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

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.

Figure 1

Identification of a point mutation in the GCSFR cDNA of an SCN patient hyporesponsive to G-CSF. (A) Partial nucleotide sequence of the mutant and wild-type GCSFR cDNA, with the relative position of the corresponding P206H mutation in the encoded G-CSF-R indicated. (B) Analysis of fibroblast chromosomal DNA for the presence of the mutation. The relevant genomic region of the GCSFR gene is presented (top), along with the relative positions of the primers used for PCR (GRFWI6 and GRRV15). The BsiHkAI restriction sites are also indicated, with the new site introduced by the mAR mutation shown in parentheses. Digestion of a wild-type allele predictively generates two fragments, a and b, whereas digestion of a mutant allele generates three fragments, a′, a′′, and b, as indicated. At the bottom are the results of BsiHkAI digestion of PCR products generated from either the patient (AR) or a normal individual (WT).

Figure 1

Identification of a point mutation in the GCSFR cDNA of an SCN patient hyporesponsive to G-CSF. (A) Partial nucleotide sequence of the mutant and wild-type GCSFR cDNA, with the relative position of the corresponding P206H mutation in the encoded G-CSF-R indicated. (B) Analysis of fibroblast chromosomal DNA for the presence of the mutation. The relevant genomic region of the GCSFR gene is presented (top), along with the relative positions of the primers used for PCR (GRFWI6 and GRRV15). The BsiHkAI restriction sites are also indicated, with the new site introduced by the mAR mutation shown in parentheses. Digestion of a wild-type allele predictively generates two fragments, a and b, whereas digestion of a mutant allele generates three fragments, a′, a′′, and b, as indicated. At the bottom are the results of BsiHkAI digestion of PCR products generated from either the patient (AR) or a normal individual (WT).

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.

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.

Figure 3

Effect of the P206H mutation on G-CSF–mediated growth and differentiation. (A) Cell proliferation of 32D[WT] and 32D[mAR] cells on 100 ng/ml G-CSF, as indicated. Data represent the mean growth of three clones. (B) Morphological features of representative 32D[WT] cells in the presence of IL-3, and 32D[WT] or 32D[mAR] cells after 7 d exposure to G-CSF, as indicated. For the 32D[mAR] clone, the positions of an apoptotic cell and a differentiated cell are indicated with a light and bold arrow, respectively.

Figure 3

Effect of the P206H mutation on G-CSF–mediated growth and differentiation. (A) Cell proliferation of 32D[WT] and 32D[mAR] cells on 100 ng/ml G-CSF, as indicated. Data represent the mean growth of three clones. (B) Morphological features of representative 32D[WT] cells in the presence of IL-3, and 32D[WT] or 32D[mAR] cells after 7 d exposure to G-CSF, as indicated. For the 32D[mAR] clone, the positions of an apoptotic cell and a differentiated cell are indicated with a light and bold arrow, respectively.

Figure 4

Effect of P206H mutation on G-CSF–mediated STAT activation. (A) Kinetics of STAT activation in 32D.cl8.6 cells expressing wild-type G-CSF-R (WT) or P206H mutant (mAR) receptor. Serum- and growth factor–deprived cells were incubated at 37°C without factor or with 100 ng/ml G-CSF for the times indicated. Nuclear extracts were prepared and incubated with ³²P-labeled double-stranded m67 or β-cas oligonucleotides, as indicated. The relative position and identity of STAT complexes are shown: S1, STAT1; S3, STAT3; S5, STAT5. (B) G-CSF dose–response of STAT activation in 32D[WT] and 32D[mAR] cells as described in A, except after stimulation for 15 min with the indicated concentrations of G-CSF.

Figure 4

Effect of P206H mutation on G-CSF–mediated STAT activation. (A) Kinetics of STAT activation in 32D.cl8.6 cells expressing wild-type G-CSF-R (WT) or P206H mutant (mAR) receptor. Serum- and growth factor–deprived cells were incubated at 37°C without factor or with 100 ng/ml G-CSF for the times indicated. Nuclear extracts were prepared and incubated with ³²P-labeled double-stranded m67 or β-cas oligonucleotides, as indicated. The relative position and identity of STAT complexes are shown: S1, STAT1; S3, STAT3; S5, STAT5. (B) G-CSF dose–response of STAT activation in 32D[WT] and 32D[mAR] cells as described in A, except after stimulation for 15 min with the indicated concentrations of G-CSF.

Figure 5

Binding of G-CSF to wild-type and P206H mutant receptors. FACS^® analysis of 32D parental (light gray shaded, dotted line), 32D[WT] (dark gray shaded, no line), or 32D[mAR] (open, bold line) clones with either α–G-CSF-R antiserum or biotinylated G-CSF, as indicated.

Figure 6

Analysis of signaling from the wild-type G-CSF-R at saturating and nonsaturating G-CSF concentrations. (A) Growth of 32D[WT] cells at either 100 or 0.3 ng/ml G-CSF, as indicated. (B) STAT activation in 32D[WT] cells at either 100 or 0.3 ng/ml G-CSF, as indicated, performed as described in the legend to Fig. 4 A.

Figure 6

Analysis of signaling from the wild-type G-CSF-R at saturating and nonsaturating G-CSF concentrations. (A) Growth of 32D[WT] cells at either 100 or 0.3 ng/ml G-CSF, as indicated. (B) STAT activation in 32D[WT] cells at either 100 or 0.3 ng/ml G-CSF, as indicated, performed as described in the legend to Fig. 4 A.

Figure 7

Analysis of 32D cells coexpressing wild-type and mutant receptors. (A) FACS^® analysis of 32D[WT^Neo], 32D[WT^Neo/WT^Puro], and 32D[WT^Neo/mAR^Puro] cells with α–G-CSF-R antiserum, as described in the legend to Fig. 2 A. In the bottom panels, the dotted line represents the G-CSF-R expression level of the original 32D[WT^Neo] clone. (B) Growth of 32D[WT^Neo/WT^Puro] cells (filled circles) or 32D[WT^Neo/mAR^Puro] cells (filled triangles) at G-CSF concentrations of either 100 ng/ml (solid line) or 1 ng/ml (dashed line). The growth curve of the original 32D[WT^Neo] clone at 100 ng/ml G-CSF is included for comparison (open squares, dotted line). (C) G-CSF–induced STAT5 activation in 32D[WT^Neo/WT^Puro] cells (WT) or 32D[WT^Neo/mAR^Puro] cells (mAR), as described in the legend to Fig. 4 B.

Figure 7

Analysis of 32D cells coexpressing wild-type and mutant receptors. (A) FACS^® analysis of 32D[WT^Neo], 32D[WT^Neo/WT^Puro], and 32D[WT^Neo/mAR^Puro] cells with α–G-CSF-R antiserum, as described in the legend to Fig. 2 A. In the bottom panels, the dotted line represents the G-CSF-R expression level of the original 32D[WT^Neo] clone. (B) Growth of 32D[WT^Neo/WT^Puro] cells (filled circles) or 32D[WT^Neo/mAR^Puro] cells (filled triangles) at G-CSF concentrations of either 100 ng/ml (solid line) or 1 ng/ml (dashed line). The growth curve of the original 32D[WT^Neo] clone at 100 ng/ml G-CSF is included for comparison (open squares, dotted line). (C) G-CSF–induced STAT5 activation in 32D[WT^Neo/WT^Puro] cells (WT) or 32D[WT^Neo/mAR^Puro] cells (mAR), as described in the legend to Fig. 4 B.

Figure 7

Analysis of 32D cells coexpressing wild-type and mutant receptors. (A) FACS^® analysis of 32D[WT^Neo], 32D[WT^Neo/WT^Puro], and 32D[WT^Neo/mAR^Puro] cells with α–G-CSF-R antiserum, as described in the legend to Fig. 2 A. In the bottom panels, the dotted line represents the G-CSF-R expression level of the original 32D[WT^Neo] clone. (B) Growth of 32D[WT^Neo/WT^Puro] cells (filled circles) or 32D[WT^Neo/mAR^Puro] cells (filled triangles) at G-CSF concentrations of either 100 ng/ml (solid line) or 1 ng/ml (dashed line). The growth curve of the original 32D[WT^Neo] clone at 100 ng/ml G-CSF is included for comparison (open squares, dotted line). (C) G-CSF–induced STAT5 activation in 32D[WT^Neo/WT^Puro] cells (WT) or 32D[WT^Neo/mAR^Puro] cells (mAR), as described in the legend to Fig. 4 B.

Figure 8

Model for receptor–ligand complexes formed with WT and mAR (P206H) mutant G-CSF-Rs.