The Erythropoietin Receptor Transmembrane Region Is Necessary for ...

MOLECULAR

AND

CELLULAR BIOLOGY, JUlY 1992, p. 2949-2957

Vol. 12, No. 7

0270-7306/92/072949-09$02.00/0 Copyright © 1992, American Society for Microbiology

The Erythropoietin Receptor Transmembrane Region Is Necessary for Activation by the Friend Spleen Focus-Forming Virus gp55 Glycoprotein LEONARD I. ZON, JEAN-FRAN(OIS MOREAU, JAH-WON KOO, BERNARD MATHEY-PREVOT, AND ALAN D. D'ANDREA*

Department of Pediatrics, Division of Hematology-Oncology, The Children's Hospital, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 Received 22 January 1992/Accepted 3 April 1992

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55.

Erythropoietin (EPO) is the major glycoprotein regulator of mammalian erythropoiesis. The erythropoietin receptor (EPO-R) is a 507-amino-acid type 1 membrane-spanning protein (16). The EPO-R can be activated to signal cell proliferation by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein (7, 20, 26). There is no amino acid similarity between EPO and gp55, and the interaction between the EPO-R and these molecules has not been well defined. Because gp55 has only a twoamino-acid cytoplasmic tail (3, 12, 36) and because EPO-R mutants lacking the cytoplasmic tail coimmunoprecipitate with gp55 (17), gp55 must bind to the EPO-R via the transmembrane region or the extracytoplasmic region (Fig. 1). The transmembrane region of gp55 from the polycythemia strain of SFFV appears to determine activation of EPO-independent cell proliferation (11, 35). Srinivas et al. (32) have recently demonstrated that a gp55 polypeptide lacking the transmembrane region is nonleukemogenic, though the molecular basis for the transmembrane region requirement has not been established. Other evidence suggests that the amino-terminal, extracytoplasmic region of gp55 mediates EPO-R activation (1, 23, 25). This region of gp55 is derived from the amino terminus of the gp7O envelope protein of the leukemogenic mink cell focus-forming (MCF) virus (9) and from ecotropic envelope sequence, separated by a proline-rich region (Fig. 1). The MCF gp7O itself has recently been shown to interact with the EPO-R (24). These results, taken together, suggest that both the transmembrane region and extracytoplasmic region of gp55 could potentially participate in the interaction with the EPO-R. The EPO-R is a member of the cytokine receptor superfamily (5, 6, 13) that includes the receptors for many of the *

interleukins, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and several other growth factors. Members of this superfamily contain four conserved cysteine residues and the motif WSXWS in the extracytoplasmic region and contain a single transmembrane region (Fig. 1). In contrast, the cytoplasmic regions of the superfamily members differ. The cytoplasmic region of some cytokine receptors, such as the EPO-R and the interleukin-2 receptor (IL-2R), is required for signal transduction. These two receptors share amino acid similarities in a region which is required for ligand-mediated proliferation (15). The cytoplasmic region of other receptors, such as the IL-6R and the GM-CSF receptor, is not required for signal transduction. These so-called a-subunit receptors (29) interact with a , signal transduction subunit. Other members of the superfamily, such as the AIC2A polypeptide of the highaffinity IL-3R (21, 22), may participate in signal transduction, although this has not been formally shown. Interestingly, the AIC2A cytoplasmic domain has amino acid homology to the cytoplasmic domain of the EPO-R and the IL-2R (19). Chimeric receptors have previously been used to map functional domains of members of the tyrosine kinase receptor superfamily. For instance, a chimeric epidermal growth factor-insulin receptor polypeptide binds to epidermal growth factor and induces insulin receptor tyrosine kinase activity (34). Functional chimeric receptors of the cytokine receptor superfamily have not been previously described. In this study, we have generated functional chimeric receptors of the cytokine receptor superfamily containing regions of the EPO-R and the related IL-3R. These chimeric receptors were used to identify the transmembrane region of the EPO-R as a critical region for gp55 activation and to demonstrate that the extracytoplasmic region of the EPO-R specifically interacts with gp55.

Corresponding author. 2949

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EPO-R FIG. 1. Schematic representation of the EPO-R/gp55 interaction. The EPO-R contains four cysteine residues (Cl through C4) which are conserved among members of the cytokine receptor superfamily and a unique fifth cysteine residue (C5). The WSXWS motif is conserved among members of the cytokine receptor superfamily. gp55 is a chimeric protein composed of an amino terminus derived from the MCF viral envelope protein and a membrane anchor derived from the ecotropic helper virus, Friend murine leukemia virus.

MATERIALS AND METHODS Cells and cell culture. Ba/F3 cells (17) were maintained in RPMI 1640 medium supplemented with 10% (vol/vol) fetal calf serum (FCS) and 10% (vol/vol) conditioned medium from WEHI-3B cells as a source of IL-3 (IL-3 medium). COS-1 cells were grown as previously described (16). Plasmid construction. A full-length AIC2A cDNA was isolated by reverse transcription of mRNA from Ba/F3 cells and subsequent polymerase chain reaction (PCR) using specific primers (21). Chimeric receptor cDNAs were generated by PCR, using the cDNAs for the murine EPO-R (16), the murine AIC2A polypeptide (21), and the human IL-6R (37) as templates. The EPO-R/IL-3R A junction was made at proline residue 248 of the EPO-R and proline 432 of AIC2A. The EPO-R/IL-3R B junction was made at arginine residue 273 of the EPO-R and arginine 446 of AIC2A. The EPO-R/ IL-3R C junction was made at tryptophan residue 232 of the EPO-R and at tryptophan residue 427 of AIC2A. The EPOR/IL-6R junction was made at proline residue 248 of the EPO-R and proline residue 355 of the IL-6R. A soluble, secreted EPO-R (sEPO-R) was generated by PCR, introducing a stop codon (TAG) at amino acid position 248 of the EPO-R. The cysteine point mutations of the EPO-R were generated by the M13 mutagenesis method (Bio-Rad), resulting in conversion of each extracytoplasmic cysteine residue, Cys-28 (Cl), Cys-38 (C2), Cys-66 (C3), Cys-82 (C4), and Cys-180 (C5), to serine residues. The chimeric receptor and point mutant cDNAs, sEPO-R cDNA, and cysteine mutant EPO-R cDNAs were confirmed by

dideoxy DNA sequence analysis and subcloned into the mammalian expression vector, PXM. DNA transfection of Ba/F3 cells. Ba/F3 cells (106 cells) were transfected by coelectroporation with a PXM-chimeric receptor cDNA (linearized with NdeI) and with pSV2neo (linearized with AccI), as previously described (17). Selection with G418 (1.5 mg/ml) in 10% WEHI-conditioned medium was initiated 48 h after electroporation. Selected cells were subcloned by limiting dilution, and individual subclones were expanded in G418/IL-3 medium and analyzed for the presence of the wild-type or chimeric EPO-R polypeptides by metabolic labeling and immunoprecipitation as previously described (17). Coimmunoprecipitation of EPO-R and gp55 in COS cell extracts. COS-1 cells were cotransfected by the DEAEdextran method with the PXM-chimeric EPO-R cDNA and an expression plasmid (PXM-gp55) encoding the gp55 polypeptide as previously described (16). Briefly, both PXMchimeric EPO-R cDNA and PXM-gp55 (2 ,ug of each) were cotransfected into a 10-cm plate of COS-1 cells. Three days after transfection, the cells were metabolically labeled with [35S]methionine, the labeled proteins were extracted in lysis buffer (1% Triton X-100), and immunoprecipitation was performed with a goat polyclonal antienvelope antiserum (26). EPO-dependent growth characteristics of Ba/F3 subclones. Individual subclones expressing the EPO-R or chimeric receptor polypeptides were screened for growth in RPMI 1640 medium supplemented with 10% FCS and several concentrations of recombinant human EPO (without supplemental WEHI-conditioned medium) (EPO medium). Cell growth and viability were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MIT) assay as previously described (17). Electroporations with each chimeric mutant were performed three times. When resulting transfectants were isolated and tested for EPO responsiveness, identical results were obtained each time. gp55-dependent growth characteristics of Ba/F3 subclones. Subclones expressing the EPO-R or chimeric receptor polypeptides were infected with a high-titer (SFFV) retroviral supernatant encoding the gp55 polypeptide. Approximately 5 x 105 Ba/F3 cells, growing in RPMI medium supplemented with WEHI-conditioned medium (IL-3 medium), were infected as previously described (26). Forty-eight hours after infection, the cells were washed twice with HBS (Hanks balanced salt solution, 20 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid [HEPES], pH 7.4) and resuspended in RPMI 1640 with 10% FCS without EPO or IL-3 (plain medium). The infected cells were screened for growth and viability in this unsupplemented medium by using the MIT reduction assay (17). In addition, the SFFV-infected Ba/F3 subclones were grown for 72 h in IL-3 medium. These infected cells were screened for productive SFFV infection by metabolic labeling and immunoprecipitation of the gp55 polypeptide, using the goat polyclonal antienvelope antiserum previously described (26). Binding of EPO to the cell surface EPO-R. Ba/F3 subclones (107 cells of each) were incubated with the indicated concentrations of radiolabeled EPO (specific activity, 1,000 to 1,500 cpm/fmol) in 200 ,u of RPMI 1640 medium with 10% FCS for 10 h at 4°C. The radiolabeled EPO was prepared by the iodine monochloride method as previously described (16), and it retained full biological activity. After sedimentation of cells through dibutylphthalate oil, cell-associated radioactivity was measured. Specific binding was obtained by subtracting the radioactivity bound to the parent (untransfected)

VOL. 12, 1992

ERYTHROPOIETIN RECEPTOR AND gp55 INTERACTION

Ba/F3 cells (nonspecific binding) from the radioactivity bound to the Ba/F3 subclones tested (total binding). Nonspecific binding was less than 20% of total binding. Each EPO concentration was run in triplicate. Binding of EPO to the sEPO-R. Conditioned medium containing sEPO-R was incubated with increasing concentrations of radiolabeled EPO at 37°C for 1 h in the absence (total binding) or presence (nonspecific binding) of excess (100 nM) cold EPO. Immunoprecipitation was performed by using anti-EPO-R antiserum plus protein A-Sepharose. 12511 labeled EPO immunoprecipitated by the anti-EPO-R antiserum was counted from the pellet, using an Auto-GAMMA counter (Packard). Specific binding (total minus nonspecific, done in triplicate) was calculated. Cell surface localization. Ba/F3 cells expressing the wildtype EPO-R or the cysteine point mutants (107 cells of each subclone) were incubated in 200 ,ul of plain medium with or without 5 Rl of anti-EPO-R (anti-amino-terminal) rabbit serum. Incubation was for 10 h at 4°C. The cells were washed twice in HBS and incubated in 200 ,ul of plain medium, with radioiodinated protein A (90 ,uCi/,ug, 50,000 cpm; New England Nuclear) as a second label. Cells were washed twice in HBS. 1251 in the cell pellet was counted, using an Auto-GAMMA counter (Packard). Specific binding was calculated by subtracting the counts bound to the parent (untransfected) Ba/F3 cells (nonspecific binding) from the radioactivity bound to the Ba/F3 subclones tested (total binding). Each subclone was measured in triplicate. RESULTS Construction and expression of chimeric receptor cDNAs. Initially, we used PCR to generate chimeric receptor cDNAs containing regions of EPO-R and the related cytokine receptor for IL-3 (AIC2A) (21). The AIC2A polypeptide does not interact with EPO or gp55. Although it shares some amino acid similarity with the EPO-R in its cytoplasmic region, it has not formally been shown to have signal transducing activity. The predicted chimeric receptor polypeptides are shown schematically in Fig. 2. One chimera (EPO-R/IL-3R A) contains the extracytoplasmic region of the EPO-R and the transmembrane and cytoplasmic regions of AIC2A. A second chimera (EPO-R/IL-3R B) contains the extracytoplasmic and transmembrane regions of the EPO-R and the cytoplasmic region of AIC2A. These two chimeras differ only in their 24-amino-acid transmembrane regions. EPO-RI IL-3R C contains the extracytoplasmic region of the EPO-R to the WSXWS motif and the remaining extracytoplasmic, transmembrane, and cytoplasmic regions of the IL-3R. We also generated a chimeric receptor containing the extracytoplasmic region of the EPO-R and the transmembrane and cytoplasmic regions of the human IL-6R ot chain (37). These regions of the IL-6R can be deleted without loss of IL-6induced signal transducing activity (33); a ligand-bound soluble IL-6R a chain transduces a signal through the gpl30 ,B chain. The chimeric cytokine receptor cDNAs were ligated into the expression vector PXM, which functions both in Ba/F3 cells and in COS cells (17). Ba/F3 subclones were generated by cotransfection with linearized chimeric receptor cDNAs and pSV2neo, selected in G418, and subcloned by limiting dilution. To confirm that the G418-resistant Ba/F3 subclones expressed the chimeric receptor polypeptides, representative subclones were analyzed by immunoprecipitation (Fig. 2). All three EPO-R/IL-3R chimeric receptors have an electrophoretic mobility of 85 kDa (lanes 2 to 4), suggesting

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FIG. 2. Immunoprecipitation of EPO-R and chimeric cytokine receptor polypeptides from Ba/F3 cell transfectants. Ba/F3 cells were electroporated with plasmid PXM, containing the cDNA encoding either wild-type EPO-R (lane 7), the -91 hypersensitive mutant (lane 5), chimera A (lane 2 and 6), chimera B (lane 3), chimera C (lane 4), or EPO-R/IL-6R (lane 1), or were mock transfected. Ba/F3 subclones, isolated by limiting dilution, were expanded and labeled for 60 min with [35S]methionine. Labeled proteins were extracted in 1% Triton X-100, and immunoprecipitation was performed with a polyclonal antiserum directed against the amino terminus of the EPO-R as previously described (38). The structure of the predicted polypeptide is shown schematically below each lane. Sizes are indicated in kilodaltons.

that they are identical with respect to carbohydrate processing. The EPO-R/IL-6R chimera has a molecular size of 29 kDa, consistent with the short cytoplasmic domain of this polypeptide (lane 1). As controls, immunoprecipitation analysis was performed on Ba/F3 cells expressing the 66-kDa wild-type EPO-R (lane 7) and a truncated 38-kDa EPO-R (lane 5) which has been previously shown to be hypersensitive to EPO (17). Chimeric receptors containing the AIC2A cytoplasmic region confer EPO-dependent growth in Ba/F3 transfectants. We have previously shown that expression of the wild-type EPO-R polypeptide converts the IL-3-dependent cell line Ba/F3 to EPO-dependent growth, using an MTT reduction assay for cell proliferation (17). As shown in Fig. 3A, all three EPO-R/IL-3R chimeric receptors (A, B, and C) conferred EPO-dependent growth which was indistinguishable from that conferred by the wild-type EPO-R. In contrast, Ba/F3 cells containing the EPO-R/IL-6R chimeric receptor and mock-transfected Ba/F3 cells did not grow in the presence of EPO. As previously described, the -91 EPO-R was hypersensitive to EPO (17); these cells showed half-maximal MTT reduction at 1 mU of EPO per ml. All other EPOresponsive subclones tested showed half-maximal MTT reduction at 10 mU of EPO per ml. To demonstrate that these differences in EPO-dependent cell growth were not due to differences in cell surface EPO binding, we next examined the EPO binding properties of the Ba/F3 subclones transfected with the various chimeric re-

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TABLE 1. Number of EPO-Rs expressed on Ba/F3 transfectant cells expressing wild-type, mutant, and chimeric EPO-Ra No. of EPO-R cDNA

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transfected

o

EPO-R/cell

0 None ............................................ 1,180 ................................ Wild type ............ -91 truncated mutant ............................................ 1,024 EPO-R/IL-3R A ....................... ..................... 1,560 EPO-R/IL-3R B ............................................ 1,668 EPO-R/IL-3R C ............................................ 1,380 EPO-R/IL-3R A ....................... ..................... 1,823 EPO-R/IL-6R ............................................ 1,788

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FIG. 3. Evidence that the transmembrane region of the EPO-R is for activation by gp55. (A) EPO-dependent growth characteristics of Ba/F3 subclones expressing the EPO-R and EPO-R/ IL-3R chimeras. The IL-3-dependent cell line Ba/F3 was coelectroporated with the indicated cDNAs and pSV2neo, and stable transfectants were selected in G418 as described previously (17). The isolated Ba/F3 subclones were analyzed for EPO-dependent growth by using the MTT reduction assay (17). Ba/F3 subclones expressed either the EPO-R (0), the -91 truncated, hypersensitive mutant (A), EPO-R/IL-3R A (+), EPO-R/IL-3R B (M), EPO-R/ IL-3R C (0), EPO-R/IL-3R A' (A), EPO-R/IL-6R ([), or no heterologous protein (ffl). (B) gp55-activated growth of Ba/F3 subclones expressing the EPO-R and EPO-R chimeras. Ba/F3 subclones expressed the heterologous proteins indicated. Ba/F3 subclones A and A' differ in the expression level of the EPO-R/IL-3R A polypeptide (see text). The Ba/F3 subclones were infected in IL-3 medium by using a high-titer retrovirus encoding gp55 (26). Two days postinfection, the cells were washed and transferred to medium without IL-3 or EPO supplementation and were analyzed by the MTT reduction assay. O.D., optical density. necessary

ceptors (Table 1). For these binding studies, the cells

were

incubated with six concentrations of radiolabeled EPO (specific activity, 1,000 to 1,500 cpm/fmol). Each subclone had a single affinity for radiolabeled EPO, with Kds ranging from 200 to 300 pM. On the basis of the amount of EPO bound at the saturating concentration (1,000 pM), the number of cell surface EPO-Rs per cell was calculated. Ba/F3 cells expressing the wild-type and -91 truncated EPO-R had approximately 1,000 EPO binding sites per cell surface, consistent with previous reports (17). Ba/F3 cells expressing any of the three EPO-R/IL-3R chimeric receptors (A, B, or C) or the EPO-R/IL-6R chimera had a higher number of EPO binding

sites, ranging from 1,380 to 1,823 sites per cell surface, demonstrating that these chimeric receptors are efficiently transported to the cell surface. The transmembrane region of the EPO-R is required for a cellular response to gp55. We next infected the Ba/F3 subclones, growing in IL-3 medium, with a high-titer retrovirus encoding gp55 and assayed conversion of the cells to factorindependent growth (26). Following infection, all subclones expressed similar levels of gp55 polypeptide by immunoprecipitation with a goat polyclonal antiserum against the envelope protein (data not shown). The infected cells were washed and transferred to medium without supplemental growth factor (plain medium). Figure 3B shows that only the Ba/F3 cells stably expressing the wild-type EPO-R, the -91 EPO-R mutant, and the EPO-R/IL-3R B chimera were activated by gp55 to grow in plain medium. gp55-infected Ba/F3 subclones expressing the chimeric receptors A and C remained dependent on IL-3 or EPO for growth. Retroviral infection of the Ba/F3 subclones was performed four times, and identical results were obtained each time. To ensure that expression level of chimeric receptor polypeptide did not account for these differences in gp55induced proliferation, we analyzed multiple Ba/F3 subclones expressing the EPO-R/IL-3R chimeric polypeptides and performed five independent infections with the gp55-containing retrovirus; identical results were obtained each time. One representative subclone (A') expressed EPO-R/IL-3R A at a high level (Fig. 2, lane 2 versus lane 6) and had a higher number of EPO binding sites at the cell surface than did other subclones (Table 1). Despite this relatively high level of expression, these cells could not be converted to EPOindependent growth by coexpression with gp55 (Fig. 3B). As controls, mock-transfected and the EPO-RIIL-6R-transfected Ba/F3 cells always remained dependent on IL-3 for growth after infection with SFFV. Only those receptors which contained the transmembrane region of the EPO-R (EPO-R, -91 mutant, and EPO-R/IL-3R B) were activated by gp55, resulting in factor-independent growth of Ba/F3. Finally, to further confirm the transmembrane requirement for gp55 activation, we also stably expressed the EPO-R/IL-3R chimeric receptors (A, B, and C) in an IL-2dependent T-lymphocyte line, CTLL-2. All three polypeptides confer EPO-dependent growth, but again, only EPOR/IL-3R B was activated by gp55 (data not shown). The extracytoplasmic region of the EPO-R specifically interacts with gp55. Previous studies have demonstrated that


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gp55 forms a stable complex with the EPO-R in Friend virus-infected cells (10, 26). To test for physical interaction between gp55 and the chimeric receptors, we used a previously described (17) rapid coimmunoprecipitation assay in COS cells (Fig. 4). In this assay, COS cell monolayers were cotransfected with a chimeric receptor cDNA and an expression vector which encodes the SFFV gp55 polypeptide (PXM-gp55). An immunoprecipitation was then performed with an antiserum directed against gp55. The wild-type EPO-R (66 kDa) coimmunoprecipitated with gp55 (Fig. 4, lane EPO-R). The wild-type AIC2A (110 kDa) does not coimmunoprecipitate with gp55 (lane IL-3R), although it does immunoprecipitate with an anti-AIC2A antiserum from the same lysate. All three EPO-R/IL-3R chimeras (A, B, and C) coimmunoprecipitate with gp55 (lanes A, B, and C respectively). Of note, the expression levels and coimmunoprecipitation of the chimeric receptor polypeptides (A, B, and C) and of the gp55 polypeptide differ in these three lanes. For instance, the EPO-R/IL-3R B chimera band intensity is greater in lane B, and the gp55 band intensity is greater in lane C. Despite these quantitative differences in COS cells, no AIC2A polypeptide coimmunoprecipitated with gp55, confirming that the interaction between the EPO-R and gp55 is specific in this assay. Also, the expression level of chimeric receptor and gp55 does not affect the function of these receptors in Ba/F3 cells (Fig. 3). Even low levels of the EPO-R/IL-3R B chimera and gp55 result in constitutively growing Ba/F3 cells (14a). On the basis of these results for COS cells, the EPO-R must interact with gp55 in the extracytoplasmic region. Membrane anchorage of the EPO-R is required for gp55

FIG. 5. Biosynthesis and secretion of a truncated EPO-R and binding to radiolabeled EPO. (A) Carbohydrate processing and secretion into media of the truncated form of the EPO-R. The cDNA encoding the secreted EPO-R mutant was transfected into COS cell monolayers. The COS cells were metabolically labeled for 30 min with [35S]cysteine, washed, and resuspended in fresh growth media with unlabeled cysteine (chase period). At the indicated times, the radiolabeled proteins which were cell associated (lanes 1, 3, 5, 7, and 9) or secreted into the medium (lanes 2, 4, 6, 8, and 10) were analyzed by immunoprecipitation with the anti-amino-terminal antiEPO-R antiserum. Period of chase was for the indicated times. (B) Evidence that membrane anchorage of the EPO-R is necessary for gp55 binding. The cDNAs encoding the wild-type EPO-R (lanes 1 and 3) and the sEPO-R (lanes 2 and 4) were transfected into COS cells with (lanes 3 and 4) or without (lanes 1 and 2) the cDNA for gp55. Immunoprecipitation was performed as described above. Sizes are indicated in kilodaltons.

interaction. To test whether the extracytoplasmic domain alone is sufficient for gp55 ahd EPO binding, we next generated sEPO-R which lacked a transmembrane region. The sEPO-R cDNA was transfected into COS-1 cells, and expression was assayed by pulse-chase analysis (Fig. SA). sEPO-R polypeptide existed in three forms (24, 26, and 28 kDa), analogous to the unglycosylated, high-mannose, and complex-carbohydrate form of the full-length EPO-R (38). The unglycosylated (24-kDa) form and the high-mannose (26-kDa) form were found in cell lysates (Fig. 5A, lanes 1, 3, 5, 7, and 9). The complex-carbohydrate form (28 kDa) began to accumulate in the supernatant after 1 h (Fig. SA, lane 4). After a 4-h chase period, 50% of the labeled complexcarbohydrate form was secreted into the supernatant fraction (Fig. SA, lane 10). An indirect immunoprecipitation assay was used to demonstrate that the sEPO-R was capable of binding EPO. By using this approach (data not shown), radiolabeled EPO was incubated with COS cell supernatants containing the sEPO-R. The anti-N-terminal EPO-R antiserum specifically immunoprecipitated a complex of sEPO-R bound to 125I-EPO. We next examined whether the sEPO-R will bind to gp55. COS-1 cells were transfected with sEPO-R cDNA alone or with the gp55 expression plasmid. As shown in Fig. 5B (lane

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55

2), the 24- and 26-kDa forms of the sEPO-R polypeptide immunoprecipitated with antiserum to the EPO-R but did not coimmunoprecipitate with gp55 antiserum (Fig. 5B, lane 4). Therefore, the extracytoplasmic domain of the EPO-R is necessary for gp55 binding (Fig. 4, lane A) but is not sufficient for gp55 binding (Fig. SB, lane 4); membrane anchorage of the EPO-R is also necessary for a productive gp55 interaction to occur. Analysis of cysteine point mutants of the EPO-R for EPO binding and gp55 binding. We next determined whether the transmembrane domain of the EPO-R is sufficient for gp55 activation. For this purpose, we generated point mutations of the extracellular EPO-R region, leaving the transmembrane region intact. Several point mutations were informative, particularly those involving the five cysteine residues of the extracytoplasmic region (Cl through C5) (Fig. 1). Four of these cysteine residues are conserved among members of the cytokine receptor superfamily. As shown for the human growth hormone receptor (HGH-R) (14, 18), intramolecular disulfide bonds are formed between Cl and C2 and between C3 and C4. A fifth cysteine, C5, is unique to the EPO-R. cDNAs encoding all five cysteine mutant polypeptides were transfected into Ba/F3 cells. Full-length EPO-R mutant receptors were evident by immunoprecipitation and had the same electrophoretic mobility as did the wild-type EPO-R (data not shown). Ba/F3 subclones expressing these mutant EPO-Rs were isolated and assayed for EPO binding (Fig. 6A) and for cell surface localization (Fig. 6B). Only the wild-type EPO-R polypeptide and the C5 mutant were transported to the cell surface, bound EPO at the cell surface, and conferred EPO-dependent and gp55-dependent cell growth (Fig. 7). Mutations of any one of the other four conserved cysteine residues (Cl through C4) resulted in EPO-R polypeptides which failed to confer either EPO-dependent or gp55-dependent cell growth. We also performed radiolabeled EPO binding studies on membrane preparations from Ba/F3 subclones (31) expressing wild-type or cysteine mutant EPO-R polypeptides. Only membranes prepared from Ba/F3 cells expressing the wild-type EPO-R or the C5 mutant demonstrated specific radiolabeled EPO binding (data not shown). Mutant polypeptides Cl through C4, enriched in these membrane preparations, still failed to bind EPO. In contrast, when the four mutant EPO-R polypeptides (Cl through C4) were coexpressed with gp55 in COS cells (Fig. 6C), they were found to coimmunoprecipitate with the envelope protein. These results confirm that the cysteine residues are not required for gp55 binding. All four cysteine mutants (Cl through C4) which fail to translocate to the cell surface coimmunoprecipitate with gp55, confirming our previous results that the bulk of the EPO-R/gp55 interaction occurs in an intracellular compartment (38). Although mutants Cl through C4 each contain a wild-type EPO-R transmembrane region and can bind and coimmunoprecipitate with gp55, they are not activated by gp55 to signal cell

of cell surface EPO-R polypeptide by using an antiserum to the N terminus of the EPO-R followed by radiolabeled protein A as a second label. Specific binding (done in triplicate) is shown. (C) COS cells were cotransfected with gp55 and EPO-R (mutant cDNAs), and the heterologous polypeptides were assayed for coimmunoprecipitation. cDNAs transfected encoded either mutant Cl (lanes 1 and 6), mutant C2 (lanes 2 and 7), mutant C3 (lanes 3 and 8), mutant C4 (lanes 4 and 9), or gp55 (lanes 5 through 10). Immunoprecipitation was with either the anti-amino-terminal EPO-R antiserum or the anti-gp7O antiserum. presence

.. ,;

FIG. 6. Characterization of EPO-R cysteine mutants for EPO binding, cell surface localization, and gp55 binding. (A) The cDNAs encoding wild-type (wt) EPO-R and the five cysteine mutants were stably transfected into Ba/F3 cells. Ba/F3 subclones were analyzed for cell surface EPO binding by using a 1 nM (saturating) concentration of radiolabeled EPO. Specific binding (done in triplicate) is shown. (B) The same Ba/F3 subclones were analyzed for the

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VI/T 7

I9

EPO-R EPO-R EPO-R IL-3R IL-3R IL-3R A B C

2955

IL-3R

ci

HR I

C5 sEPO-R EPO6R IL-6R

UI" Hl N 0, X zi_____uzvzizrizuzr +

+

III +

+

+

-

+

+

+

I

Surface Localization +

+

+

+

+

-

+

NA

+

NA

gp55 Binding

+

+

+

+

+

+

+

-

+

EPO Proliferation

+

+

+

+

+

-

+

gp55 Proliferation

+

+

-

+

_

6

EPO Binding

z

_ +

FIG. 7. Analysis of chimeric and mutant EPO-R polypeptides by multiple assays. All assays were performed in stable Ba/F3 transfectants except for the gp55 binding assay, which was performed in COS cells. Results for mutants C2, C3, and C4 (not shown) were identical to those shown for mutant Cl. WT, wild type; NA, not applicable.

growth. Mutant polypeptides Cl through C4 failed to bind EPO and failed to translocate to the cell surface, probably because of incorrect protein folding; however, their use in these studies has formally dissociated gp55 binding from gp55 activation. DISCUSSION

Using functional chimeric cytokine receptors, we have demonstrated that the transmembrane region of the EPO-R is necessary for gp55 activation. In accord with these data, the transmembrane regions of other membrane proteins contain functional information for signalling. For instance, a single amino acid mutation in the transmembrane region of the neu oncogene product, a tyrosine kinase cell surface receptor, has been shown to confer constitutive activation (4). In addition, transmembrane interactions mediate dimerization of the T-cell receptor complex (28) and of glycophorin A (8). Three potential models could explain the requirement of the transmembrane region of the EPO-R for gp55 activation. First, there may be a direct physical interaction between the EPO-R and the gp55 transmembrane regions. One face of the gp55 transmembrane a helix may bind to a face of the EPO-R transmembrane a helix. This direct binding of the gp55 would thereby lock the EPO-R into a constitutively activated conformation. Interestingly, the major amino acid differences between the gp55 polypeptides of the anemia and polycythemia strains of Friend SFFV reside in the transmembrane region of gp55 (11). Direct binding between gp55 and the EPO-R in the transmembrane region could therefore

account for the different phenotypes of these viruses in vivo.

In addition, a discrete binding domain in the transmembrane region of the EPO-R may be only one component of a larger structure that includes the extracellular domains of the EPO-R and gp55 as well. Second, the transmembrane domain of the EPO-R may not directly interact with gp55 but instead may alter EPO-R biosynthesis and processing. Contrary to this model, our results do not demonstrate a difference in the chimeric receptor expression level or cell surface localization compared with the wild-type EPO-R (Table 1). Third, the transmembrane domain may be required for gp55-induced but not EPO-induced signal transduction. It is interesting that the EPO-R/IL-3R A (and C) chimera is activated by EPO but not by gp55. This dissociation of EPO-induced and gp55-induced growth may be explained, at least in part, by our recent observation that EPO activates the EPO-R at the cell surface, while gp55 activates the EPO-R in a subcellular location (38). Our results also demonstrate that the extracytoplasmic region of the EPO-R interacts with gp55, but that it must be anchored to the membrane in order for binding to occur. Chimeras EPO-R/IL-3R A, B, and C (Fig. 4) and chimera EPO-R/IL-6R (data not shown) all specifically associate and coimmunoprecipitate with gp55, while the soluble EPO-R does not (Fig. 5B). Cells expressing the cysteine EPO-R mutants (Cl through C4), which each contain an intact transmembrane region and bind to gp55, are not activated by gp55. This finding suggests that the proper folding of the EPO-R extracytoplasmic region is critical for a productive gp55/EPO-R activation event. This proper conformation may also be required for transport of the EPO-R polypeptide

2956

ZON ET AL.

to the cell surface and for EPO binding. Further studies will be required to identify the critical amino acid residues of the extracytoplasmic region which are required for gp55 binding and EPO binding and to determine whether these binding sites are shared by the two molecules. Casadevall et al. (10) have recently demonstrated cross-linked molecular complexes containing EPO-R, EPO, and gp55, suggesting that the EPO and gp55 binding sites are distinct. Studies of the chimeric cytokine receptors described in this report have provided additional information regarding the structure and function of the superfamily members. First, this is the first demonstration that chimeric receptors of the cytokine receptor superfamily are functional for signal transduction. Second, these studies have demonstrated that the cytoplasmic region of the AIC2A polypeptide contains a signal transduction region which can substitute for the critical cytoplasmic region of the EPO-R. The AIC2A polypeptide, expressed alone in IL-2-dependent CTLL-2 cells, does not confer IL-3 responsiveness (22). This may be due to the lack of IL-3R subunits in CTLL-2 cells. Expression of AIC2A in these cells results in a low affinity for IL-3 (Kd = 10 nM). Substitution of the extracytoplasmic region of AIC2A with the extracytoplasmic region of the EPO-R results in EPO-induced signal transduction through the AIC2A cytoplasmic region. As expected, the cytoplasmic region of the IL-6R ot chain cannot substitute for the signalling regions of the EPO-R or AIC2A. Chimeric receptors with different cytoplasmic regions may therefore be useful in demonstrating function of the signal transduction domains of other cytokine receptors. Third, these studies have delineated the EPO binding and gp55 binding sites of the EPO-R. Since EPO-R/IL-3R C binds EPO but does not bind IL-3, the EPO binding site must be present amino terminal to the WSXWS motif of the EPO-R and may reside in the region from Cl (Cys-28) to C4 (Cys-82). This result is consistent with what has been found for the prolactin receptor (30) and the G-CSF receptor (19), which have ligand binding sites in the amino terminus. Finally, our results obtained with the chimeric polypeptides are consistent with certain aspects of the crystallographic structure of the HGH-R (18), a member of the cytokine receptor superfamily. According to this structure, the HGH-R has two distinct domains which contribute to ligand binding and which promote receptor dimerization. Further studies will be required to determine whether EPO and gp55 promote EPO-R dimerization. The gp55/EPO-R transmembrane interaction in Ba/F3 cells appears to mediate the constitutive activation of the EPO-R and mitogenesis. In addition, the extracytoplasmic regions of these polypeptides interact, providing a potential mechanism for both the specificity and modulation of the signalling process. Several recent studies demonstrate that this interaction between the EPO-R and gp55 accounts for the early stage of Friend virus-induced erythroleukemia (2, 27). In addition, the interaction between the EPO-R and gp55 persists, even during the late (leukemogenic) stage of Friend disease (26). One prediction of our results, therefore, is that interference with the transmembrane interaction of gp55 and the EPO-R could convert these cells to a nontransformed

EPO-dependent state. ACKNOWLEDGMENTS We thank D. Nathan, S. Lux, and G. Fasman for helpful discussions and C. Stiles for reviewing the manuscript. The IL-6R cDNA was generously supplied by T. Taga and T. Kishimoto. This work was supported in part by Physician-Scientist Award NIH-HL02347 and by a grant from the Robert Wood Johnson

MOL. CELL. BIOL.

Pharmaceutical Research Institute to L.I.Z. L.I.Z., B.M.-P., and A.D.D. were also supported by grant P01 HL32262-10 from the National Institutes of Health. A.D.D. is a Lucille P. Markey Scholar, and this work was supported in part by a grant from the Lucille P. Markey Charitable Trust. REFERENCES 1. Adachi, A., K. Sakai, N. Kitamura, S. Nakanishi, 0. Niwa, M. Matryama, and A. Ishimoto. 1984. Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA. J. Virol. 50:813-821. 2. Aizawa, S., Y. Suda, Y. Furuta, T. Yagi, N. Takeda, N. Watanabe, M. Nagayashi, and Y. Ikawa. 1990. Env-derived gp55 gene of Friend spleen focus forming virus specifically induces neoplastic proliferation of erythroid progenitor cells. EMBO J. 9:2107-2116. 3. Amanuma, H., A. Katori, M. Obata, N. Sagata, and Y. Ikawa. 1983. Complete nucleotide sequence of the gene for the specific glycoprotein gp55 of Friend spleen focus-forming virus. Proc. Natl. Acad. Sci. USA 80:3913-3917. 4. Bargman, C., M. Hung, and R. A. Weinberg. 1986. Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649657. 5. Bazan, J. F. 1989. A novel family of growth factor receptors: a common binding domain in the growth hormone, prolactin, erythropoietin and IL-6 receptors, and the P75 IL-2 receptor beta chain. Biochem. Biophys. Res. Commun. 164:788-793. 6. Bazan, J. F. 1990. Structural design and molecular evolution of a cytokine receptor superfamily. Proc. NatI. Acad. Sci. USA

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tal structure of the complex. Science 255:306-312. 19. Fukunaga, R., E. Ishizaka-Ikeda, C.-X. Pan, Y. Seto, and S. Nagata. 1991. Functional domains of the granulocyte colonystimulating factor receptor. EMBO J. 10:2855-2865. 20. Hoatlin, M. E., S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak, and D. Kabat. 1990. Activation of erythropoietin receptors by Friend viral gp55. Proc. Natl. Acad. Sci. USA 87:99859989. 21. Itoh, N., S. Yonehara, J. Schreurs, D. M. Gorman, K. Maruyamna, A. Ishii, I. Yahara, K.-I. Arai, and A. Miyajima. 1990. Cloning of an interleukin-3 receptor gene: a member of a distinct receptor gene family. Science 247:324-327. 22. Kitamura, T., K. Hayashida, S. Sakamaki, T. Yokota, K.-i. Arai, and A. Miyajima. 1991. Reconstitution of functional receptors for human granulocyte/macrophage colony-stimulating factor GM-CSF: evidence that the protein encoded by the AIC2B cDNA is a subunit of the murine GM-CSF receptor. Proc. Natl. Acad. Sci. USA 88:5082-5086. 23. Koch, W., W. Zimmermann, A. Oliff, and R. Friedrich. 1984. Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences. J. Virol. 45:828-840. 24. Li, J.-P., and D. Baltimore. 1991. Mechanism of leukemogenesis induced by the MCF murine leukemia viruses. J. Virol. 65:24082414. 25. Li, J. P., R. K. Bestwick, C. Spiro, and D. Kabat. 1987. The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor. J. Virol. 61:2782-2792. 26. Li, J.-P., A. D. D'Andrea, H. F. Lodish, and D. Baltimore. 1990. Activation of cell growth by binding of Friend spleen focusforming virus gp55 glycoprotein to the erythropoietin receptor. Nature (London) 343:762-764. 27. Longmore, G. D., and H. F. Lodish. 1991. An activating mutation in the murine erythropoietin receptor induces erythroleukemia in mice: a cytokine receptor superfamily oncogene. Cell 67:1089-1102. 28. Manolios, N., J. S. Bonifacino, and R. D. Klausner. 1990. Transmembrane helical interactions and the assembly of the T

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