The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC-transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fabantitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4°C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20°C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20°C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.Transferrin, an iron-binding serum protein, and its receptor on the plasma membrane are considered to be involved in a major pathway for the transport of iron into cells. Superficially, the receptor appears to act as a cation transporter; however, the detailed mechanism of the cellular internalization of iron is still unclear. Initial studies using ~3q-labeled transferrin and cell fractionation found transferfin on the surface of reticuiocytes (l). In more recent work, using ascites tumor cells, ferrocyanide staining of transferrin and ferritinconjugated antibody to transferrin also showed that transferfin is localized on the cell surface (2). In contrast, there is evidence that transferrin is pinocytosed into reticulocytes and normoblasts (3)(4)(5). In these studies, ferritin-conjugated antibodies to transferrin (3, 4) and transferrin-colloidal gold complexes (5) were employed to visualize the localization of transferrin.Experiments demonstrating that transferrin co-purifies with clathrin in h u m a n placental tissues (6, 7) imply that the mechanism of transferrin uptake into cells might be similar to that of many peptides, i.e., asialoglycoprotein receptor, yolk proteins, a-2 macroglobulin, and the hormones, insulin and epidermal growth factor (8, 9). Many studies of r...

Risk of type 1 diabetes at 3 years is high for initially multiple and single Ab+ IT and multiple Ab+ NT. Genetic predisposition, age, and male sex are significant risk factors for development of Ab+ in twins.

Human chromosome 3 has been identified as responsible for expression of the transferrin receptor in mouse-human lymphocyte hybrids. The receptor was detected by immunoprecipitation with anti-human receptor antibody of "MI-labeled cells. This method also detected a similar 94,000-dalton protein in mouse cells. A radioimmunoassay developed for the human transferrin receptor measured 10% crossreactivity with the mouse protein. The two proteins were distinguished by NaDodSO4/ polyacrylamide gel patterns of partial proteolytic digests of the immunoprecipitated proteins. Mouse-human hybrids were generated by fusing a mouse thymoma (BW5147) cell line to either concanavalin A-or pokeweed mitogen-activated human peripheral blood lymphocytes or a mouse myeloma (NS-1) to uncultured human peripheral blood lymphocytes. Each hybrid was karyotyped with respect to both mouse and human chromosomes. In every case, expression of the human transferrin receptor correlated only with human chromosome 3.The genes for a number of human cell surface proteins have been assigned to specific chromosomes and chromosome regions by studying somatic cell hybrids between human and rodent cells (1-4). Several of these genes have been assigned to the human X chromosome (1, 2, 5-7); others have been assigned to the autosomes (3,4,(8)(9)(10)(11).In most cases, the antigens have been defined only by reactivity with human cells and mouse-human hybrid clones containing certain human chromosomes (1, 5, 10) although, in a few, the marker molecules have been characterized as well (7, 9).In this paper, we describe expression of the human transferrin receptor in mouse-human lymphocyte cell hybrids and assign the gene for this receptor to human chromosome 3. Assignment is based on identification of the transferrin receptor by its molecular and immunochemical properties. Partial digestion patterns were used to distinguish the human transferrin receptor from the mouse transferrin receptor. We have also identified and characterized molecular and immunochemical properties of the mouse transferrin receptor.MATERIALS gle's medium (GIBCO)/15% newborn calf serum (GIBCO)/ 0.1 mM hypoxanthine/0.4 tuM aminopterine/13 tLM thymidine containing penicillin at 100 pug/ml and streptomycin at 100 units/ml] and cloned by limiting dilution on human fibroblast feeders. A hypoxanthine/aminopterin/thymidine-sensitive BALB/c myeloma (NS-1) was fused with human peripheral blood lymphocytes, and the hybrids were cultured and cloned as T-cell hybrids.The hybrid clones and the cell lines were cultured in Dulbecco's modified Eagle's medium/15% newborn calfserum, the human erythroid cell line K562 was cultured in RPMI 1640 (GIBCO)/10% fetal calf serum, and BeWo cells were cultured in BeWo medium (12). All cultures were grown in the presence of penicillin (100 tug/ml) and streptomycin (100 units/ml) (GIBCO).Karyotypes and Clones. Chromosome preparations were made from the hybrid clones on multiple occasions (see Table 2) and once from the BW5147 and NS-1 cell lines. Giemsa bands...

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Hybrid cells have been recovered from selective culture medium after fusion of concanavalin-A-activated human lymphocytes with an AKR mouse thymoma (BW 5147). After 6 months of culture twenty-seven out of forty-nine clones still contained human chromosomes. Human chromosome 6 was present in 89% of these clones, and human X in 70%. Clones from one hybrid line contained several human chromosomes. In twelve of the clones carrying human chromosomes, the rosetting with sheep erythrocytes (SRBC) was 3 times as high as in the BW 5147 cell line. All these clones carried the human chromosome 6, and eight clones contained the human X chromosome as well. In some of these clones (25%) chromosome 6 was the only human one present. In the two clones in which human chromosome 6 was completely missing, the rosetting with SRBC was at the level of the BW line. We therefore suggest that genes on human chromosome 6 are responsible for rosetting with SRBC.