Intracellular pools of transferrin receptors result from constitutive internalization of unoccupied receptors.

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Abstract
In HeLa cells the majority of transferrin (Tf) receptors are found within the endocytic apparatus, with only 20% of receptors exposed at the cell surface. Receptor distribution is unaltered by the presence or absence of Tf. The mechanism responsible for the cellular distribution of receptors was explored by selectively inactivating receptors within the endocytic apparatus. This was accomplished by employing Tf-horseradish peroxidase conjugates. Peroxidase-catalyzed oxidation of diaminobenzidine within an endosome destroys Tf receptor activity. Using such conjugates, we have demonstrated that the majority of internal Tf receptors could be inactivated when less than 6.0% of receptors were occupied by the conjugate at steady state. This result indicates that occupied and unoccupied receptors are in the same compartment. Furthermore, horseradish peroxidase that was internalized by fluid-phase pinocytosis inactivated intracellular Tf receptors in the absence of Tf; this indicates that the presence of internal receptors is ligand independent. Following exposure of cells to the conjugate, receptor inactivation was proportional to the percentage of the endocytic cycle traversed by the conjugate--that is, the rate of ligand accumulation was the same as the rate of endosomal Tf receptor inactivation. When the Tf-horseradish peroxidase conjugate and 125I-labeled Tf were internalized simultaneously, both ligands were found in the same compartment. However, if the two ligands were administered as separate pulses and the period between pulses was as short as 1 min, the ligands remained separate within the cell. Together these results demonstrate that the intracellular pool of Tf receptors reflects the constitutive internalization of unoccupied Tf receptors, which, once internalized, remain segregated.