Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells

W Stoorvogel, H J Geuze, J M Griffith, A L Schwartz, G J Strous

    Research output: Contribution to journalArticleAcademicpeer-review


    We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.

    Original languageEnglish
    Pages (from-to)2137-48
    Number of pages12
    JournalJournal of Cell Biology
    Issue number6
    Publication statusPublished - Jun 1989


    • Asialoglycoprotein Receptor
    • Asialoglycoproteins
    • Biological Transport
    • Carcinoma, Hepatocellular
    • Cell Compartmentation
    • Cross-Linking Reagents
    • Endocytosis
    • Endopeptidase K
    • Hexosephosphates
    • Humans
    • Liver
    • Liver Neoplasms
    • Macromolecular Substances
    • Mannosephosphates
    • Microscopy, Electron
    • Receptor, IGF Type 2
    • Receptors, Cell Surface
    • Receptors, Immunologic
    • Receptors, Transferrin
    • Serine Endopeptidases
    • Time Factors
    • Transferrin
    • Tumor Cells, Cultured


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