 30--60 min after antigen binding to surface Ig on splenic B cells. Maximal numbers of complexes are assembled by 2 h in a process that is sensitive to acidic vesicle inhibitors but not to inhibitors of protein synthesis. The processed antigen-I-E k complexes have a relatively short half-life of 2--4 h and are disassembled or degraded within 8 h after antigen is first internalized. The disassembly or degradation of the processed antigen-I-E k complexes requires acidic vesicle function, and in the presence of an acidic vesicle inhibitor the complexes are long lived. Thus, using a biochemical assay to monitor processed antigen-I-E k complexes, we find that, in B cells, processed antigen is relatively rapidly associated in acidic vesicles with preexisting MHC class II molecules, and the complexes are disassembled 4-6 h later in processes that also require acid vesicle function.
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