Figure 5.
Figure 5. Ubiquitination and degradation of the EpoR intracellular domain occur at the cell surface. (A) Epo-induced ubiquitination of the EpoR. UT-7 cells were incubated for 105 minutes with 50 μM LLnL, and 10 U/mL Epo was added to cell samples after 15, 45, 75, or 95 minutes of incubation with LLnL. Cells were thus incubated for the same time (105 minutes) with LLnL and for the indicated times with Epo. At the end of the incubation, whole cell lysates (WCL) were analyzed by Western blot (WB) using C-20 anti-EpoR antibodies (i), and C-236 anti-EpoR immunoprecipitates were analyzed using antiubiquitin (Ub) antibodies (ii). Numbers 1 and 2 indicate the mature and maturing forms of the EpoR, respectively; bracket, ubiquitinated EpoRs; arrowheads, nonspecific bands. (B) Tyrosine-phosphorylated EpoRs are ubiquitinated. UT-7 cells were preincubated for 15 minutes with LLnL and incubated for 10 minutes with 10 U/mL Epo (lane 1) or without Epo (lane 2). Cell lysates were prepared, and EpoRs were immunoprecipitated using C-236 anti-EpoR antibodies. Immunoprecipitates were dissociated by boiling in buffer containing 1% SDS and 50 mM DTT. DTT was removed by chromatography through Sephadex G50 using spin columns, and phosphotyrosine-containing proteins were immunoprecipitated. Immunoprecipitated proteins were analyzed by Western blot using C-20 anti-EpoR antibodies. (C) Ubiquitination of cell surface EpoRs. UT-7 cells were preincubated for 15 minutes with LLnL and stimulated for 10 minutes with 10 U/mL Epo. After washing to remove free Epo, cells were incubated for 30 minutes at 4°C with anti-Epo antibodies and washed to remove unbound antibodies. Cells were then solubilized, and immune complexes were recovered using protein G Sepharose beads (lane 1). As controls, EpoRs were immunoprecipitated with C-236 anti-EpoR antibodies from cells pretreated with LLnL for 15 minutes and stimulated (lane 3) or not (lane 2) for 10 minutes with 10 U/mL Epo. All immunoprecipitates were analyzed by Western blots using anti-EpoR antibodies. Symbols are as in panel A. (D) Proteasome-mediated EpoR degradation in methyl β cyclodextrine (MBCD)–treated cells. UT-7 cells were preincubated for 30 minutes with cycloheximide to prevent replacement of degraded EpoRs by newly synthesized receptors, either alone (control) or in combination with MBCD to prevent internalization or MBCD and lactacystin to inhibit both internalization and proteasome activity. Cells were then stimulated with 10 U/mL Epo, and whole cell lysates were prepared at the indicated times and analyzed by Western blotting using C-20 anti-Epo antibodies. Symbols are as in panel A. (E) Densitometric scanning of the experiment presented in panel D. After scanning, the intensity of band 1 was determined using the ImageJ software. For each panel, the intensity of band 1 at t = 0 was set at 100%, and the intensity of this band at later incubation times was expressed relative to this value.

Ubiquitination and degradation of the EpoR intracellular domain occur at the cell surface. (A) Epo-induced ubiquitination of the EpoR. UT-7 cells were incubated for 105 minutes with 50 μM LLnL, and 10 U/mL Epo was added to cell samples after 15, 45, 75, or 95 minutes of incubation with LLnL. Cells were thus incubated for the same time (105 minutes) with LLnL and for the indicated times with Epo. At the end of the incubation, whole cell lysates (WCL) were analyzed by Western blot (WB) using C-20 anti-EpoR antibodies (i), and C-236 anti-EpoR immunoprecipitates were analyzed using antiubiquitin (Ub) antibodies (ii). Numbers 1 and 2 indicate the mature and maturing forms of the EpoR, respectively; bracket, ubiquitinated EpoRs; arrowheads, nonspecific bands. (B) Tyrosine-phosphorylated EpoRs are ubiquitinated. UT-7 cells were preincubated for 15 minutes with LLnL and incubated for 10 minutes with 10 U/mL Epo (lane 1) or without Epo (lane 2). Cell lysates were prepared, and EpoRs were immunoprecipitated using C-236 anti-EpoR antibodies. Immunoprecipitates were dissociated by boiling in buffer containing 1% SDS and 50 mM DTT. DTT was removed by chromatography through Sephadex G50 using spin columns, and phosphotyrosine-containing proteins were immunoprecipitated. Immunoprecipitated proteins were analyzed by Western blot using C-20 anti-EpoR antibodies. (C) Ubiquitination of cell surface EpoRs. UT-7 cells were preincubated for 15 minutes with LLnL and stimulated for 10 minutes with 10 U/mL Epo. After washing to remove free Epo, cells were incubated for 30 minutes at 4°C with anti-Epo antibodies and washed to remove unbound antibodies. Cells were then solubilized, and immune complexes were recovered using protein G Sepharose beads (lane 1). As controls, EpoRs were immunoprecipitated with C-236 anti-EpoR antibodies from cells pretreated with LLnL for 15 minutes and stimulated (lane 3) or not (lane 2) for 10 minutes with 10 U/mL Epo. All immunoprecipitates were analyzed by Western blots using anti-EpoR antibodies. Symbols are as in panel A. (D) Proteasome-mediated EpoR degradation in methyl β cyclodextrine (MBCD)–treated cells. UT-7 cells were preincubated for 30 minutes with cycloheximide to prevent replacement of degraded EpoRs by newly synthesized receptors, either alone (control) or in combination with MBCD to prevent internalization or MBCD and lactacystin to inhibit both internalization and proteasome activity. Cells were then stimulated with 10 U/mL Epo, and whole cell lysates were prepared at the indicated times and analyzed by Western blotting using C-20 anti-Epo antibodies. Symbols are as in panel A. (E) Densitometric scanning of the experiment presented in panel D. After scanning, the intensity of band 1 was determined using the ImageJ software. For each panel, the intensity of band 1 at t = 0 was set at 100%, and the intensity of this band at later incubation times was expressed relative to this value.

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