The f3c Component of the Granulocyte- Macrophage Colony-Stimulating Factor (GM-CSF)/ 3 (IL-3)/IL-5 Interacts with a Hybrid GM- CSF/ Receptor to Influence Proliferation and 3-Globmi mRNA Expression

Paul T. Jubinsky, Yayoi Shikama, Andrew Laurie, David G. Nathan, Martin Carroll, and Colin A. Sieff Divisions of Pediatric Hematology and Oncology, Dana-Farber Cancer Institute and Children's Hospital, Boston, Massachusetts, U.S.A. Department of Hematology and Oncology, Beth Israel Hospital, Boston, Massachusetts, U.S.A. Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, U.S.A.

ABSTRACT Background: The interaction of different members of Results: Scatchard analysis showed that both GMER + the hematopoietic family may be (c and GMER + (3tr bound hGM-CSF with high affinity relevant to the increased proliferation and the failure of (Kd 40 pM to 65 pM). Proliferation assays showed that differentiation that characterizes the myeloid leukemias. the maximum growth of cells expressing GMER + /3c We recently demonstrated that a chimeric receptor was identical to that of cells with GMER alone. However, (GMER) that is composed of the extracellular and trans- proliferation of the cells that expressed GMER + ftr was membrane domains of the human granulocyte-macro- reduced by 80-95% of GMER. Dose-response curves phage colony-stimulating factor (GM-CSF) receptor a- showed that the concentration of GM-CSF required for chain (GMRa) and the cytoplasmic domain of the mu- half-maximal growth was 0.5-5.0 pM for GMER + j3c rine erythropoietin receptor mEpoR binds hGM-CSF and 0.5-5 nM for GMER and GMER + f3tr. The EpoR with low affinity (3 nM) and confers both proliferative cytoplasmic domain of GMER also undergoes ligand- and differentiation signals to stably transfected murine inducible tyrosine phosphorylation. However, the ty- Ba/F3 cells. rosine phosphorylation did not correlate with growth in Materials and Methods: To investigate whether the cells expressing (3tr. Coexpression of (3c with GMER in common (3-subunit of the GM-CSF receptor (3c) can in- Ba/F3 cells grown in hGM-CSF markedly enhanced teract with GMER, either the entire (3-subunit or a mu- f3-globin mRNA expression. tant, truncated (3-subunit that completely lacks the cy- Conclusions: These results indicate that ,Bc can trans- toplasmic domain (f3tr) was introduced into Ba/F3 cells duce a unique signal in association with GMER to influ- that express GMER, and the binding of GM-CSF as well ence both proliferative and differentiation signal path- as proliferation and differentiation responses were mea- ways. sured.

INTRODUCTION several hematopoietic growth factors (HGFs). For The process by which hematopoietic stem cells example, erythroid progenitors require interleu- differentiate along one or another specific cell- kin 3 (IL- 3) or granulocyte-macrophage colony- lineage involves the action of a combination of stimulating factor (GM-CSF) and Steel factor during the early phases of their growth, while Address correspondence and reprint requests to: Paul T. later during differentiation erythropoietin (Epo) Jubinsky, Dana-Farber Cancer Institute, 44 Binney Street, is required for the developing erythroid cells to Boston, MA 02115, U.S.A.

766 Copyright © 1996, Molecular Medicine, 1076-1551/96/$10.50/0 Molecular Medicine, Volume 2, Number 6, November 1996 766-773 P. T. Jubinsky et al.: 8c, GMER, Proliferation, and f3-Globin Expression 767 proliferate and mature into hemoglobin-contain- lular and transmembrane domain of the hGM- ing erythrocytes (1,2). All of these HGFs act by CSF receptor a-chain ligated to the complete binding to specific receptors on the cell surface. cytoplasmic tail of the mEpoR, was transfected The GM-CSF receptor (GMR) is comprised of a into Ba/F3 cells as previously described (1). unique a-chain (GMRa) (3,4) that binds its li- Ba/F3 GMER cells were then electroporated with gand with low affinity and a common (3-chain either f3c or ,Btr along with the hygromycin re- (also shared with the IL-3 and IL-5 receptors) sistance (8), or with (3c or ,3tr in the pREP-4 that does not bind ligand alone but is essential for vector (Invitrogen, San Diego, CA, U.S.A.) that high-affinity binding (5). The first step in cell carries the hygromycin resistance gene. These activation is the ligand-induced heterodimeriza- cells were maintained in the 800 ,ug/ml hygro- tion of the GMRa and 13-subunits (6). The cyto- mycin (Calbiochem, La Jolla, CA, U.S.A.). Ba/F3 plasmic domains of both subunits appear to be cells that express GMRa2 plus fc (Ba/F3-GMR + essential for GM-CSF-stimulated proliferation ,3c) were obtained as previously described (8). (7), although the proliferative role of the cyto- All of these murine cell lines are factor depen- plasmic domain of GMRa is still uncertain (8). dent and were grown in RPMI medium supple- To determine if the cytoplasmic region of an mented with glutamine and 10% fetal calf serum HGF receptor can transmit a specific proliferative (FCS; Sigma Chemical Co., St. Louis, MO, and differentiative signal, we stably expressed a U.S.A.). This medium was supplemented with hybrid receptor, GMER (9), which consists of the purified recombinant mIL-3 (kindly provided by extracellular and transmembrane regions of Steve Gillis, Immunex, Seattle, WA, U.S.A.) for hGMRa and the cytoplasmic domain of the mu- the Ba/F3 wt cells and Ba/F3 cells transfected rine (m) EpoR into Ba/F3 cells (10), a murine with GMER + (3tr, 3 U/ml purified recombinant IL-3-dependent cell with erythroid features human Epo (R & D Systems) for Ba/F3-EpoR (11). We found that these cells proliferated in cells, or 1 nM purified recombinant Chinese hGM-CSF and expressed increased levels of cell hamster ovary (CHO) hGM-CSF (kindly pro- surface glycophorin, an early erythroid marker vided by Steven Clark, Genetics Institute, Cam- (9). Control cells that expressed hGMRa + ,3c bridge, MA, U.S.A.) for cells that expressed proliferated in response to GM-CSF but did not GMER, or 10 pM hGM-CSF for cells that ex- express glycophorin, thus establishing a role for pressed GMER + ,3c or GMRa + f3c. the cytoplasmic region of the hybrid receptor in the glycophorin response. Since IL-3 and GM- CSF have a synergistic role with Epo in erythro- Immunoprecipitation poiesis, we stably expressed either f3c or a cyto- Equal numbers of parental Ba/F3 wt and plasmically truncated f3c (,Btr) in Ba/F3 cells that transfected cells were washed and incubated in contained GMER to determine if f3c could affect methionine- and cysteine-free RPMI medium GMER signaling. We show here that O3c increases supplemented with 2% FCS for 1 hr, after which the binding affinity and proliferative response of 200 ,tCi/ml 35S methionine/cysteine (New En- GMER to hGM-CSF. In addition, coexpression of gland Nuclear, Boston, MA, U.S.A.) was added GMER with ,Bc markedly increases 13-globin and the cells were incubated for a further 5-6 hr mRNA in GMER cells stimulated with hGM-CSF. at 370C. The metabolically labeled cells were In contrast, despite high-affinity binding, expres- stimulated with mIL-3 or hGM-CSF at 1 nM for sion of ,Btr acts as a dominant negative on cell 10 min, washed three times with phosphate- proliferation without leading to increased f3-glo- buffered saline (PBS), containing phosphatase bin mRNA levels. inhibitors (10 mM ,B glycerophosphate, 2 mM sodium pyrophosphate, 1 mM NaF, 1 mM EDTA, and 1 mM sodium orthovanadate, all from Sigma) and solubilized in lysis buffer that con- MATERIALS AND METHODS tained 1% NP40 (Sigma), 150 mM NaCl, 50 mM Tris, pH 8.0, with protease inhibitors (10 ,g/ml Cell Lines and Cultures leupeptin, 0.2 U/ml aprotinin, 10 ,ug/ml leupep- Ba/F3 wild-type cells (Ba/F3 wt) and Ba/F3 tin, and 2 mM phenylmethylsulfonyl fluoride, all cells that express the mEpoR (Ba/F3-mEpo) from Sigma) and phosphatase inhibitors (as were a gift of A. D'Andrea (Dana-Farber Cancer above). After centrifugation (168,000 X g for 15 Institute, Boston, MA, U.S.A.) (12). GMER, a min at 4°C), the supernatants were incubated hybrid receptor consisting of the entire extracel- with monoclonal antibody to phosphotyrosine 768 Molecular Medicine, Volume 2, Number 6, November 1996

(4G10, kindly provided by Brian Druker, Oregon GM-CSF at 40C for 18 hr. The cells were then Health Sciences University, Portland, OR, U.S.A.) centrifuged through 100% FCS with 0.04% so- overnight at 4°C. A sepharose beads dium azide at 4°C and the cell pellets counted in a were added (25 ,lI packed volume), and after 1.5 -y counter (Packard Instrument Company, Meri- hr at 40C the beads were washed three times in den, CT, U.S.A.). Nonspecific binding, which was lysis buffer. For the double immunoprecipitation never more than 25% of total binding, was deter- experiments the bound were eluted by mined by counting the pellet from cells that were boiling for 2 min in solubilization buffer (50 mM incubated in 150-fold excess of unlabeled GM-CSF triethanolamine, 0.4% sodium dodecyl sulfate for 30 min before the addition of labeled factor. The (SDS), 100 mM NaCl, 2 mM EDTA, and 2 mM binding data were subjected to Scatchard analysis mercaptoethanol) with the subsequent addition using the LIGAND program (15). of iodoacetamide and Triton X-100, added to final concentrations of 10 ,uM and 10%, respec- Thymidine Incorporation tively. The eluates were then incubated with rab- bit polyclonal antibody to the C terminus of ,3c Ba/F3 wt cells and cells that expressed for 4 hr at 4°C. This antibody specifically detects GMER or GMRa + /3c or f3tr were washed three f3c by immunoprecipitation and Western blot (13). times and plated at 3 x 104 cells/well in triplicate Bound proteins were collected with protein A in 160 ,ul of the appropriate media in microtiter sepharose as above, eluted by boiling in Laemmli wells for 72 hr at 37°C in a 5% CO2 incubator. sample buffer that contained 5% ,B-mercapto- Three hours and 15 min before harvesting, 1 ,uCi ethanol, and subjected to SDS polyacrylamide gel of [3H]thymidine (New England Nuclear) was electrophoresis (7-15% acrylamide gradient) added to each well. Cells were harvested on filter and autoradiography after treatment with auto- paper by an automated harvester (Skatron, radiographic enhancer (New England Nuclear). Tranby, Norway) and counted in a ,3 counter Exposure times were 5-14 days. For immuno- (Wallac, Gaithersburg, MD, U.S.A.). blotting, the primary antiphosphotyrosine/pro- tein A beads were boiled in Laemmli sample Northern Hybridization buffer with 5% ,B-mercaptoethanol, and the elu- RNA was prepared by guanidinium/CsCl ates were subjected to PAGE, as above. The sep- method (16). Ten micrograms per lane were arated proteins were then transferred to nylon loaded on a 1% agarose-formaldehyde gel and membrane (Hoefer, San Francisco, CA, U.S.A.), RNA was separated by electrophoresis at 150 V for immunoblotted with a 1:50 dilution of rabbit 3 hr. RNA was transferred by capillary transfer polyclonal antibodies to the mEpoR C-terminus overnight using 20X SSC to Duralon (Stratagene, (14) (gift of Alan D'Andrea, DFCI), and washed, La Jolla, CA, U.S.A.) nylon membrane. RNA was and the bound antibodies were detected with cross-linked by UV using a Stratalinker (Strat- horseradish peroxidase secondary antibodies and agene) according to the manufacturer's instruc- enhanced chemiluminescence, as described by tions. DNA probes were labelled with 32p using the manufacturers (ECL Western Blotting-Amer- random primed labelling (Boehringer-Mannheim, sham, Arlington Heights, IL, U.S.A.). Indianapolis, IN, U.S.A.). Blots were prehybridized for 30 min at 68°C in a hybridization oven using a GM-CSF Binding to Cells commercial hybridization solution (Quik-hyb, Stratagene) and hybridized for 2 hr at 68°C. Blots Radioiodinated GM-CSF was purchased from were washed in 2X SSC/0.1% SDS at room tem- New England Nuclear (DuPont Company, New perature for a low-stringency wash, 0.1 x England Nuclear Research Products, Wilmington, SSC/0. 1% SDS at 60°C for a high-stringency wash, DE, U.S.A.). The 125I-GM-CSF had a specific activ- and exposed to film. ity of 4.9 X 1018 cpm/mol. Before the binding of 1251-GM-CSF, all factor-dependent cells were cul- tured for 24 hr or more in GM-CSF-free RPMI RESULTS media containing 10% WEHI-conditioned media, followed by 6 hr in RPMI containing 10% FCS. The Both f8c and f8tr Convert Binding of cells (1-4 X 106) were washed and then resus- hGM-CSF in Ba/F3-GMER Cells from Low pended in binding media RPMI with 4% FCS, 30 to High Affinity mM HEPES pH 7.4, 0.04% sodium azide that con- Cell surface expression of GMER alone or with tained the indicated concentration of radiolabeled each of the 13-subunits was characterized by P. T. Jubinsky et al.: 1c, GMER, Proliferation, and f-Globin Expression 769

v .vA2 GMER Kd=5nM Q 0.01 5 0 3600/cell 0 E 0.0 00 0

0.00 5 %..w

0 .0 *_0.I- 0 0.; 3 GMER+pc Kd=4OpM 0.22!5 5000 600/cell I- B LL 4000 - | | 0.1 5. GMER+ptr

3000 - 0.07)5 .E 2000 - 0 1000 - 0.,5

,GMER+Ptr Kd=65pM - 0.375- 10 10'4 10 ' 10100 " 10 '° 101 108 1lo' 1300/cell A 0.2?5 Human GM-CSF Concentration (M)

0.12 A AA FIG. 2. ,Bc confers a high-affinity hGM-CSF proliferative response on GMER, whereas f8tr acts as a dominant negative 0 2E-11 4E-11 6E-11 8E-11 Tritiated thymidine incorporation assay of Ba/F3- GMER (D), Ba/F3-GMER + (3c (@), and Ba/F3- Bound(M) GMER + ,Btr (U). The upper panel shows that Ba/F3-GMER + f3c have the same maximum prolif- FIG. 1. Scatchard analysis of equilibrium bind- eration as Ba/F3-GMER but a 3-log increase in ing of radiolabeled hGM-CSF to Ba/F3 cells that hGM-CSF sensitivity. Ba/F3-GMER + ,3tr prolifera- tion is markedly inhibited; the upper plot is replot- express GMER without or with j8c or .8tr ted with an expanded y-axis in the lower panel to The upper panel shows low-affinity binding to show that the half-maximal proliferative response is Ba/F3-GMER, while the middle and lower panels similar to Ba/F3-GMER. The data are from one of show that ,Bc or (tr can convert this binding to high three experiments. affinity. The data are from one of two experiments.

,Bc Confers a High-Affinity Proliferative binding assays with radiolabeled hGM-CSF. The Response while f3tr Acts as a Dominant results were analyzed by the LIGAND program Negative and are presented in Fig. 1 as Scatchard plots. As To examine whether coexpression of these two illustrated in the top panel, Ba/F3-GMER (3-subunits affected GMER-induced proliferation, showed low-affinity binding to hGM-CSF and a a growth assay was performed, as shown in Kd of 5 nM, with 3600 binding sites per cell. Fig. 2. The closed circles in the top panel show Ba/F3-GMER + fc (middle panel) bound to that Ba/F3-GMER + ,Bc cells responded to hGM- hGM-CSF with a Kd of 40 pM and expressed 600 CSF and demonstrated the same growth rate as high-affinity receptors per cell. Ba/F3-GMER + Ba/F3-GMER cell, shown as open squares. How- /3tr, shown in the bottom panel, bound GM-CSF ever, in contrast to Ba/F3-GMER cells, the half- with a Kd of 65 pM and 1300 high-affinity bind- maximal growth of these Ba/F3-GMER + ,Bc was ing sites/cell. There was no significant difference obtained at a 3 log lower concentration of hGM- in affinity between cells expressing GMER + ,3c CSF, identical to Ba/F3 cells that stably express and GMER + 3tr. These data indicate that the hGMRa2 + fc (not shown). In contrast, as majority of GMER were associated with O3c as a shown by the closed squares, the maximum high-affinity complex, since no low-affinity com- growth of Ba/F3-GMER + ,Btr was strikingly re- ponent was detected. duced to 3-20% of the maximum growth ob- 770 Molecular Medicine, Volume 2, Number 6, November 1996

x4 x01 FIG. 3. Tyrosme phosphorylation of A receptor subunits (A) Double immunoprecipitation of Ba/ G;IU-C,SF (n-M). o 1 O 1 F3-GMER + 3c cells before (0) and after -200 (1) hGM-CSF stimulation. Anti-phospho- tyrosine immunoprecipitates were sub- jected to a second immunoprecipitation - 97 with rabbit polyclonal antibodies to the C terminus of 13c. Background phosphor- - 66 ylation is increased after hGM-CSF stim- ulation, similar to control Ba/F3-GMRa + /3c cells. (B) Immunoprecipitation/ Western analysis of Ba/F3-GMER (1), Ba/F3-GMER + ,Bc (2), and Ba/F3- GMER + f3tr (3) before (0) and after (1) stimulation with 0.1 or 20 nM hGM- 1 _ 2 3 CSF. Cellular proteins were immuno- B precipitated with 4G10 antibodies to phosphotyrosine, subjected to SDS poly- acrylamide gel electrophoresis, and G.cFW(nu) 0.120 0 0120 00t2 0 1Epo(WmI) transferred to a nylon membrane. West- ern analysis was then carried out using antibodies to the C terminus of the GMEl _ mEpoR. A dose-related induction of phosphorylation was seen in each case, similar to control Ba/F3-EpoR cells stim- ulated with Epo, shown on the right. These data are from one of three repre- = ! lI1-~ - sentative experiments. served with Ba/F3-GMER cells with or without study phosphorylation of the EpoR cytoplasmic full-length ,Bc. In the bottom panel of Fig. 2, the domain of GMER, the cells were stimulated with expanded y-axis emphasizes that the half-maxi- 0, 0.1, or 20 nM of hGM-CSF at 370C for 10 min, mal concentration of GM-CSF for Ba/F3- immunoprecipitated first with anti-phosphoty- GMER + ftr is identical to that of Ba/F3-GMER rosine antibody and then blotted with an anti- alone, indicating that the observed growth was body to the C terminus of the EpoR. Figure 3B induced through the low-affinity GMER subunit. shows that the GMERs in all three cell lines were All three cell lines showed similar growth curves tyrosine-phosphorylated in response to hGM- in response to murine IL-3 (not shown). CSF. This result is similar to the Epo-stimulated tyrosine phosphorylation of control cells that ex- Receptor Tyrosine Phosphorylation Does press wild-type EpoR, as shown on the right. Not Correlate with Cell Growth Note that the cells with GMER + B3tr did not show reduced intensity of phosphorylation, de- To see whether a correlation exists between cell spite remarkably reduced proliferation. This sug- growth and the pattern of receptor phosphory- gests that there is no correlation between cell lation, tyrosine phosphorylation of each of the proliferation and induction of tyrosine phos- receptor subunits was examined. Tyrosine phos- phorylation of GMER and that the inhibition of phorylation of the (-subunit was measured by proliferation induced by f3tr occurs distal to the double immunoprecipitation with and without phosphorylation of tyrosine on GMER. GM-CSF stimulation. Anti-phosphotyrosine im- munoprecipitants were subjected to a second im- munoprecipitation with antibodies to the C ter- ,Bc Increases 18-Globin mRNA in minus of f3c. An hGM-CSF-dependent increase Ba/F3-GMER Cells in tyrosine phosphorylation of the ,3c-subunit in To determine whether (Bc affects the differentia- Ba/F3-GMER + j3c was observed (Fig. 3A) and tion function of GMER, we incubated cells in was indistinguishable from the induced tyrosine mIL-3, hGM-CSF, or a combination of both fac- phosphorylation of f3c in Ba/F3-GMRa + ,3c. To tors for 7 days before RNA extraction and North- P. T. Jubinsky et al.: 1c, GMER, Proliferation, and 13-Globin Expression 771

FIG. 4. Northern analysis of 3-glo- bin mRNA Cells were cultured in hGM-CSF, mIL-3, GMER GMER+Ac GMER+Dtr , or both, as indicated, and the RNA ex- huGM-CSF + ++ ++ + tracted. The samples were electropho- resed, transferred to nylon, and hybrid- mulL-3 + + + + + + ized to a random-labeled ,3-globin probe. The greatest increase in f3-globin mRNA was seen in GMER + 3c cells stimulated 28S. with hGM-CSF; mIL-3 had no effect and appeared to down-modulate hGM-CSF- induced f3-globin mRNA accumulation. 18s- Much lower levels of ,B-globin mRNA were seen with the GMER, GMER + Mtr, P globin-* and control GMRa + f3c cells stimulated with hGM-CSF. The data represent one of two experiments.

ern blot analysis. Figure 4 shows that ,B-globin GMER-mediated proliferation is stimulated by mRNA levels were markedly increased in cells GM-CSF in the low affinity dose range and that the that expressed GMER + ,3c after incubation in truncated ,Bc subunit acts as a dominant negative hGM-CSF. In contrast, the same cells incubated by inhibiting maximal proliferation, with no effect in mIL-3 showed no increase in ,B-globin mRNA. on the half-maximal GM-CSF concentration, are There was no increase in f3-globin mRNA expres- consistent with this dimerization hypothesis but do sion in GMER + ,Btr cells, demonstrating that the not prove it. We believe that the truncation mutant increase in ,3-globin message was mediated may interfere with the formation of GMER ho- through the cytoplasmic domain of the (3-chain modimers and does so efficiently, since the pres- and not via a protein that interacts with the ence of (tr leads to high-affinity GM-CSF binding. extracytoplasmic or transmembrane portion of GM-CSF induced tyrosine phosphorylation O3c. Only slight levels of ,B-globin message were of the Epo cytoplasmic domain of GMER in all detected in cells transfected with GMRa + ,Bc, three cell lines, GMER, GMER + ,Bc, and GMER + indicating the importance of the cytoplasmic com- ,Btr. The finding that tyrosine phosphorylation is ponent of GMER in the differentiation signal. unimpaired in GMER + ,Btr despite greatly reduced proliferation implies that receptor phosphoryla- tion is not directly coupled to cell proliferation. DISCUSSION This finding is consistent with the observation The data reported here show, first, that /3c con- that a cytoplasmic truncation mutant of EpoR verts the low-affinity binding and proliferation of that lacks the 91 C-terminal amino acids, includ- a hybrid GMER receptor to high-affinity binding ing seven of eight cytoplasmic tyrosines, is hy- and proliferation. This result is analogous to the persensitive to Epo and that EpoR tyrosine phos- role of (c when coexpressed with the wild-type phorylation has a negative regulatory role (12). GMRa (5) or a truncated receptor (7). We also Ba/F3 cells transfected with mEpoR have show that a complete cytoplasmic truncation been shown to have high levels of ,3-globin mutant of the (3-chain, B3tr, while supporting high- mRNA accumulation after growth in Epo (11), affinity binding of GM-CSF to the GMER/P com- indicating that an the EpoR is important for this plex, greatly inhibits the proliferation of Ba/F3- differentiation response. This result was not ob- GMER cells but does not affect the GM-CSF served by Maruyama et al. (21), suggesting that induced tyrosine phosphorylation of the EpoR cy- Ba/F3 cells may exhibit variable responsiveness, toplasmic domain of GMER. Last, and surprisingly, possibly depending on receptor expression. How- f3c also plays a role in the accumulation of P-globin ever Maruyama et al. did demonstrate that in these GMER cells after incubation in hGM-CSF. mEpo-responsive TSA8 erythroleukemia cells Studies with a constitutive active EpoR transfected with an extracellular domain epider- (17,18) and with a cytoplasmic truncated EpoR mal growth factor (EGF) /intracellular domain (19,20) suggest that dimerization may be impor- Epo hybrid receptor displayed EGF-stimulated tant in EpoR signaling. Our observations that growth and induction of f-globin protein syn- 772 Molecular Medicine, Volume 2, Number 6, November 1996 thesis. The experiments reported here, however, extracellular and transmembrane domains of fc suggest that the cytoplasmic domain of EpoR are sufficient to act as a high-affinity converter of alone in GMER is insufficient for P3-globin induc- a hybrid GMER receptor. The cytoplasmic domain tion in Ba/F3 cells, but it can be made competent of ,Bc is necessary for high-affinity proliferation, by introduction of a complete O3c-subunit. Fur- and a truncated (3-subunit greatly inhibits this re- thermore, the cytoplasmic domain of EpoR is sponse. Coexpression of GMER and /3c leads to essential, because cells that express GMRa + ,Bc accumulation of 1-globin transcripts, and this func- show only background levels of 3-globin. Inter- tion depends on the erythropoietin receptor cyto- estingly, species-specific interactions must occur plasmic domain of the hybrid. The f3c appears to act between the extracellular and or transmembrane as an accessory protein in this context. Whether it portion of GMER and 13c, otherwise one would might fulfill the same role in normal Epo-respon- expect that cells transfected with GMER alone sive cells is the subject of current studies. would induce f3-globin by interacting with en- dogenous murine P3. Lack of interaction of GMER and murine 13 probably accounts for the low- affinity binding of GM-CSF to GMER. ACKNOWLEDGMENTS Could ,Bc be important for differentiation in PTJ is a recipient of a Clinical Oncology Career cells that express normal EpoR? It is difficult to test Development Award from the American Cancer this idea in Ba/F3 cells because they express en- Society. This work was supported by U.S. Public dogenous murine 13c. There is published evidence Health Service Award GM 13452, RO1 CA45559, to suggest that the EpoR may indeed associate and the Charles H. Hood Foundation. with another protein. First, partially purified complexes of Epo and EpoR show an associated 130 kD protein that contains phosphotyrosine (pp 130) (22). Both the size and the ligand-in- REFERENCES duced tyrosine phosphorylation of pp 130 are con- 1. Sieff CA, Emerson SG, Donahue RE, et al. sistent with its being ,Bc, which is 134 kD and (1985) Human recombinant granulocyte-mac- which becomes phosphorylated on tyrosine after rophage colony-stimulating factor: A multilin- GM-CSF or IL-3 binding. There is evidence, how- eage hematopoietin. Science 230: 1171-1173. ever, that this pp 130 kD protein is JAK2, a non- 2. Martin FH, Suggs SV, Langley KE, et al. that is phosphorylated on (1990) Primary structure and functional ex- tyrosine after Epo stimulation and associates with pression of rat and human the receptor (23). Recently, we were able to dem- DNAs. Cell 63: 203-211. onstrate associated ,Bc in EpoR immunoprecipita- 3. Gearing DP, King JA, Gough NM, Nicola NA. tion experiments (24). Second, it has been reported (1989) Expression cloning of a receptor for that hybrid receptors that comprise the extraceliu- human granulocyte-macrophage colony- lar domain of EpoR and the transmembrane and stimulating factor. EMBO J. 8: 3667-3676. intracellular domains of the hIL-2R or mIL-3Rf3 4. Crosier KE, Wong GG, Mathey-Prevot B, can induce /3-globin in Ba/F3 cells Nathan DG, Sieff CA. (1991) A functional (25). It is difficult to explain this induction of isoform of the human granulocyte-macro- ,B-globin gene expression without invoking the phage colony-stimulating factor receptor action of a protein accessory to the extracellular contains a unique cytoplasmic domain. Proc. domain of EpoR. An alternative explanation could Natl. Acad. Sci. U.S.A. 88: 7744-7748. be that some Ba/F3 clones express (or are induced 5. Hayashida K, Kitamura T, Gorman DM, Arai to express) endogenous EpoR (20,26), and it is K-I, Yokota T, Miyajima A. (1990) Molecular possible that the hybrid receptors associate with cloning of a second subunit of the receptor endogenous EpoR in the Ba/F3 cells and in the for human granulocyte-macrophage colony- murine erythroleukemia cells tested in these stimulating factor (GM-CSF): Reconstitution studies. We are currently evaluating analogous of a high-affinity GM-CSF receptor. Proc. chimeras of EpoR/,Bc and EpoR/GMRa in Ba/F3 Natl. Acad. Sci. U.S.A. 87: 9655-9659. and CTLL cells to test this possibility. Finally, 6. Miyajima A, Mui AL-F, Ogorochi T, Sakamaki observations by Hanazono et al. that Epo can K. (1993) Receptors for granulocyte-macroph- induce tyrosine phosphorylation of f3c suggest age colony-stimulating factor, interleukin-3, that EpoR and /3c functionally interact (27). and interleukin-5. Blood 82: 1960-1974. In conclusion, we provide evidence that the 7. Weiss M, Yokoyama C, Shikama Y, Naugle P. T. Jubinsky et al.: fc, GMER, Proliferation, and f-Globin Expression 773

C, Druker B, Sieff CA. (1993) Human gran- 17. Yoshimura A, Longmore G, Lodish HF. ulocyte-macrophage colony-stimulating fac- (1990) Point mutation in the exoplasmic do- tor receptor signal transduction requires the main of the erythropoietin receptor resulting proximal cytoplasmic domains of the a and 1B in hormone-independent activation and tu- subunits. Blood 82: 3298-3306. morigenicity. Nature 348: 647-649. 8. Sakamaki K, Miyajima I, Kitamura T, Miya- 18. Watowich SS, Yoshimura A, Longmore GD, jima A. (1992) Critical cytoplasmic domains Hilton DJ, Yoshimura Y, Lodish HF. (1992) of the common beta subunit of the human Homodimerization and constitutive activa- GM-CSF, IL-3 and IL-5 receptors for growth tion of the erythropoietin receptor. Proc. signal transduction and tyrosine phosphory- Natl. Acad. Sci. U.S.A. 89: 2140-2144. lation. EMBO J. 11: 3541-3549. 19. Barber DL, DeMartino JC, Showers MO, 9. Jubinsky PT, Nathan DG, Wilson DJ, Sieff D'Andrea AD. (1994) A dominant negative CA. (1993) A low-affinity human granulo- erythropoietin (EPO) receptor inhibits EPO- cyte-macrophage colony-stimulating factor/ dependent growth and blocks F-gp55-de- murine erythropoietin hybrid receptor func- pendent transformation. Mol. Cell. Biol. 14: tions in murine cell lines. Blood 81: 587-59 1. 2257-2267. 10. Palacios R, Steinmetz M. (1985) IL3-depen- 20. Nakamura Y, Nakauchi H. (1994) A trun- dent mouse clones that express B-220 sur- cated erythropoietin receptor and cell death: face antigen contain Ig in germ-line A reanalysis. Science 264: 588-589. configuration, and generate B lymphocytes 21. Maruyama K, Miyata K, Yoshimura A. (1994) in vivo. Cell 41: 727-734. Proliferation and erythroid differentiation 11. Liboi E, Carroll M, D'Andrea AD, Mathey- through the cytoplasmic domain of the ery- Prevot B. (1993) Erythropoietin receptor sig- throid receptor. J. Biol. Chem. 269: 5976-5980. nals both proliferation and erythroid-specific 22. Yoshimura A, Lodish HF. (1992) In vitro differentiation. Proc. Natl. Acad. Sci. U.S.A. 90: phosphorylation of the erythropoietin recep- 11351-11355. tor and an associated protein, pp 130. Mol. 12. D'Andrea AD, Yoshimura A, Youssoufian H, Cell. Biol. 12: 706-715. Zon LI, Koo J-W, Lodish H. (1991) The cy- toplasmic region of the erythropoietin recep- 23. Witthuhn BA, Quelle FW, Silvennoinen 0, tor contains nonoverlapping positive and et al. (1993) JAK2 associates with the eryth- negative growth-regulatory domains. Mol. ropoietin receptor and is tyrosine phosphor- Cell. Biol. 11: 1980-1987. ylated and activated following stimulation 13. Shikama Y, Barber DL, D'Andrea A, Sieff with erythropoietin. Cell 74: 227-236. CA. (1996) A constitutively activated chi- 24. Jubinsky PT, Sieff C, Nathan DG. (1995) The meric receptor confers factor-de- ,B chain of the IL-3 receptor functionally as- pendent growth in hematopoietic cell lines. sociates with the erythropoietin receptor. Blood 88: 455-464. Blood 86: 15a. 14. Yoshimura A, D'Andrea AD, Lodish HF. 25. Chiba T, Nagata Y, Kishi A, et al. (1993) (1990) Friend spleen focus-forming virus Induction of erythroid-specific gene expres- glycoprotein gp55 interacts with the eryth- sion in lymphoid cells. Proc. Natl. Acad. Sci. ropoietin receptor in the endoplasmic retic- U.S.A. 90: 11593-11597. ulum and affects receptor metabolism. Proc. 26. Damen J, Mui AL-F, Hughes P, Humphries Natl. Acad. Sci. U.S.A. 87: 4139-4143. K, Krystal G. (1992) Erythropoietin-induced 15. Munson PJ, Rodbard D. (1980) LIGAND: A tyrosine phosphorylations in a high erythro- versatile computerized approach for charac- poietin receptor-expressing lymphoid cell terization of ligand-binding systems. Anal. line. Blood 80: 1923-1932. Biochem. 107: 220-239. 27. Hanazono Y, Sasaki K, Hiroyuki N, Yazaki Y, 16. Chirgwin JM, Przybila AE, MacDonald RJ, Hirai H. (1995) Erythropoietin induces ty- Rutter WJ. (1979) Isolation of biological ac- rosine phosphorylation of the ,B chain of the tive ribonucleic acid from a source enriched GM-CSF receptor. Biochem. Biophys. Res. Com- in ribonuclease. Biochemistry 18: 5294-5299. mun. 208: 1060-1066.

Contributed by D. G. Nathan. Accepted on August 14, 1996.