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Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment.

Ward AC, van Aesch YM, Gits J, Schelen AM, de Koning JP, van Leeuwen D, Freedman MH, Touw IP - J. Exp. Med. (1999)

Bottom Line: Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF).Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment.Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hematology, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands. ward@hema.fgg.eur.nl

ABSTRACT
Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

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Expression of wild-type and P206H mutant G-CSF-Rs in myeloid 32D cells, and characterization of their G-CSF responsiveness. (A) Flow cytometric analysis of G-CSF-R expression on parental 32D.cl8.6 cells (32D) and 32D.cl8.6 cells expressing either wild-type (32D[WT]) or P206H mutant (32D[mAR]) receptors. Cells were either stained with biotinylated mouse anti–human G-CSF-R antibodies followed by PE-conjugated streptavidin, biotinylated antistreptavidin, and finally PE-conjugated streptavidin (open), or without the anti–G-CSF-R step (shaded). (B) G-CSF dose–response of representative 32D[WT] and 32D[mAR] clones in a 24-h thymidine-incorporation assay, as indicated. Data are expressed relative to the response to IL-3.
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Figure 2: Expression of wild-type and P206H mutant G-CSF-Rs in myeloid 32D cells, and characterization of their G-CSF responsiveness. (A) Flow cytometric analysis of G-CSF-R expression on parental 32D.cl8.6 cells (32D) and 32D.cl8.6 cells expressing either wild-type (32D[WT]) or P206H mutant (32D[mAR]) receptors. Cells were either stained with biotinylated mouse anti–human G-CSF-R antibodies followed by PE-conjugated streptavidin, biotinylated antistreptavidin, and finally PE-conjugated streptavidin (open), or without the anti–G-CSF-R step (shaded). (B) G-CSF dose–response of representative 32D[WT] and 32D[mAR] clones in a 24-h thymidine-incorporation assay, as indicated. Data are expressed relative to the response to IL-3.

Mentions: To study the functionality of the P206H mutant G-CSF-R, designated mAR, we replaced the extracellular region of the wild-type G-CSF-R with that of the mutant form. Expression vectors containing the WT and mAR GCSFR cDNAs were introduced into the IL-3–dependent murine myeloid cell line 32D.cl8.6, which does not express endogenous G-CSF-R. Expression levels of the different G-CSF-R proteins in the transfectants were determined by flow cytometry using anti–G-CSF-R antiserum (Fig. 2 A). Several independent clones were obtained expressing approximately equivalent levels of wild-type and P206H mutant receptors (32D[WT] and 32D[mAR], respectively), and used in subsequent analyses. To directly test whether the P206H mutation could contribute to the G-CSF hyporesponsiveness observed in the patient, we analyzed the sensitivity of the WT and mAR clones to G-CSF by measuring DNA synthesis in 3H-TdR incorporation assays in response to titrated doses of cytokine (Fig. 2 B). This revealed a large right shift in dose–response for the 32D[mAR] clones. However, even at maximal G-CSF concentrations, 32D[mAR] clones failed to reach the same level of 3H-TdR incorporation as those expressing the wild-type receptor. These data establish that the P206H mutation causes hyporesponsiveness to G-CSF in myeloid cells.


Novel point mutation in the extracellular domain of the granulocyte colony-stimulating factor (G-CSF) receptor in a case of severe congenital neutropenia hyporesponsive to G-CSF treatment.

Ward AC, van Aesch YM, Gits J, Schelen AM, de Koning JP, van Leeuwen D, Freedman MH, Touw IP - J. Exp. Med. (1999)

Expression of wild-type and P206H mutant G-CSF-Rs in myeloid 32D cells, and characterization of their G-CSF responsiveness. (A) Flow cytometric analysis of G-CSF-R expression on parental 32D.cl8.6 cells (32D) and 32D.cl8.6 cells expressing either wild-type (32D[WT]) or P206H mutant (32D[mAR]) receptors. Cells were either stained with biotinylated mouse anti–human G-CSF-R antibodies followed by PE-conjugated streptavidin, biotinylated antistreptavidin, and finally PE-conjugated streptavidin (open), or without the anti–G-CSF-R step (shaded). (B) G-CSF dose–response of representative 32D[WT] and 32D[mAR] clones in a 24-h thymidine-incorporation assay, as indicated. Data are expressed relative to the response to IL-3.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195597&req=5

Figure 2: Expression of wild-type and P206H mutant G-CSF-Rs in myeloid 32D cells, and characterization of their G-CSF responsiveness. (A) Flow cytometric analysis of G-CSF-R expression on parental 32D.cl8.6 cells (32D) and 32D.cl8.6 cells expressing either wild-type (32D[WT]) or P206H mutant (32D[mAR]) receptors. Cells were either stained with biotinylated mouse anti–human G-CSF-R antibodies followed by PE-conjugated streptavidin, biotinylated antistreptavidin, and finally PE-conjugated streptavidin (open), or without the anti–G-CSF-R step (shaded). (B) G-CSF dose–response of representative 32D[WT] and 32D[mAR] clones in a 24-h thymidine-incorporation assay, as indicated. Data are expressed relative to the response to IL-3.
Mentions: To study the functionality of the P206H mutant G-CSF-R, designated mAR, we replaced the extracellular region of the wild-type G-CSF-R with that of the mutant form. Expression vectors containing the WT and mAR GCSFR cDNAs were introduced into the IL-3–dependent murine myeloid cell line 32D.cl8.6, which does not express endogenous G-CSF-R. Expression levels of the different G-CSF-R proteins in the transfectants were determined by flow cytometry using anti–G-CSF-R antiserum (Fig. 2 A). Several independent clones were obtained expressing approximately equivalent levels of wild-type and P206H mutant receptors (32D[WT] and 32D[mAR], respectively), and used in subsequent analyses. To directly test whether the P206H mutation could contribute to the G-CSF hyporesponsiveness observed in the patient, we analyzed the sensitivity of the WT and mAR clones to G-CSF by measuring DNA synthesis in 3H-TdR incorporation assays in response to titrated doses of cytokine (Fig. 2 B). This revealed a large right shift in dose–response for the 32D[mAR] clones. However, even at maximal G-CSF concentrations, 32D[mAR] clones failed to reach the same level of 3H-TdR incorporation as those expressing the wild-type receptor. These data establish that the P206H mutation causes hyporesponsiveness to G-CSF in myeloid cells.

Bottom Line: Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF).Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment.Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor.

View Article: PubMed Central - PubMed

Affiliation: Institute of Hematology, Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands. ward@hema.fgg.eur.nl

ABSTRACT
Severe congenital neutropenia (SCN) is a heterogeneous condition characterized by a drastic reduction in circulating neutrophils and a maturation arrest of myeloid progenitor cells in the bone marrow. Usually this condition can be successfully treated with granulocyte colony-stimulating factor (G-CSF). Here we describe the identification of a novel point mutation in the extracellular domain of the G-CSF receptor (G-CSF-R) in an SCN patient who failed to respond to G-CSF treatment. When this mutant G-CSF-R was expressed in myeloid cells, it was defective in both proliferation and survival signaling. This correlated with diminished activation of the receptor complex as determined by signal transducer and activator of transcription (STAT) activation, although activation of STAT5 was more affected than STAT3. Interestingly, the mutant receptor showed normal affinity for ligand, but a reduced number of ligand binding sites compared with the wild-type receptor. This suggests that the mutation in the extracellular domain affects ligand-receptor complex formation with severe consequences for intracellular signal transduction. Together these data add to our understanding of the mechanisms of cytokine receptor signaling, emphasize the role of GCSFR mutations in the etiology of SCN, and implicate such mutations in G-CSF hyporesponsiveness.

Show MeSH
Related in: MedlinePlus