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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
Published on: July 31, 2012
The beta chain of the interleukin-3 receptor functionally associates with the erythropoietin receptor
P T Jubinsky1, O I Krijanovski, D G Nathan
1Division of Pediatric Hematology, Dana-Farber Cancer Institute and Children's Hospital, Boston, MA, USA.
Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors share a common beta chain (beta(c)), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and beta(c) may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine beta(c). This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine beta. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect beta-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against beta and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against beta, showed that EpoR and beta coimmunoprecipitate. These data show that the beta chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.
Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors share a common beta chain (beta(c)), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and beta(c) may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine beta(c). This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine beta. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect beta-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against beta and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against beta, showed that EpoR and beta coimmunoprecipitate. These data show that the beta chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.
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