Abstract
During terminal differentiation of murine erythroleukemic (MEL) cells, the number of surface transferrin binding sites per cell decreases dramatically, while steady-state ligand uptake and immunoblotting studies demonstrate that the total number of transferrin receptors per cell remains constant. Since the amount of protein per cell decreases 4-fold during this 4-day period, the amount of transferrin receptor protein, relative to total soluble cell protein, increases 4-fold during this time, suggesting continued synthesis of the receptor. Supporting this, we show that the amount of transferrin receptor transcript in equal amounts of total cell RNA also increases as differentiation proceeds. Uninduced cells maintain 52% of the total transferrin binding sites on the cell surface, whereas only 22% of the receptors are on the surface in 4-day induced cells. All ligand endocytosed by either uninduced or induced cells at 37 degrees C is rapidly and completely exocytosed from the cells, suggesting that all of the cellular receptors are cycling. These studies suggest that, during MEL cell differentiation, an increasing fraction of transferrin receptors are localized to the cell interior, but are nevertheless cycling to the cell surface. This observed redistribution is due to altered kinetic parameters of the receptor. Receptor-bound 125I-labeled transferrin ligand has been followed through a single endocytic cycle. Ligand internalization occurs much more rapidly in induced cells (t1/2 = 2.9 min) than in uninduced cells (t1/2 = 6.9 min). The rates for ligand movement back out to the cell surface and its subsequent release into the medium in both uninduced and induced cells are quite similar.
Highlights
During terminal differentiationof murine erythro- Specific membrane receptors for transferrin were initially leukemic (MEL) cells, the number of surface transfer- demonstrated on immature erythroid cells, whichrequire large rin binding sites per cell decreases dramatically, while amounts of iron for hemoglobin production (Jandl and Katz, steady-state ligand uptake ainmdmunoblotting studies 1963).during erythroid differentiation, the compodemonstrate that thetotal number of transferrin recep- sition of the plasma membrane undergoes significant alterators per cell remains constant
The mechanism by which the transferrin receptor is cleared from the erythroid cell during maturation is unknown
The transferrin receptor has been characterized in several ferrin binding sites on the cell surface, whereas only cell types; it is a glycoprotein covalently associated with fatty
Summary
Immunological Detection of the Transferrin Receptor-The total number of transferrin receptors present in MEL cells was examined byWestern blotting of total cell proteins from equal numbersof cells. - autoradiography (Fig. l),both theuninduced and theinduced cells exhibited a single band of M , 95,000 present in equal amounts regardless of the extentof cell differentiation, indicating that the totalnumber of transferrin receptors per cell remained constant throughout the differentiation period. Equal amounts of total soluble cell protein (200pg)were examined by immunoblotting techniques and the amount of transferrin receptor in each band increased as differentiation progressed. Probing Northern blots of equal amounts of total cellular RNA with human transferrinreceptor cDNA revealed the 28 and 15 S rRNA bands as well as a 4.9-kb putative transferrin receptor transcript. Since these cells have 3.2 X lo5surface receptors, 48% of the receptors for transferrin were localized intracellularly. Further,thetotal number of cellular transferrin molecules, andthus functional transferrin receptors, re-
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