At pH 5.4, apotransferrin (iron-free transferrin) binds to cell-surface transferrin receptors to the same extent and with the same affinity as does diferric transferrin at pH 7.0. Apotransferrin is quickly dissociated from its receptor when the pH is raised to 7.0. These and other results strongly support a simple model that explains the cycling of transferrin during a single cycle of receptor-mediated endocytosis. Diferric transferrin binds to cell-surface receptors, and the transferrin-receptor complex is endocytosed. The pH of the endocytic vesicle is lowered to 5.5 or below; this causes dissociation of iron from the transferrin-receptor complex, but apotransferrin remains bound to its receptor. The iron remains within the cell, and the apotransferrin-receptor complex is recycled to the cell surface. Upon encountering the neutral pH of the medium, apotransferrin is dissociated from the cell.During receptor-mediated endocytosis, ligands bind to specific cell-surface receptors and are internalized in membranelimited endocytic vesicles (1, 2). Most ligands studied to date are degraded within the cell; examples include asialoglycoproteins, insulin, epidermal growth factor, low density lipoprotein, and human choriogonadotropin (3-8). Although the exact pathway followed by such ligands is not known, many receptor-ligand complexes enter the cell through clathrin-coated pits and vesicles. Dissociation of receptor and ligand then occurs, probably in a prelysosomal vesicle (9)(10)(11)(12)(13)(14), and the ligand is transported in a series of vesicles to lysosomes, wherein it is degraded (1,2,4,(6)(7)(8). Less is known about the pathway taken by the receptor. In several systems the receptor is not degraded but recycles to the cell surface; such is the case for the receptors to asialoglycoprotein (15), insulin (16,17), a2 macroglobulin (18), mannose (19), mannose 6-phosphate (20) and low density lipoprotein (21).The fate of transferrin is different. Transferrin (22), a major mammalian serum glycoprotein, transports iron from sites of absorption and storage to tissue cells. The first step of iron delivery involves binding of ferrotransferrin to specific cellsurface receptors (23) that are found on all growing cells. This iron-loaded transferrin is subsequently internalized by endocytosis, and the iron is delivered to the cell (24-28). However, apotransferrin (iron-free transferrin) is not degraded within the cell, but is exocytosed intact into the medium (ref. 29; unpublished data). Is apotransferrin dissociated from its receptor within the cell? If so, how does it escape degradation by the lysosome and how is it secreted into medium? Or does transferrin remain bound to its receptor in endocytic vesicles? If so, how and when is apotransferrin released from its receptor into the culture medium?Endocytic vesicles which contain a2-macroglobulin or transferrin are acidic, with a pH of about 5 (12, 30). In this paper we demonstrate the unique pattern of binding of transferrin and apotransferrin to its receptor as a f...

Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes and is coupled to their association with detergent-resistant membrane domains. Finally, clathrin-independent endocytosis requires dynamin and is specifically regulated by Rho family GTPases. These results define novel properties of receptor-mediated endocytosis and establish that the IL2 receptor is efficiently internalized through this clathrin-independent pathway.

The mechanism by which T cell antigen receptors (TCR) accumulate at the immunological synapse has not been fully elucidated. Since TCRs are continuously internalized and recycled back to the cell surface, we investigated the role of polarized recycling in TCR targeting to the immunological synapse. We show here that the recycling endosomal compartment of T cells encountering activatory antigen-presenting cells (APCs) polarizes towards the T cell-APC contact site. Moreover, TCRs in transit through recycling endosomes are targeted to the immunological synapse. Inhibition of T cell polarity, constitutive TCR endocytosis, or recycling reduces TCR accumulation at the immunological synapse. Conversely, increasing the amount of TCRs in recycling endosomes before synapse formation enhanced their accumulation. Finally, we show that exocytic t-SNAREs from T cells cluster at the APC contact site and that tetanus toxin inhibits TCR accumulation at the immunological synapse, indicating that vesicle fusion mediated by SNARE complexes is involved in TCR targeting to the immunological synapse.

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.