1. Activation of the Erythropoietin Receptor Is Not Required for Internalization of Bound Erythropoietin
by Diana L. Beckman, Lilie L. Lin, Mary E. Quinones, and Gregory D. Longmore
Blood
Volume 94(8):
October 15, 1999
©1999 by American Society of Hematology

2. Schematic diagram of EPO-R variants and their level of expression in 32D cells.
Schematic diagram of EPO-R variants and their level of expression in 32D cells. (A) Stick figure of the murine EPO-R depicting the various truncated EPO-R isoforms generated (arrows) represents the wild-type EPO-R (lacking the signal peptide). The dark box represents the transmembrane domain. The gray boxes represent Box1 and Box2 domains. Specific point mutations constructed are designated. EPO-R(YF) is a full-length EPO-R in which all 8 intracellular (cytoplasmic) tyrosine residues (Y) were changed to phenylalanines. The positions of the tyrosine residues (horizontal line) relative to the sites of truncation are shown. TM, transmembrane domain. (B) Immunoblot analysis of detergent soluble extracts from 32D clones containing the various EPO-R isoforms. All lanes were loaded with a detergent soluble extract from 7.5 × 105 cells. A polyclonal rabbit antisera against the extracellular N-terminal peptide of the murine EPO-R was used. Molecular mass standards (in kilodaltons) are depicted on the right.
Diana L. Beckman et al. Blood 1999;94:
©1999 by American Society of Hematology

3. 32D cells containing the EPO-R reconstitute EPO internalization and receptor downregulation. 125I-EPO internalization studies.
32D cells containing the EPO-R reconstitute EPO internalization and receptor downregulation. 125I-EPO internalization studies. Solid symbols represent the percentage of total bound counts at time 0 that were internalized. Open symbols represent the percentage of total bound counts at time 0 that remained surface bound. Open symbols and a broken line represent the percentage of total bound counts at time 0 that were released into the medium. Standard deviations for each point were within 10% of the value plotted and are not shown. At each time point, the summation of internalized EPO, surface-bound EPO, and counts in the medium equaled the amount of 125I-EPO bound at time 0. Internalization studies were performed on 3 different clones and were performed 2 times for each clone. Data presented are from a single representative clone of each. 32D.Wild-type EPO-R(1-483) cells, squares; 32D.Neo cells (no EPO-R), circles.
Diana L. Beckman et al. Blood 1999;94:
©1999 by American Society of Hematology

4. A membrane-proximal 69 amino acid domain of the EPO-R cytoplasmic tail is the minimal domain required for internalization of EPO and receptor downregulation. 125I-EPO internalization studies.
A membrane-proximal 69 amino acid domain of the EPO-R cytoplasmic tail is the minimal domain required for internalization of EPO and receptor downregulation. 125I-EPO internalization studies. (A) The percentage of total bound counts at time 0 that were internalized. (B) The percentage of total bound counts at time 0 that remained surface bound. Standard deviations for each point were within 10% of the value plotted and are not shown. At each time point, the summation of internalized EPO, surface-bound EPO, and counts in the medium equaled the amount of 125I-EPO bound at time 0. Internalization studies were performed on 2 clones for each EPO-R isoform and were performed 2 times for each clone. Data presented are from a single representative clone of each. (□) Wild-type EPO-R(1-483); (▪) EPO-R(1-411); (▵) EPO-R(1-373); (○) EPO-R(1-321); and (•) EPO-R(1-252).
Diana L. Beckman et al. Blood 1999;94:
©1999 by American Society of Hematology

5. Effect of cytoplasmic domain deletion on receptor internalization.
Effect of cytoplasmic domain deletion on receptor internalization. Cell surface glycoproteins were labeled with [3H]galactose, and receptor internalization was determined as described in Materials and Methods. The values shown are the average of 2 independent determinations. (□) The percentage of surface EPO-R internalized in 40 minutes at 37°C in the absence of EPO (−EPO); (▪) the percentage of surface EPO-R internalized in 40 minutes at 37°C in the presence of EPO (+EPO). Total cell surface glycoproteins internalization was determined from anti–EPO-R immunoprecipitates of lysates from (3H)galactose-labeled parental 32D cells that do not contain an EPO-R (total surface GP).
Diana L. Beckman et al. Blood 1999;94:
©1999 by American Society of Hematology

6. 32D cells containing EPO-R(W282R) or EPO-R(YF) do not proliferate in response to EPO. (•) MTT assay of 32D clones containing wild-type EPO-R; (○) EPO-R(1-373); (▵) EPO-R(W282R); and (□) EPO-R(YF).
32D cells containing EPO-R(W282R) or EPO-R(YF) do not proliferate in response to EPO. (•) MTT assay of 32D clones containing wild-type EPO-R; (○) EPO-R(1-373); (▵) EPO-R(W282R); and (□) EPO-R(YF). Data presented are from a single representative clone of each.
Diana L. Beckman et al. Blood 1999;94:
©1999 by American Society of Hematology

7. EPO-R and JAK2 tyrosine phosphorylation in 32D cells containing variants of the EPO-R.
EPO-R and JAK2 tyrosine phosphorylation in 32D cells containing variants of the EPO-R. (A) Cells were washed in RPMI and placed in OptiMEM (GIBCO) in the absence of serum and growth factors. After 6 hours, cells were stimulated with 50 U/mL EPO (+) for 7 minutes or were not stimulated (−). Cell extracts from 7.5 × 105 cells were separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies. Lanes 1 and 2, 32D.EPO-R(1-483), wild-type cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells. Molecular mass standards (in kilodaltons) are on the left. (B) Cell extracts from starved cells (0 EPO) or cells stimulated with 1 U/mL EPO or 50 U/mL EPO were immunoprecipitated with antisera against the N-terminal peptide of the murine EPO-R. Bound products were washed and separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies (upper panel). The blot was then stripped and reprobed with antisera against the EPO-R (lower panel); arrowheads on the left indicate the position of wild-type (wt) EPO-R, EPO-R(W282R) and EPO-R(YF), IgG, and EPO-R(1-373), respectively. Lanes 1 through 3, 32D.EPO-R(1-483) wt cells; lanes 4 through 6, 32D.EPO-R(1-373) cells; lanes 7 and 8, 32D.EPO-R(YF) cells; and lanes 9 and 10, 32D.EPO-R(W282R) cells. (C) Cell extracts from starved cells (−) or cells stimulated with 50 U/mL EPO (+) were immunoprecipitated with antisera against JAK2. Bound products separated by 8% SDS-PAGE under reducing conditions and transferred to Hybond membranes, and immunoblotting was performed with antiphosphotyrosine antibodies (upper panel); the arrowhead on the left identifies the position of JAK2. The blot was then stripped and reprobed with antisera against JAK2 (lower panel). Lanes 1 and 2, 32D.EPO-R(1-483) wt cells; lanes 3 and 4, 32D.EPO-R(1-373) cells; lanes 5 and 6, 32D.EPO-R(YF) cells; and lanes 7 and 8, 32D.EPO-R(W282R) cells.
Diana L. Beckman et al. Blood 1999;94:
©1999 by American Society of Hematology

8. Neither EPO-induced activation of JAK2 tyrosine kinase nor tyrosine phosphorylation of the EPO-R was required for EPO internalization and receptor downregulation. 125I-EPO internalization studies.
Neither EPO-induced activation of JAK2 tyrosine kinase nor tyrosine phosphorylation of the EPO-R was required for EPO internalization and receptor downregulation. 125I-EPO internalization studies. Solid lines represent the percentage of total bound counts at time 0 that were internalized. Broken lines represent the percentage of total bound counts at time 0 that remained surface bound. Standard deviations for each point were within 10% of the value plotted and, thus, are not shown. Internalization studies were performed at least 3 times for each clone. Data presented are from a single representative clone of each. (□) Wild-type EPO-R(1-483); (▪) EPO-R(W282R); and (○) EPO-R(YF).
Diana L. Beckman et al. Blood 1999;94:
©1999 by American Society of Hematology