Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells.
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.
Stoorvogel
W
W
Laboratory of Cell Biology, University of Utrecht Medical School, The Netherlands.
Geuze
H J
HJ
Griffith
J M
JM
Schwartz
A L
AL
Strous
G J
GJ
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins / Q000378:metabolism*; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002451:Cell Compartmentation; D003432:Cross-Linking Reagents; D004705:Endocytosis; D019286:Endopeptidase K; D006600:Hexosephosphates / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008360:Mannosephosphates / Q000378:metabolism*; D008854:Microscopy, Electron; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000494:pharmacology; D013997:Time Factors; D014168:Transferrin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D003432:Cross-Linking Reagents; D006600:Hexosephosphates; D046911:Macromolecular Substances; D008360:Mannosephosphates; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D011990:Receptors, Transferrin; D014168:Transferrin; C027693:mannose-6-phosphate; D012697:Serine Endopeptidases; D019286:Endopeptidase K