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Roles of the cytoplasmic domains of the α and β subunits of human granulocyte- macrophage colony-stimulating factor receptor  Akihiko Muto, PhDa, Sumiko.

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Presentation on theme: "Roles of the cytoplasmic domains of the α and β subunits of human granulocyte- macrophage colony-stimulating factor receptor  Akihiko Muto, PhDa, Sumiko."— Presentation transcript:

1 Roles of the cytoplasmic domains of the α and β subunits of human granulocyte- macrophage colony-stimulating factor receptor  Akihiko Muto, PhDa, Sumiko Watanabe, MSa, Tohru Itoh, MSa, Atsushi Miyajima, PhDb, Takashi Yokota, PhDa, Ken-ichi Arai, MD, PhDa  Journal of Allergy and Clinical Immunology  Volume 96, Issue 6, Pages (December 1995) DOI: /S (95) Copyright © 1995 Mosby, Inc. Terms and Conditions

2 FIG. 1 Structure and signal transduction of GMR.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

3 FIG. 1 Structure and signal transduction of GMR.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

4 FIG. 2 Schematic representation of mutant α subunits.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

5 FIG. 3 Functional analyses of the cytoplasmic domain of the α subunit. A, hGM-CSF-dependent growth of Ba/F3 transfectants was measured by the MTT assay, as described in Methods. B, Activation of the c-fos promoter through mutant hGMR was measured by the transient transfection assay. C, Induction of tyrosine phosphorylation of cellular proteins in total cell lysate (upper panel) and of the immunoprecipitated β subunit (middle and lower panels). Samples were prepared from cells stimulated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) 10 ng/ml hGM-CSF for 5 minutes. Phosphorylated proteins were analyzed by Western blotting with an anti-phosphotyrosine (anti-PY) antibody (4G10) (upper and middle panels) and anti-β antibody (lower panel). Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

6 FIG. 3 Functional analyses of the cytoplasmic domain of the α subunit. A, hGM-CSF-dependent growth of Ba/F3 transfectants was measured by the MTT assay, as described in Methods. B, Activation of the c-fos promoter through mutant hGMR was measured by the transient transfection assay. C, Induction of tyrosine phosphorylation of cellular proteins in total cell lysate (upper panel) and of the immunoprecipitated β subunit (middle and lower panels). Samples were prepared from cells stimulated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) 10 ng/ml hGM-CSF for 5 minutes. Phosphorylated proteins were analyzed by Western blotting with an anti-phosphotyrosine (anti-PY) antibody (4G10) (upper and middle panels) and anti-β antibody (lower panel). Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

7 FIG. 3 Functional analyses of the cytoplasmic domain of the α subunit. A, hGM-CSF-dependent growth of Ba/F3 transfectants was measured by the MTT assay, as described in Methods. B, Activation of the c-fos promoter through mutant hGMR was measured by the transient transfection assay. C, Induction of tyrosine phosphorylation of cellular proteins in total cell lysate (upper panel) and of the immunoprecipitated β subunit (middle and lower panels). Samples were prepared from cells stimulated with (lanes 2, 4, and 6) or without (lanes 1, 3, and 5) 10 ng/ml hGM-CSF for 5 minutes. Phosphorylated proteins were analyzed by Western blotting with an anti-phosphotyrosine (anti-PY) antibody (4G10) (upper and middle panels) and anti-β antibody (lower panel). Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

8 FIG. 4 The cytoplasmic domain of the α subunit is required for activation of but not association with the β subunit. The β subunits expressed in Ba/F3-α,β (lanes 1 to 4) and Ba/F3-α328,β (lanes 5 to 8) cells were immunoprecipitated with an anti-α (lanes 1, 2, 5, and 6) or anti-β (lanes 3, 4, 7, and 8) antibody after stimulation of cells with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) 10 ng/ml hGM-CSF for 5 minutes at 37° C. The immunoprecipitates were analyzed by Western blotting with (A) anti-β and (B) anti-phosphotyrosine (anti-PY) antibodies. IP, Immunoprecipitate. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

9 FIG. 5 Schematic representation of chimeric receptors. a.a., Amino acid; TM, transmembrane. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

10 FIG. 5 Schematic representation of chimeric receptors. a.a., Amino acid; TM, transmembrane. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

11 FIG. 6 Northern blot analysis of immediate early genes in Ba/F3 transfectants. Factor-depleted Ba/F3 clones were stimulated with either 1 ng/ml mIL-3 or 5 ng/ml hGM-CSF. After incubation for 30 minutes with mIL-3 as a control (lane C) or for indicated times (0, 15, 30, 60, and 120 minutes) with hGM-CSF (lanes 0 to 120), total RNA was extracted, and 10 μg of each RNA was analyzed by Northern blotting, as described in Methods. The names of transfectants and detected mRNAs are given at the top and left of the figure, respectively. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

12 FIG. 6 Northern blot analysis of immediate early genes in Ba/F3 transfectants. Factor-depleted Ba/F3 clones were stimulated with either 1 ng/ml mIL-3 or 5 ng/ml hGM-CSF. After incubation for 30 minutes with mIL-3 as a control (lane C) or for indicated times (0, 15, 30, 60, and 120 minutes) with hGM-CSF (lanes 0 to 120), total RNA was extracted, and 10 μg of each RNA was analyzed by Northern blotting, as described in Methods. The names of transfectants and detected mRNAs are given at the top and left of the figure, respectively. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

13 FIG. 7 Long-term growth of Ba/F3 transfectants by hGM-CSF. Continuously growing cells were harvested and washed with factor-free RPMI 1640 medium supplemented with 10% FCS and resuspended in the same medium containing 0.25 ng/ml mIL-3 (filled symbols) or 3 ng/ml hGM-CSF (open symbols) at 5 × 104 cells/ml at day 0. The number of trypan blue-resistant cells was counted every 24 hours. The vertical and horizontal axes indicate cell number and days of culture, respectively. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. Number sign (#) indicates clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

14 FIG. 7 Long-term growth of Ba/F3 transfectants by hGM-CSF. Continuously growing cells were harvested and washed with factor-free RPMI 1640 medium supplemented with 10% FCS and resuspended in the same medium containing 0.25 ng/ml mIL-3 (filled symbols) or 3 ng/ml hGM-CSF (open symbols) at 5 × 104 cells/ml at day 0. The number of trypan blue-resistant cells was counted every 24 hours. The vertical and horizontal axes indicate cell number and days of culture, respectively. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. Number sign (#) indicates clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

15 FIG. 7 Long-term growth of Ba/F3 transfectants by hGM-CSF. Continuously growing cells were harvested and washed with factor-free RPMI 1640 medium supplemented with 10% FCS and resuspended in the same medium containing 0.25 ng/ml mIL-3 (filled symbols) or 3 ng/ml hGM-CSF (open symbols) at 5 × 104 cells/ml at day 0. The number of trypan blue-resistant cells was counted every 24 hours. The vertical and horizontal axes indicate cell number and days of culture, respectively. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. Number sign (#) indicates clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

16 FIG. 7 Long-term growth of Ba/F3 transfectants by hGM-CSF. Continuously growing cells were harvested and washed with factor-free RPMI 1640 medium supplemented with 10% FCS and resuspended in the same medium containing 0.25 ng/ml mIL-3 (filled symbols) or 3 ng/ml hGM-CSF (open symbols) at 5 × 104 cells/ml at day 0. The number of trypan blue-resistant cells was counted every 24 hours. The vertical and horizontal axes indicate cell number and days of culture, respectively. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. Number sign (#) indicates clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

17 FIG. 7 Long-term growth of Ba/F3 transfectants by hGM-CSF. Continuously growing cells were harvested and washed with factor-free RPMI 1640 medium supplemented with 10% FCS and resuspended in the same medium containing 0.25 ng/ml mIL-3 (filled symbols) or 3 ng/ml hGM-CSF (open symbols) at 5 × 104 cells/ml at day 0. The number of trypan blue-resistant cells was counted every 24 hours. The vertical and horizontal axes indicate cell number and days of culture, respectively. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. Number sign (#) indicates clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

18 FIG. 8 Dose requirement of hGM-CSF-dependent cell proliferation of Ba/F3 cells. Proliferation of Ba/F3 transfectants was determined by MTT assay after culture for 24 hours in the presence of 0 to 100 ng/ml hGM-CSF. As a control, cells were cultured with 1 ng/ml mIL-3. Horizontal axis indicates concentrations of hGM-CSF, and vertical axis indicates relative MTT reduction value normalized to values for cells incubated with 1 ng/ml mIL-3. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. All values are averages of samples analyzed in triplicate, and standard deviations are shown as error bars. Number sign (#) indicates the clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

19 FIG. 8 Dose requirement of hGM-CSF-dependent cell proliferation of Ba/F3 cells. Proliferation of Ba/F3 transfectants was determined by MTT assay after culture for 24 hours in the presence of 0 to 100 ng/ml hGM-CSF. As a control, cells were cultured with 1 ng/ml mIL-3. Horizontal axis indicates concentrations of hGM-CSF, and vertical axis indicates relative MTT reduction value normalized to values for cells incubated with 1 ng/ml mIL-3. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. All values are averages of samples analyzed in triplicate, and standard deviations are shown as error bars. Number sign (#) indicates the clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

20 FIG. 8 Dose requirement of hGM-CSF-dependent cell proliferation of Ba/F3 cells. Proliferation of Ba/F3 transfectants was determined by MTT assay after culture for 24 hours in the presence of 0 to 100 ng/ml hGM-CSF. As a control, cells were cultured with 1 ng/ml mIL-3. Horizontal axis indicates concentrations of hGM-CSF, and vertical axis indicates relative MTT reduction value normalized to values for cells incubated with 1 ng/ml mIL-3. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. All values are averages of samples analyzed in triplicate, and standard deviations are shown as error bars. Number sign (#) indicates the clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

21 FIG. 8 Dose requirement of hGM-CSF-dependent cell proliferation of Ba/F3 cells. Proliferation of Ba/F3 transfectants was determined by MTT assay after culture for 24 hours in the presence of 0 to 100 ng/ml hGM-CSF. As a control, cells were cultured with 1 ng/ml mIL-3. Horizontal axis indicates concentrations of hGM-CSF, and vertical axis indicates relative MTT reduction value normalized to values for cells incubated with 1 ng/ml mIL-3. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. All values are averages of samples analyzed in triplicate, and standard deviations are shown as error bars. Number sign (#) indicates the clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

22 FIG. 8 Dose requirement of hGM-CSF-dependent cell proliferation of Ba/F3 cells. Proliferation of Ba/F3 transfectants was determined by MTT assay after culture for 24 hours in the presence of 0 to 100 ng/ml hGM-CSF. As a control, cells were cultured with 1 ng/ml mIL-3. Horizontal axis indicates concentrations of hGM-CSF, and vertical axis indicates relative MTT reduction value normalized to values for cells incubated with 1 ng/ml mIL-3. A, Ba/F3-α,β. B, Ba/F3-α/β,β/α. C, Ba/F3-α,β/α. D, Ba/F3-α/β,β. E, Ba/F3-α/β cells. All values are averages of samples analyzed in triplicate, and standard deviations are shown as error bars. Number sign (#) indicates the clone number. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

23 FIG. 9 Induction of tyrosine phosphorylation in response to hGM-CSF in Ba/F3 transfectants. After factor depletion at the concentration of 4 × 106 cells/ml, cultures were additionally incubated for 10 minutes with 1 ng/ml mIL-3 (lane 2), 5 ng/ml hGM-CSF (lanes 3, 5, 7, and 9), or no factor (lanes 1, 4, 6, and 8). The cells were harvested and lysed in Laemmli's solution at 2 × 107 cells/ml. Ten microliters of total cell extracts corresponding to 2 × 105 cells from Ba/F3-α,β (lanes 1 to 3), Ba/F3-α/β,β/α (lanes 4 and 5), Ba/F3-α,β/α (lanes 6 and 7), and Ba/F3-α/β,β (lanes 8 and 9) were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (8%); and tyrosine-phosphorylated proteins were analyzed by Western blotting with anti-phosphotyrosine antibody 4G10. Sizes of proteins are shown in kilodaltons on the left. Positions of hGM-CSF-induced tyrosine-phosphorylated proteins are indicated by arrowheads on the right. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

24 FIG. 10 Structure of GM-CSF and its association with GMR. A, Tertiary structure of GM-CSF. GM-CSF, as well as other cytokines, has four α helices, which are numbered from the N-terminal. B, Association of GM-CSF with GMR. The first and fourth helices associate with the β and α subunits of GMR, respectively. Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

25 FIG. 11 Possible models of the activation mechanism of GMR.
Journal of Allergy and Clinical Immunology  , DOI: ( /S (95) ) Copyright © 1995 Mosby, Inc. Terms and Conditions

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