Cytoplasmic Domains of the Human Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF) Receptor β Chain (hβc) Responsible for Human GM-CSF-induced Myeloid Cell Differentiation*

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates differentiation, survival, and proliferation of myeloid progenitor cells. The biologic actions of GM-CSF are mediated by its binding to the α and β subunits of the GM-CSF receptor (GM-CSFRα and βc, respectively). To determine whether identical regions of the βc protein mediate both cell growth and differentiation, we expressed cDNA constructs encoding the human wild-type (897 amino acids) and truncated βc (hβc) subunits along with the wild-type human GM-CSFRα subunit in the murine WT19 cell line, an FDC-P1-derived cell line that differentiates toward the monocytic lineage in response to murine GM-CSF. Whereas the WT19 cell line carrying the C-terminal deleted hβc subunit of 627 amino acids was still able to grow in human GM-CSF (hGM-CSF), 681 amino acids of the hβc were necessary for cell differentiation. The addition of hGM-CSF to WT19 cell lines containing the hβc627 subunit stimulated the phosphorylation of ERK (extracellular signal-regulated kinase) and induced the tyrosine-phosphorylation of SHP-2 and STAT5, suggesting that the activation of these molecules is insufficient to mediate the induction of differentiation. A point mutation of tyrosine 628 to phenylalanine (Y628F) within hβc681 abolished the ability of hGM-CSF to induce differentiation. Our results indicate that the signals required for hGM-CSF-induced differentiation and cell growth are mediated by different regions of the hβc subunit.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) 1 is secreted by activated T cells, endothelial cells, fibroblasts, mast cells, B cells, and macrophages and plays an important role in promoting differentiation, survival, and proliferation of colony-forming unit-granulocyte-macrophage (CFU-GM) progenitor cells. GM-CSF also enhances the function of mature neutrophils, monocytes, and eosinophils, and stimulates the burst-promoting activity for BFU-E (1)(2)(3). In addition, in murine models, alveolar proteinosis, a disease caused by excessive secretion of protein into the alveolar spaces, is associated with the deletion of the GM-CSF gene, suggesting that GM-CSF is essential for alveolar macrophage functions (4,5).
The biologic actions of GM-CSF are mediated by its binding to a specific receptor consisting of ␣ and ␤ subunits, both of which are members of the type I cytokine receptor family (6,7). The ␣ subunit (GM-CSFR␣) binds GM-CSF with low affinity (6). The ␤ subunit does not bind GM-CSF itself, but it forms a high affinity receptor in combination with the ␣ subunit (7). The ␤ chain is referred to as the common ␤ chain (␤c) because it is also utilized by interleukin 3 (IL-3) and interleukin 5 (IL-5) (8,9).
We have recently reported that the cytoplasmic domain of the human GM-CSFR␣ (hGM-CSFR␣) is indispensable for cellular differentiation induced by human GM-CSF (hGM-CSF) (10). In addition, the cytoplasmic domain of the human ␤c (h␤c) has recently been demonstrated to be essential for hGM-CSFmediated myeloid cell differentiation (11). However, the exact mechanism of differentiation by GM-CSF has not been clearly elucidated. In the present study, we examined the role of ␤c in GM-CSF-mediated cell differentiation by using the murine WT19 cell line, an FDC-P1-derived cell line that uniformly differentiates toward the monocytic lineage in response to murine GM-CSF (mGM-CSF), but grows without differentiation in the presence of murine IL-3 (mIL-3) (10,12). By expressing the wild-type and mutated h␤c constructs along with the wild-type hGM-CSFR␣ in WT19 cells, we demonstrate the following results. 1) Mutation of the two prolines in the box 1 region of h␤c, which are necessary for Jak2 activation, results in the loss of hGM-CSF-induced cell differentiation. 2) The h␤c C-terminal truncation mutants demonstrate that 681 amino acids of the h␤c are sufficient to mediate hGM-CSF-induced differentiation, but the truncation of the h␤c subunit to amino acid 627 abolishes this differentiation response. In contrast, 559 amino acids of the h␤c are sufficient for hGM-CSF-induced cell growth. 3) Within the h␤c681 subunit, a point mutation of tyrosine 628 to phenylalanine abolishes the ability of h␤c681 to induce differentiation. 4) The activation of either ERK or STAT5 by hGM-CSF does not correlate with the ability of hGM-CSF to induce myeloid cell differentiation. These results demonstrate that hGM-CSF-mediated cell growth and differentiation require distinct regions of the h␤c cytoplasmic domain. MgCl 2 , 1 mM EGTA, 100 mM NaF, 10 mM NaPP i , 1 mM Na 3 VO 4 , 1 mM phenylmethanesulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin) at 10 8 cells/ml. The lysates were separated on SDS-polyacrylamide gels and then electrotransferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked for 2 h in 2% bovine serum albumin-TBST (20 mM Tris-HCl, pH 7.6, 0.15 M sodium chloride, 0.1% Tween 20), incubated with primary antibodies in TBST for 1 h, washed three times with TBST, and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or rabbit immunoglobulin (Amersham Pharmacia Biotech) diluted 1:10,000 in TBST. After three washes in TBST, the blot was developed with the enhanced chemiluminescence system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Immunoprecipitation-The cell lysates were incubated with the indicated antibody for 2 h at 4°C, followed by protein A-Sepharose beads (Amersham Pharmacia Biotech) for an additional 1 h at 4°C. The beads were washed 3 times in PLC lysis buffer, suspended in SDS-sample buffer, and heated at 95°C for 5 min. The eluted proteins were applied to an SDS-polyacrylamide gel and detected by Western blotting.
GST Fusion Protein and Affinity Purification-GST-MycN262 (a generous gift from Dr. E. M. Blackwood, University of California, San Diego, La Jolla, CA) was described previously (15). GST-MycN262 fusion protein was induced with 0.2 mM isopropyl-␤-D-thiogalactopyranoside and purified over glutathione-Sepharose according to the manufacturer recommendations (Amersham Pharmacia Biotech).
Immune Complex Kinase Assay-The cell lysates (1 ϫ 10 7 cells/ sample) were incubated with 0.4 g of polyclonal anti-ERK2 antibody for 2 h at 4°C, followed by protein A-Sepharose beads (Amersham Pharmacia Biotech) for an additional 1 h. The beads were washed 3 times in PLC lysis buffer and then once in kinase buffer (20 mM HEPES, pH7.9, 10 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM sodium vanadate, 10 mM ␤-glycerophosphate, and 1 mM dithiothreitol). The kinase reaction was initiated by the addition of 40 l of kinase buffer with 20 M ATP, 5 Ci of [␥-32 P]ATP, and 5 g of GST-MycN262 and was allowed to proceed for 15 min at 30°C. The reaction was terminated by the addition of 2ϫ SDS sample buffer. Samples were boiled and resolved by SDS-PAGE, and the fixed gel was exposed to an x-ray film and the PhosphorImager (Molecular Dynamics).

Expression of Wild-type and Mutant h␤c in WT19 Cells-To
study the role of h␤c in hGM-CSF-mediated differentiation, we constructed a series of cytoplasmic deletion and point mutants of the h␤c subunit (Fig. 1A). These mutant h␤c subunits were transfected into WT19 cells expressing the wild-type hGM-CSFR␣ subunit (Fig. 1, B and C). The amino acid length of each h␤c mutant represents the number of amino acids from the N terminus including the leader sequence. As evaluated by Western blotting and flow-cytometry, the expression level of each mutant h␤c subunit in these clonal cell lines (Fig. 2, A and B) was found to be similar. The mutations introduced into the h␤c cytoplasmic domain did not affect the high affinity ligand binding as reported by others (16) (data not shown). At least three clones were isolated expressing each h␤c mutant and were analyzed in the following experiments (see below).
Induction of Monocytic Differentiation through h␤c in WT19 Cells-Parental WT19 cells growing in mIL-3 demonstrated a myeloblastic morphology including rounded nuclei, fine chromatin, and basophilic cytoplasm. In response to mGM-CSF, the cells demonstrated monocytic characteristics: an indented nucleus with fine stranded appearance, increased cytoplasm containing a variable number of vacuoles, and larger total cell size. WT-19 cells treated with mGM-CSF demonstrated significantly increased surface expression of F4/80, a marker of monocyte differentiation (17). As previously reported (10), hGM-CSF treatment of WT19␣␤wt cells expressing hGM-CSFR␣ and the wild-type h␤c subunits induced monocytic differentiation of WT19 cells, as measured by changes in morphology and the surface expression of F4/80 (Fig. 3, A and B).
The type I cytokine receptors share amino acid sequence homology in a membrane proximal region called the box 1 region (18,19). The box 1 region contains conserved prolines that are essential for Jak2 activation by various cytokines (20). To test whether these prolines are necessary for hGM-CSFinduced differentiation, they (Pro-479 and -481) were mutated to alanines and this receptor (h␤cPA) was expressed along with the wild-type hGM-CSFR␣ in WT19 cells. The addition of hGM-CSF to this cell line (WT19␣␤PA) failed to induce differentiation as measured by morphology and the lack of induction of F4/80 expression (Fig. 3, A and B), indicating that the prolines of the box 1 region are essential for hGM-CSF-mediated differentiation.
To determine which regions of h␤c subunit are required for hGM-CSF-mediated cell differentiation and growth, additional truncation mutants were generated, and stable cell lines were isolated. Clear morphological differentiation was observed in WT19 cell lines expressing h␤c subunits of 832, 796, or 681 amino acids in length when stimulated by hGM-CSF (Fig. 3A, Table I). In these cell lines, hGM-CSF induced the increase of F4/80 surface expression (Fig. 3B, Table I). In contrast, WT19 cells expressing h␤c subunit containing 627 or fewer amino acids did not exhibit morphological differentiation or increased F4/80 expression in response to hGM-CSF (Fig. 3, Table I). These results suggest that the region between amino acids 627 and 681 of h␤c is essential for hGM-CSF-induced cell differentiation of WT19 cells.
As phosphorylated tyrosines are important for the induction of cell growth and differentiation by several growth factor receptors (12,(21)(22)(23), the possibility that tyrosine residues of h␤c681 were involved in the differentiation signal was examined next. Only one tyrosine (Tyr-628) is found between amino acids 627 and 681 of h␤c subunit. To determine whether Tyr-628 is involved in differentiation signals, it was mutated to phenylalanine in the truncated h␤c681. First, to examine if Tyr-628 of h␤c is phosphorylated in response to hGM-CSF in WT19 cells, WT19␣␤681 and WT19␣␤681Y628F cells were stimulated with hGM-CSF, and the immunoprecipitated h␤c was probed with either the anti-phosphotyrosine antibody or the anti-h␤c antibody (Fig. 4). The anti-h␤c immunoblot demonstrates that the expression levels of the two receptors in WT19 cells was similar, whereas the anti-phosphotyrosine immunoblot demonstrates that the hGM-CSF-mediated tyrosine phosphorylation of h␤c681Y628F was 10% of that of h␤c681. Therefore, Tyr-628 is the major tyrosine-phosphorylated site within the h␤c681 subunit.
Addition of hGM-CSF to clonal cell lines expressing this mutant, h␤c681Y628F, failed to induce monocytic differentiation in response to hGM-CSF (Fig. 3, A and B). In comparison, when this identical point mutation was introduced into the full-length h␤c subunit, h␤cY628F, and expressed in WT19 cells, the cells differentiated normally in response to hGM-CSF (Fig. 3 A and B). These findings demonstrated that although Tyr-628 plays an important role in hGM-CSF-mediated cell differentiation in the truncated 681 amino acid receptor, the C-terminal region of the wild-type h␤c may transmit a similar differentiation signal.
Proliferation and Differentiation Signals Are Mediated by Different Regions of h␤c-GM-CSF regulates both cell differentiation and proliferation of myeloid progenitor cells. To inves-tigate whether differentiation and proliferation signals are generated by different or identical regions of the h␤c subunit, the growth of each of the above mutants was examined at low concentrations (1-40 pg/ml) of hGM-CSF (Fig. 5). The growth response to hGM-CSF of WT19 cells containing h␤c796 was similar to ␣␤wt cells. Cells containing the h␤c627, h␤c681, and h␤c681Y628F transfectants grew at a reduced rate when compared with ␣␤wt cells incubated with comparable concentrations of hGM-CSF. Similar results were obtained with WT19 cells containing h␤c559 and 592 (data not shown). However, ␣␤PA cells did not show any growth response to hGM-CSF (data not shown). Therefore, hGM-CSF-mediated signals for proliferation and differentiation are controlled by different regions of the h␤c subunit.
Activation of the MAP Kinase Pathway Is Not Sufficient to Stimulate hGM-CSF-induced Differentiation-To determine which hGM-CSF-induced signals are important for growth and/or differentiation of WT19 cells, the signaling pathways activated by hGM-CSF were examined. Shc and SHP-2, when phosphorylated on tyrosines by GM-CSF stimulation, bind to the SH2-containing Grb2 protein (24,25) and mediate the activation of the Ras-ERK pathway. hGM-CSF-induced tyrosine-phosphorylation of Shc, and SHP-2 was equivalent in WT19␣␤wt, ␣␤681, ␣␤681Y628F and ␣␤627 cell lines, suggesting that these proteins are similarly regulated in cells that are or are not capable of undergoing hGM-CSF-induced differentiation (Fig. 6, A and B). In contrast, cells that contain the box 1 proline mutant (h␤cPA) were incapable of both Shc and SHP-2 phosphorylation (Fig. 6, A and B).
ERK phosphorylation, a downstream event of Shc and SHP-2 phosphorylation, was examined in WT19␣␤ cells by immunoblotting with an antibody specifically raised against phosphorylated forms of ERKs (Fig. 6C). hGM-CSF addition to these WT19 clones induced ERK phosphorylation in ␣␤681Y628F and ␣␤627 cells to a level similar to that seen in ␣␤wt and ␣␤681 cells (Fig. 6C). ERK phosphorylation was not seen in clones containing h␤c473 or h␤cPA, which has two prolines in the box 1 domain replaced with alanines. As the phosphorylation of tyrosine residues in the activation loop of ERKs occurs with the kinase activation of these proteins (26), these data suggest that activation of ERKs does not correlate with hGM-CSF-induced differentiation.
The role of ERKs in the induction of cell differentiation is controversial (23,(27)(28)(29)(30). WT19 cells containing a C-terminal truncation mutant of hGM-CSFR␣ subunit and the wild-type h␤c subunit, hGM-CSF were capable of differentiation, but not ERK activation, in response to hGM-CSF (10). To further examine the role of the ERK pathway in the induction of differentiation, WT19 cells were treated with PD98059, a specific inhibitor of (MEK), prior to the addition of mGM-CSF. Although PD98059 addition to mGM-CSF-treated WT19 cells caused a 3-fold decrease in the activity of immunoprecipitated ERKs to phosphorylate GST-myc in vitro (Fig. 7A), this compound had no effect on the mGM-CSF-mediated differentiation of WT19 cells, as evidenced by the induction of F4/80 and morphologic changes (Fig. 7, B and C). Although the possibility that a small amount of ERK activity persists after PD98059 treatment in vivo cannot be ruled out by this experiment, these results suggest that full activation of the ERK pathway is neither sufficient nor necessary for GM-CSF-induced differentiation of WT19 cells. Tyrosine Phosphorylation of STAT5 Proteins Is Not Sufficient to Mediate hGM-CSF-induced Differentiation-An important substrate of the Jak2 proteins whose tyrosine phosphorylation is induced by the addition of hGM-CSF is STAT5 (31). To determine whether STAT5 activation is sufficient to induce the differentiated phenotype, tyrosine phosphorylation of STAT5 proteins by hGM-CSF in the mutant h␤c cell lines was examined. Stat5 consists of two proteins, Stat5a and Stat5b, encoded by separate genes, both of which have been shown to be activated by GM-CSF (31). To investigate the hGM-CSF-mediated activation of Stat5 proteins, immunoprecipitations were performed using specific antibodies against Stat5a or 5b from hGM-CSF-stimulated cell lysates followed by immunoblotting with the anti-phosphotyrosine antibody. Both Stat5a and 5b were phosphorylated on tyrosine after hGM-CSF treatment in WT19␣␤wt and 681 cells, which respond to hGM-CSF with differentiation, and in WT19␣␤627 and ␣␤681Y628F cells, which do not differentiate (Fig. 8). These results suggest that the activation of Stat5 proteins is not sufficient to induce differentiation of WT19 cells. DISCUSSION By expressing truncated forms of h␤c in WT19 cells, we demonstrated that hGM-CSF-induced morphological differentiation and the surface expression of F4/80 requires h␤c subunits of 832, 796, or 681 amino acids in length (Fig. 3, Table I). In contrast, WT19 cells expressing h␤c subunit containing 627 amino acids or less did not differentiate but were capable of growth in the presence of hGM-CSF (Fig. 3). Mutation of the two prolines in the box 1 region blocked both growth and differentiation. Thus, these results suggest that cell growth and differentiation are mediated by different regions of the receptor and that the signals generated by the box 1 region of the receptor are necessary for both cellular events. Similar results to those described above have been generated using M1 and WEHI-3B Dϩ cells as a target cell population for transfection (11). In this study, wild-type h␤c and h␤c783, but not h␤c541 or 461, could mediate hGM-CSF-induced morphological change and differentiation marker induction, whereas induction of cell differentiation by h␤c626 required a 50-to 100fold higher concentration of hGM-CSF (M1 cells). However, in the WT19 cell system, neither h␤c627 nor h␤c681Y628F could induce cell differentiation even at a very high concentration (1 g/ml) of hGM-CSF (data not shown). The results derived from M1 cells suggest that the region between amino acids 627 and 783 is necessary for the full differentiation signal, whereas our results more narrowly delineate the crucial region for differentiation to lie between 628 and 681.
The results described here demonstrate that tyrosine 628 is   highly phosphorylated after hGM-CSF treatment in vivo (Fig.  4). In the h␤c681 construct, this phosphotyrosine is essential for differentiation. Tyr-627 and its surrounding amino acids are conserved between human and mouse ␤c (SLEYLCL), suggesting that in both species this tyrosine may play an important role in differentiation. In the macrophage colony stimulating factor signaling, both Tyr-807 and Tyr-721 of macrophage colony stimulating factor receptor were shown to be necessary for monocytic differentiation and for the tyrosine phosphorylation of phospholipase C-␥ (PLC␥) (12,22). The involvement of PLC␥ in M-CSF-mediated cell differentiation was further demonstrated by the inhibition of differentiation using U-73122, a compound that acts as a specific inhibitor of PLC␥ (22). The surrounding amino acid sequence of h␤c Tyr-628 (YLCL) is homologous to the optimal binding site for the N-terminal SH2 domain of PLC␥ in a phosphopeptide binding assay (32). However, PLC␥ may not be involved in GM-CSF-induced differentiation as suggested by the following data. First, tyrosine phosphorylation of PLC␥ was not induced by addition of hGM-CSF to WT19 cells (data not shown) or another myeloid cell line treated with this hormone (33), although M-CSF induced significant tyrosine phosphorylation of PLC␥. Second, U-73122 did not inhibit the differentiation of WT19 cells by mGM-CSF at 1 M, a concentration 10 times higher than that capable of inhibiting the M-CSF-mediated differentiation (22) (data not shown).
Although the single tyrosine mutation in the 681 h␤c containing cell line had significant biologic effects, this mutation had no significant effect on differentiation in the wild-type receptor. The wild-type h␤c contains four tyrosines C-terminal to Tyr-628. One of them (Tyr-882) has a similar amino acid sequence (YLSL) to Tyr-628 (YLCL). Two of them (Tyr-766 and Tyr-842) have a leucine at the ϩ3 amino acid (YVEL and YCFL, respectively). The amino acid at ϩ3 position of the tyrosine is important in dictating the binding affinity for many SH2 domain-containing proteins (32). It is possible that a common signaling molecule binds to a number of tyrosines in the h␤c-mediating cell differentiation so that, in the wild-type receptor, mutation of a single tyrosine is not sufficient to abolish cell differentiation.
The role of ERKs in differentiation appears to be complex and may depend on the cell type and the stimulus involved. The ERK pathway has been suggested to be essential for the neurite outgrowth of PC12 cells (27,34). Also, interleukin 6 (IL-6) can induce PC12 differentiation when the cells are pretreated with nerve growth factor (35). A mutation of a specific tyrosine in gp130, the IL-6 receptor signaling subunit involved in ERK activation, abolishes the induction of cell differentiation by IL-6 (23). Our results suggest that the ERK pathway may not be involved in hGM-CSF-mediated differentiation of WT19 cells. 1) Although Tyr-628 of h␤c was shown to be a binding site for SHP-2 SH2 domains (36), it was not necessary for hGM-CSFinduced tyrosine phosphorylation of SHP-2 or ERK activation (Fig. 6) (36). 2) PD98059, a specific inhibitor of MEK, blocked activation, but this compound had no significant effect on differentiation.
3) The expression of a constitutively active mutant of MEK in WT19 cells did not induce differentiation. 2 Thus, activation of ERK does not appear to be sufficient or necessary for hGM-CSF-induced differentiation of WT19 cells.
Activation of Stat proteins by several cytokines has been suggested to be involved in cell differentiation (37)(38)(39)(40)(41)(42). In WT19 cells transfected with hGM-CSFR␣ and wild-type h␤c, hGM-CSF induced tyrosine phosphorylation of both isoforms of Stat5, but not other Stat proteins (Stat1, Stat3, and Stat6 examined). Tyr-628 was not necessary for the hGM-CSF-mediated tyrosine phosphorylation of either Stat5a or 5b. In addition, both h␤c628 and h␤c681Y628F could induce tyrosine phosphorylation of Stat5a and 5b in response to hGM-CSF equivalent to that seen in WT19 cells containing the wild-type receptor (Fig. 8). These results suggest that Stat5 activation is not sufficient for the induction of cellular differentiation. In a recent report, using chimeric receptors of erythropoietin and IL-3, a delayed activation of Stat5 was suggested to be important for the erythroid differentiation induction of BaF/3 cells (43). However, we did not see a delay in the time course of Stat5 activation in WT19 transfectants which differentiate in response to hGM-CSF in contrast to those cell lines which are capable of only growth (data not shown). Thus, none of the signals examined (STAT5 activation along with SHP-2 and Shc phosphorylation) are sufficient to modulate differentiation.
In conclusion, our data suggest that cell differentiation and cell growth induced by hGM-CSF are mediated by different regions of the h␤c subunit. Attempts are underway to define the novel signals generated by amino acids 627-681 of the h␤c which control the differentiated phenotype.