The objective of this work is to determine the mechanism by which reticulocytes and earlier red cells, remodel their plasma membranes during the maturation to erythrocytes. Mature red cells from different species do not retain precisely the same functions found in their respective reticulocytes. (That is, loss of function is species specific). Little is known about the cellular processing and targeting for this selective loss. The long-term objective is to establish the mechanism by which the selected proteins, such as the transferrin receptor or nucleoside transporter, are recognized and removed from the plasma membrane. Since the nucleoside transporter is retained in some species, are there differences between lost and retained transporters? Since the externalization process can occur after new mRNA formation has ceased, these events are either programmed much earlier and are selectively activated or the regulatory """"""""factors"""""""", such as heme, accumulate in the cell till a critical concentration is reached. An avian cell line, infected with a temperature sensitive, erythroblastic virus will be employed to study the early events in differentiation, under controlled conditions, to determine when transferrin receptor (and other functions) begin to be lost from the cell. The methods employed to detect these functions (and others) are ongoing in the laboratory. The experiments will examine the levels of total Hb synthesis and transferrin receptor (TFR) synthesis before and after induction. Induction requires a change to 42 degrees C plus the addition of hemin or anemic chicken serum. The fate of the TFR and protein synthesis are then followed over a period of time until the majority of the cells have matured (i.e., no further mRNA synthesis). The fate of a number of recognizable membrane proteins is also followed after release into the incubation medium. A short-term objective of the project is to assess the origin of the truncated, circulating transferrin receptor in the plasma, i.e., cleaved directly from the cell surface or from released exosomes. This new information may lead to improved assays for detection of clinical anemias and improved discrimination between effects of iron deficiency and marrow dysfunction in anemia as well as how red cells target proteins for externalization.
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