OLIGOMERIZED TRANSFERRIN RECEPTORS ARE SELECTIVELY RETAINED BY A LUMENAL SORTING SIGNAL IN A LONG-LIVED ENDOCYTIC RECYCLING COMPARTMENT

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 (Tf(10)) which was prepared by chemical cross-linking of transferrin (Tf). Tf(10) 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 Tf(10) was investigated using fluorescence and electron microscopy. Tf(10) 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 Tf(10) was TfR-associated as demonstrated by a reduction in surface TfR number when cells were incubated with Tf(10). The presence of Tf(10) within the recycling compartment did not affect trafficking of subsequently endocytosed Tf. Retention of Tf(10) within the recycling compartment did not require the cytoplasmic domain of the TfR since Tf(10) 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 Tf(10) is a long-lived organelle along the early endosome recycling pathway that remains fusion accessible to subsequently endocytosed Tf.