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Dual oncostatin M (OSM) receptors. Cloning and characterization of an alternative signaling subunit conferring OSM-specific receptor activation.

Oncostatin M (OSM) is a cytokine whose structural and functional features are similar to other members of the interleukin (IL)-6 family of cytokines (IL-6, IL-11, leukemia inhibitory factor (LIF), granulocyte colonystimulating factor, ciliary neurotrophic factor, and cardiotrophin-1), many of which utilize gp130 as a common receptor subunit. A biologically active OSM receptor has been previously described that consists of a heterodimer of leukemia inhibitory factor receptor (LIFR) and gp130. This LIFR·gp130 complex is also a functional receptor for LIF. We have cloned and characterized an alternative subunit (OSMRβ) for an OSM receptor complex (a heterodimer of gp130 and OSMRβ) that is activated by OSM but not by LIF. The signaling capability of specific receptor subunit combinations was analyzed by independent assays measuring cell proliferation or induction of acute phase protein synthesis. Our results demonstrate that both LIF and OSM cause tyrosine phosphorylation and activation of the gp130·LIFR combination, but the gp130·OSMRβ complex is activated by OSM only. OSM-induced cellular responses, initiated through low affinity binding to gp130, are mediated by two heterodimeric receptor complexes that utilize alternative signal transducing subunits that confer different cytokine specificities to the receptor complex.

Oncostatin M (OSM), 1 a cytokine produced by activated monocyte and T-lymphocyte cell lines, shares significant similarities in primary amino acid sequence and predicted secondary structure with members of the IL-6 family of cytokines including IL-6, IL-11, ciliary neurotrophic factor (CNTF), granulocyte colony-stimulating factor (G-CSF), cardiotrophin-1 (CT-1), and most notably with leukemia inhibitory factor (LIF) (1)(2)(3)(4). Similarities in gene structure among G-CSF, IL-6, OSM, and LIF suggest that these four cytokines have evolved from a common ancestral gene and are all members of a single cytokine family. The genes encoding LIF and OSM have both been localized to chromosome 22 and share identical exon organization (1,5).
LIF and OSM also share pleiotrophic biological activities. Among these are the ability to induce differentiation of the murine myeloid leukemic cell line, M1 (6), a property also shared with IL-6 and G-CSF. LIF and OSM inhibit differentiation of embryonic stem cells in vitro at comparable dose ranges (7), and both induce acute phase protein synthesis by hepatocytes (8).
Despite a number of shared biological effects of OSM and LIF, activities specific to OSM have also been reported. OSM but not LIF inhibits the growth of A375 human histiocytic melanoma cells (9 -11), as well as a variety of tumor cell lines of lung, breast, ovary, and stomach origin (12). Conversely, OSM but not LIF stimulates the growth of a number of normal fibroblast lines, rabbit vascular smooth muscle cells (13), and bovine aortic endothelial cells (14). A number of reports identify OSM as a potent growth and differentiation factor for AIDS-related Kaposi's sarcoma-derived spindle cells (15)(16)(17)(18)(19).
Biological effects of many of the IL-6 family of cytokines are mediated through cell-surface multimeric receptors involving cytokine-specific subunits and a common signaling subunit, gp130 (reviewed by 20). For example, assembly of a functional LIF receptor (21) involves ligand binding to the LIF receptor (LIFR) with low affinity followed by association with gp130 to form a high affinity signal transducing heterodimeric complex (Fig. 1).
Although gp130 functions as the high affinity converting and signaling subunit in the receptor complexes for a number of cytokines (LIF, IL-6, IL-11, CNTF, and CT-1), OSM is the only one of this group of cytokines that can bind specifically and directly to gp130 with low affinity (21,22). While OSM interaction with gp130 alone is incapable of inducing a proliferative response in permissive cells (23), association of LIFR with the low affinity OSM⅐gp130 complex forms a high affinity OSM signal transducing receptor (21) described as the LIF/OSM receptor or type I OSM receptor (24). Therefore, both OSM and LIF utilize a heterodimeric gp130⅐LIFR complex capable of transducing signaling events after either LIF or OSM binding. This signal transducing gp130⅐LIFR combination is also utilized by two other known cytokines, CT-1 (25) and CNTF. However, CNTF first binds to a specific low affinity receptor (CNTFR) before forming the high affinity CNTFR⅐LIFR⅐gp130 complex (26,27).
On a number of cell types, however, high affinity functional OSM receptors have been observed that are incapable of binding LIF and represent a second OSM-specific receptor system (24,28). Disruption of OSM/gp130 interaction with antibodies to gp130 blocks signal transduction through the OSM-specific receptor as well as through the shared receptor (23) suggesting that additional subunits are required for the OSM⅐gp130 complex to form high affinity signaling receptor complexes. The OSM-specific receptor, or type II OSM receptor (24), while utilizing the OSM⅐gp130 complex, must include a second as yet uncharacterized subunit distinct from LIFR. Interactions between OSM and various receptor subunit combinations are shown schematically in Fig. 1. This dual OSM receptor system provides an explanation for numerous OSM biological activities, some of which are shared by LIF and some of which are OSM-specific.
Utilizing the high degree of homology among the signaling components in the gp130 receptor family, we have successfully cloned, through degenerate PCR, a novel hematopoietin receptor which we call the oncostatin M-specific receptor ␤ subunit or OSMR␤. This subunit, which does not directly bind any cytokine we tested, associates with the low affinity OSM⅐gp130 complex to form a high affinity heterodimeric receptor that is capable of transducing OSM-specific signaling events.

EXPERIMENTAL PROCEDURES
Generation of PCR Library-The pool of sense orientation degenerate oligonucleotides TT(T/G)(C/A)(G/A)(G/T)(A/G)T(T/A)CG(C/G/T)(T/ A)G(T/C) and antisense oligonucleotides (A/G)CTCCA(G/T)T(C/T)(G/ A)CTCCA were used to perform PCR amplification utilizing human genomic DNA (Clontech Laboratories, Inc., Palo Alto, CA) as template. Amplification was performed in 100-l reactions with 200 ng of DNA template under standard salt conditions (Perkin-Elmer Corp.). Optimal PCR conditions were denaturation for 1 min at 94°C, annealing for 3 min at 50°C, and polymerization for 1 min at 72°C for 35 cycles. PCR products were blunted with T4 polymerase, kinased, and phenol/chloroform-extracted before separation on a 6% low melting point agarose gel. Fragments approximately 50 base pairs in length were subcloned into Bluescript SK previously linearized with EcoRV and dephosphorylated. Individual transformants were transferred to single wells in 96-well microtiter trays, amplified, and frozen in 20% glycerol at Ϫ70°C. Individual clones were amplified, and insert DNA was sequenced on an Applied Biosystems Automated DNA sequencer.
Screening of cDNA Libraries-A specific sequence oligonucleotide (5Ј-GCTGATGCCAGCCACTTCTGGAAA-3Ј) was used as a kinased oligonucleotide probe to screen previously described cDNA libraries according to standard procedures (29). Hybridization conditions were essentially as described (30). Positive isolates were identified following high stringency washing conditions (0.2 ϫ SSC, 0.1% SDS at 63°C). DNA sequences were obtained using vector-and cDNA-derived oligonucleotide primers on denatured double-stranded templates following subcloning according to standard procedures (29).
Proliferation Assay-Generation of BAF-B03 cell lines expressing gp130 and gp130/LIFR has been described (31). To create a cell line expressing gp130 and OSMR␤, full-length OSMR␤ cDNA was subcloned into the expression vector pDC411 derived from the previously described pDC409 vector (32), modified to contain the hygromycin resistance gene, and electroporated into BAF-gp130 cells. A drug-resistant BAF cell line expressing gp130 and OSMR␤ was selected by limiting dilution. To measure proliferation, transfected BAF cells were cultured in 96-well microtiter plates (1 ϫ 10 4 cells per well, 0.2 ml per well) with test samples for 72 h and pulse-labeled with [ 3 H]thymidine (0.5 mCi per well) for 5 h. Cells were harvested on glass filters, and cell-associated radioactivity was measured by a scintillation counter.
Generation of OSMR␤-specific Antibodies-A rabbit antiserum directed against OSMR␤ was obtained by nine subsequent immunizations of New Zealand White rabbits with a protein consisting of the hematopoietin domains of OSMR␤ fused to the Fc domain of human IgG (described below).
For generating mAbs, Balb/c mice were immunized three times with 15 g of OSMR␤-Fc in RIBI adjuvant (Ribi Corp., Hamilton, MT). Two weeks after the last immunization, mice were boosted intravenously with 5 g of OSMR-Fc. Three days later the mice were sacrificed, and spleen cells were fused to Ag8.653 myeloma cells (ATCC) with 50% polyethylene glycol, 10% dimethyl sulfoxide solution (Sigma). Hybridoma supernatants were screened by antibody capture assay (ABC). Briefly, 96-well plates (Flow) were coated overnight with goat antimouse Ig (Zymed) and then washed with PBS ϩ 0.05% Tween 20. Supernatants were incubated for 1 h at room temperature and then washed four times with PBS, 0.05% Tween 20. OSMR␤-Fc:biotin was then added to each well for 1 h, followed by four washes. Streptavidin was added for 15 min. The plates were washed again and developed with 3,3',5,5'-tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD). After developing, the plates were read on a plate reader at 650 nm. Positive wells were subsequently screened to remove antibodies reactive with human Ig. Positive cells were cloned and grown in bulk in culture. Supernatants were purified over a protein A column (Bio-Rad) and stored at Ϫ20°C.
Hep3B Signaling Assay-Gene splicing by overlap extension (33) was utilized to fuse the haptoglobin promoter region from nucleotides Ϫ165 to ϩ25 (34) to the open reading frame of IL-2R␣ (35) and subcloned to Bluescript SK. Five million Hep3B or HepG2 cells per well were plated in six-well culture plates (Costar) and allowed to adhere overnight. Two micrograms of DNA (equivalent amounts of each component in heterogeneous mixtures) were transfected with lipofectamine essentially as described by the manufacturer (Life Technologies, Inc.). After 48 h, expression of IL-2R␣ on the cell surface was determined essentially as described (36).
Ligand Binding to Chimeric Receptors and Transfected BAF Cells-Recombinant LIF and OSM were produced in yeast and purified as reported (37) and radioiodinated to a specific activity of 1.2-9.2 ϫ 10 15 cpm/mmol as described previously (31). DNA encoding the hematopoietin receptor domains of gp130, LIFR, and OSMR␤ was each fused to the Fc region of human IgG1 (gp130-Fc, LIFR-Fc, and OSMR␤-Fc, respectively) and expressed in CV-1/EBNA cells as described (38). Radiolabeled ligand (1 ϫ 10 Ϫ9 M) was incubated for 18 h at 4°C with 500 l of CV-1/EBNA conditioned supernatant and 25 l of protein-A Sepharose (Pharmacia Biotech Inc.) as prepared by the manufacturer's directions. Sepharose beads were washed once with PBS and purified over a 1-ml pad of 5% glucose in PBS. Radioactivity associated with the purified beads was quantitated on a gamma counter. Binding studies with BAF-BO3 cell lines expressing gp130 and gp130/OSMR␤ were performed as described previously (31).
Northern Analysis-Messenger RNA was isolated, blotted onto nylon membranes, and probed essentially as described (30). Blots were hybridized overnight with 32 P-labeled riboprobes from the entire coding region and washed using high stringency conditions (see above).

Cloning of a Novel Receptor by Degenerate PCR-Functional
receptors for IL-6 family cytokines are multisubunit complexes involving related molecules that are all members of the hematopoietin receptor superfamily. These receptors are distinguished by a 200-amino acid extracellular hematopoietin domain that is divided into two fibronectin type III modules. The amino-terminal module contains two cysteine pairs with the FIG. 1. Formation of signaling receptor complexes for LIF and OSM. Thin arrows represent low affinity binding of OSM to gp130 or LIF binding to LIFR. High affinity binding of both LIF and OSM to the gp130⅐LIFR complex and OSM binding to gp130 and a specific ␤ subunit is indicated by broad arrows. Broken arrows represent capability for signal transduction from the two functional complexes. cysteine residues within each pair separated by 8 -10 amino acids. Following a proline-rich hinge region, the second module contains a highly conserved Trp-Ser-X-Trp-Ser motif (39). Other than these features, sequence similarity is relatively low.
A subset of hematopoietin receptors (including gp130, LIFR, and G-CSFR) which share higher overall homology are also distinct from other hematopoietin receptors in having three fibronectin type III repeats separating the hematopoietin domain from the transmembrane domain (LIFR also contains a second hematopoietin domain at the amino terminus). A number of homologous regions among these receptors have been identified (37,40).
Degenerate oligonucleotides were designed from a number of homologous regions between gp130, LIFR, and G-CSFR and tested for their ability to amplify each receptor by PCR. Nucleotide sequence options in the degenerate oligonucleotides were designed to allow all possible sequence variations among the members. Successful degenerate oligonucleotides were selected for sense and antisense orientation and redesigned to allow more sequence heterogeneity by incorporating sequences from the ␥ chain of the IL-2 receptor. The regions chosen for degenerate primers are structured around amino acids 296 -300 (FRIRC) and 310 -314 (WSDWS), respectively, of human gp130 (41). These oligos were then used to amplify PCR products from human genomic DNA. After subcloning, a library of individual PCR fragments was established. To analyze the quality of the library, DNA from 12 randomly chosen isolates was sequenced. Comparison of these sequences to known hematopoietin receptors showed sequences from gp130, LIFR, and two clones encoding a novel sequence showing strong homology to LIFR.
An oligonucleotide specific for the novel sequence was used to screen three independent libraries generated from human placenta, a bone marrow stromal cell line (IMTLH), and a fibroblast cell line (WI-26 VA4). Comparison of overlapping clones Placenta 2B (Pl-2B), IMTLH-73 (IM-73), and WI-26 20B (WI-20B) ( Fig. 2A) resulted in a 3096-base pair sequence containing a complete open reading frame encoding a protein of 979 amino acids (Fig. 2B).
The predicted primary sequence includes a potential hydrophobic leader sequence (residues 1-27), the second half of a hematopoietin domain including a WSNWS motif (residues 28 -150), an Ig-like domain (residues 151-240), a second complete hematopoietin domain containing four conserved cysteine residues, and a WSEWS sequence (residues 240 -430) followed by three fibronectin type III repeats (residues 431-739) that are characteristic of this subset of hematopoietin receptors. This large extracellular domain is followed by a 22-amino acid transmembrane domain (residues 740 -761) and a 218-amino acid cytoplasmic domain.
Comparison of this novel protein to gp130 and LIFR shows considerable homology in both domain structure and primary amino acid sequence. The general structure is identical to that of LIFR except that this molecule lacks the cysteine-containing module of the amino-terminal hematopoietin domain which is present in LIFR. Considering the homology in structure and expected homology in function of an OSM specific signaling subunit, we analyzed this molecule for its ability to initiate OSM-specific signaling events.
Initiation of Cell Proliferation through OSMR␤ Signaling-The IL-3-dependent murine pre-B cell line, BAF-BO3, is commonly used to analyze the ability of cytokine receptors to mediate proliferative responses. These cells have been reported to have no gp130 and do not bind or proliferate in response to LIF or OSM (31,41). Constructs encoding the various receptor subunits were transfected into BAF cells, and lines were se-lected that demonstrated stable expression of gp130, gp130 with LIFR (31), or gp130 with OSMR␤ (BAF-gp130, BAF-gp130/LIFR, and BAF-gp130/OSMR␤, respectively). Fig. 3A shows the proliferative responses of these various cell lines after stimulation with 50 ng/ml LIF or OSM. As previously reported, untransfected or gp130 expressing BAF cells showed no proliferative response to either LIF or OSM. The BAF-gp130/LIFR cell line (expressing LIF/OSM or type I OSM receptors) is capable of proliferation after stimulation with either LIF or OSM. BAF-gp130/OSMR␤ cells demonstrate a proliferative response after OSM stimulation but show no response to LIF.
Antibodies (polyclonal and monoclonal) specific for the hematopoietin domain of OSMR␤ were tested for their ability to inhibit the OSM-specific induction of BAF-gp130/OSMR␤ proliferation. These antibodies effectively blocked the OSM-induced proliferation of these cells showing a requirement for a functional OSMR␤ subunit in the signal transducing complex. Representative data from inhibition curves with anti-OSMR␤ mAb M70 are shown in Fig. 3B. Complete inhibition of OSMinduced proliferation (up to 100 ng/ml) is achieved with antibody concentrations less than 500 ng/ml. Equilibrium binding studies were utilized to measure the binding parameters of OSM to parental BAF-B03 cells, BAF-gp130, and BAF-gp130/OSMR␤ cells. In this and in a previous report (31), binding of OSM to parental BAF and BAF-gp130 was below detectable levels. Utilizing Biosensor technology, the binding affinity of OSM to gp130 has been measured at approximately 2 ϫ 10 7 M Ϫ1 (data not shown) which may be undetectable on BAF-gp130 cells by standard Scatchard analysis. When OSMR␤ was coexpressed with gp130 in the BAF cells, 125 I-OSM binding was converted to high affinity. From an average of seven binding experiments on the BAF-gp130/OSMR␤ cells, OSM displayed a binding affinity (K a ) of 2.8 Ϯ 2.5 ϫ 10 9 M Ϫ1 with 109 Ϯ 73 total sites per cell (data not shown). This affinity is consistent with values derived from a variety of other cell types that express predominantly type II OSM receptors (19,24). In addition, binding of 125 I-LIF to the BAF-gp130/OSMR␤ cells was undetectable.
Ligand Binding to LIF and OSM Receptor Subunits-Individual receptor components were tested for their capacity to bind to LIF or OSM through ligand binding to soluble versions of each molecule. Chimeric versions of gp130, LIFR, and OSMR␤ were constructed in mammalian expression vectors with the extracellular ligand binding portions of each receptor fused to the Fc region of human IgG1 (gp130-Fc, LIFR-Fc, and OSMR␤-Fc, respectively) (38). Expression in CV-1/EBNA monkey kidney cells (42) yields supernatants containing soluble dimeric forms of each receptor subunit. Supernatants from cells transfected with individual subunits or cotransfected with combinations of subunits (resulting in expression of a mixture of homodimeric and heterodimeric forms of the receptors) were incubated with 125 I-labeled LIF or OSM. The receptor-ligand complexes were bound to protein A-agarose beads and separated from free ligand to quantitate binding. OSM bound only to gp130 and not to LIFR or OSMR␤, and LIF bound only to LIFR (Fig. 4). The LIF⅐OSM receptor complex (gp130-Fc and LIFR-Fc molecules) bound both ligands, but the combination of gp130-Fc and OSMR␤-Fc molecules (OSM-specific receptor) bound only OSM.
OSM-specific Induction of Acute Phase Protein Synthesis-The acute phase response is a complex phenomenon associated with acute inflammation that involves dramatic changes in the blood level of a variety of liver-derived plasma proteins (43). Utilizing cultured primary hepatocytes as well as hepatocyte cell lines, IL-6, LIF, OSM, CNTF, IL-11, and CT-1 have been characterized as mediators of the acute phase response causing induction of a number of proteins including haptoglobin, ␤-fibrinogen, ␣ 1 -antichymotrypsin, and ␣ 2 -macroglobulin (8, 44 -48). Two commonly utilized cell lines, HepG2 and Hep3B, induce haptoglobin protein synthesis after IL-6 stimulation demonstrating functional levels of IL-6 receptor and gp130 on both of these cell lines (45). While LIF and OSM also induce haptoglobin expression in HepG2 cells, the Hep3B cell line is nonresponsive. Hep3B cells, which do not express LIFR, become responsive to these cytokines only after transfection with LIFR cDNA to supply the missing subunit for high affinity LIF/OSM receptors (47). We utilized this system to study the signaling capabilities of receptor complexes following transfection of receptor subunits into Hep3B cells.
A reporter gene construct was designed to link the cytokine responsive element from the haptoglobin gene (49) to the coding region of the IL-2 receptor p55 subunit (IL-2R␣) causing expression of cell-surface associated IL-2R␣ to be regulated by cytokine-induced activation of the haptoglobin response elements. Using cotransfection of this reporter gene along with expression vectors encoding potential cytokine receptor components into Hep3B cells, we analyzed the functional capacities of reconstructed receptor complexes. The magnitude of the response was quantitated by measuring the amount of cytokineinducible IL-2R␣ expression.
Using this assay, we tested the ability of endogenous gp130, transfected OSMR␤, or transfected OSMR␤ supplemented with cotransfected gp130 to induce haptoglobin production when stimulated by a variety of IL-6 family cytokines. As shown in Fig. 5, Hep3B cells transfected with vector only responded only to IL-6. Expression of OSMR␤ on Hep3B cells allowed a response to both IL-6 and OSM. Supplementing these cells with transfected gp130 nearly doubled the response indicating that the endogenous level of gp130 may be a limiting factor. All other tested IL-6 family cytokines failed to induce a response through the gp130⅐OSMR␤ signaling complex.
We also tested the ability of OSMR␤ to mediate CNTF induction of acute phase protein production. Although CNTF alone does not induce haptoglobin synthesis on hepatoma cells, HepG2 cells transfected with CNTFR or Hep3B cells cotransfected with CNTFR and LIFR become responsive to CNTF regulation of haptoglobin production (47). Since CNTFR has been demonstrated to mediate CNTF responses in a released or soluble form (26), we utilized a mixture of CNTF and soluble CNTFR to analyze the ability of type II OSM receptors to mediate CNTF acute phase protein induction. While the CNTF⅐CNTFR complex was able to stimulate haptoglobin expression in HepG2 cells (approximately 3-fold), no stimulation of haptoglobin expression in Hep3B cells was detected after cotransfection and expression of either gp130, OSMR␤, or a combination of both (data not shown). Although CNTF plus soluble CNTFR is capable of inducing signal transduction in HepG2 cells which express endogenous gp130 and LIFR, Hep3B cells expressing gp130 and OSMR␤ were not able to stimulate acute phase protein induction. These results demonstrate that OSMR␤ cannot replace LIFR in the CNTF receptor complex.
Specific Immunoprecipitation of OSM Receptor Subunits-Early events in the signal transduction cascade of the gp130 family of receptors include tyrosine phosphorylation of a variety of cellular proteins including the receptor components themselves (50,51). Utilizing anti-phosphotyrosine antibodies, we analyzed the phosphorylation of specific receptor components in various cell types after stimulation with either LIF or OSM.
JAR cells (which express type I OSM receptors only) or WI26-VA4 cells (which express predominantly type II OSM receptors) (24) were either mock-stimulated or stimulated with LIF or OSM followed by immunoprecipitation with OSMR␤specific polyclonal antibody. Recovered protein was separated by SDS-PAGE, blotted to nitrocellulose, and developed with an anti-phosphotyrosine antibody (␣-Tyr(P) mAb). A phosphorylated band of approximately 180 kDa was present only in cells containing type II OSM receptors and only after OSM and not LIF stimulation (Fig. 6A). Therefore phosphorylation of this subunit appears to be mediated only through the type II-or OSM-specific receptor.
Phosphorylation of individual components of type I and type II OSM receptors was analyzed by utilizing HeLa cells (which express both type I and type II OSM receptors) either unstimulated or stimulated with OSM (Fig. 6B). Western blots developed with the ␣-Tyr(P) mAb showed a phosphorylated band of 150 kDa specifically immunoprecipitating with a polyclonal antibody against gp130. Anti-OSMR␤ antibody immunoprecipitated a 180-kDa band, and anti-LIFR antibodies immunoprecipitated a 190-kDa band; all three subunits become phosphorylated after OSM stimulation. The sizes of the immunoprecipitated LIFR and gp130 phosphoproteins correlated well with previous reports (50), and the intermediate size of the phosphoprotein purified with anti-OSMR␤ antibodies corresponded with the size predicted from the primary sequence of OSMR␤.
Tissue Distribution-Northern analysis of mRNA from a wide variety of cell types defined a single, high molecular weight species encoding OSMR␤ (Fig. 7A). Attempts to examine the tissue distribution and relative expression levels of OSM and LIF receptors by Northern blot proved unsatisfactory due to variable degradation of large mRNAs during preparation. To avoid this problem as well as obtain a more accurate semi-quantitative measure of mRNA levels, we established a PCR-based analysis using specific internal primers for OSMR␤, gp130, and LIFR. First strand cDNA from each tissue or cell source was used as PCR template to incorporate quantitative labeling into specific PCR fragments (Fig. 7B). After gel fractionation, labeled bands were quantitated and normalized for the amount of DHFR mRNA present in each cDNA and for the PCR efficiency of each template. The normalized numbers, shown in Table I, represent relative amounts of mRNA in each tissue type. The distribution of OSM receptors appears to be quite broad with the exception of hematopoietic tissues and cell lines. OSMR␤ is present at relatively high levels in all neural cells tested as well as fibroblast, epithelial, and a variety of tumor cell lines. DISCUSSION OSM signaling on responsive cells is mediated by binding to two distinct signaling receptor complexes identified as type I and type II OSM receptors. Signaling through both receptor types is initiated by OSM binding to gp130 with low affinity. The type I OSM receptor is subsequently formed by association of LIFR generating the high affinity signaling complex. This receptor complex of gp130 and LIFR is also formed by LIF and therefore constitutes a dual specificity receptor activated by both LIF and OSM. In this report we describe the cloning and characterization of OSMR␤, a receptor subunit homologous to LIFR which also forms a high affinity signaling receptor after association with the OSM⅐gp130 complex. These subunits, gp130 and OSMR␤, form the type II OSM receptor which is activated specifically by OSM. This concept of alternative signaling receptors formed by association of distinct secondary subunits has been recently described for the IL-4 and IL-13 receptors (52). In this analogous system, IL-4 binds to a specific low affinity receptor which then associates with either the IL2R␥ chain or the low affinity IL-13R. IL-4 signaling in a variety of cells has been shown to be a function of the distribution of IL-13R and IL-2R␥ (53).
The domain structure of OSMR␤ is similar to the structures within the gp130 family of cytokine receptors. The members of this family are OSMR␤, gp130, LIFR, G-CSFR, IL-12R (54), and the recently cloned leptin receptor (55). A feature that distinguishes this family from other hematopoietin receptors is the multiple fibronectin type III repeats (Fig. 8). OSMR␤ shares closest homology with LIFR (32% amino acid identity). The most obvious structural difference between LIFR and OSMR␤ is that OSMR␤ lacks the cysteine containing subdomain of the amino-terminal hematopoietin domain that is present in LIFR. Although the significance of this deletion is unknown, it is interesting to speculate that the loss of this subdomain may contribute to the loss of LIF binding capability which is the most obvious biological distinction between these subunits. Interesting from a structural point of view is that OSMR␤ is the only known example of a hematopoietin receptor where the two-subdomain structure within the hematopoietin domain is not conserved. Homology between the two molecules outside of the hematopoietin domains is lower, although the overall size of the cytoplasmic domains of the two alternative subunits (218 and 238 amino acids in OSMR␤ and LIFR, respectively) remains very similar.
Specific regions within the cytoplasmic domain of G-CSFR, LIFR, and gp130 necessary for signal transduction have been previously described (56,57). The membrane proximal Box 1 motif, shared by many of the hematopoietin receptors, and the FIG. 6. Specific immunoprecipitation of OSM and LIF receptor components. A, JAR or WI-26 cells were either left untreated or stimulated with 100 ng/ml OSM or LIF for 10 min at 37°C as indicated. Cells were lysed in 1% Triton X-100 buffer, and receptors were immunoprecipitated with polyclonal antibody specific for the hematopoietin domain of OSMR␤ followed by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, and phosphotyrosine containing proteins were visualized by Western blotting with an ␣-Tyr(P) mAb. B, HeLa cells were treated with OSM (100 ng/ml) for 10 min at 37°C, followed by lysis. Proteins immunoprecipitating with anti-gp130 immune serum, anti-OSMR␤ immune serum, or with an anti-LIFR mAb were resolved by SDS-PAGE. Phosphorylated proteins were visualized with ␣-Tyr(P) antibody as above.
FIG. 7. Analysis of OSM receptor distribution. A, total RNA Northern blots (5 g/lane) were probed with riboprobes generated from cDNA encoding the hematopoietin domain of OSMR␤. Probe hybridization was at 68°C in Starks Buffer. The blot was then washed with 0.2 ϫ SSC, 0.2% SDS at 68°C before autoradiography. B PCR primers specific for gp130, LIFR, or OSMR␤ were used to amplify first strand cDNA from various sources. Amplification was performed for 40 rounds in the presence of [ 32 P]dCTP. Radiolabeled PCR products were separated through agarose gels, blotted onto nylon membranes, and exposed to autoradiographic film. less well conserved Box 2 region are critical for the transduction of a proliferative response. These elements appear to be involved in activation of the Janus (Jak) family of tyrosine kinases (58 -61). A third region specific for the gp130 family of hematopoietin receptors, Box 3, is also required for a subset of activities involving IL-6-responsive elements (57,62). Although Box 3 is not well conserved, there is a conserved Tyr-X-X-Gln motif in which the tyrosine becomes phosphorylated after stimulation and is necessary for Stat 3 activation (63). Multiple copies of this motif are present in the carboxyl-terminal regions of many of the gp130 family of receptors, although the functional relationship between these multiple motifs has not yet been established.
Amino acid sequence comparison of G-CSFR, gp130, and LIFR to the cytoplasmic tail of OSMR␤ identify regions analogous to Box 1 (amino acids 771-777) and Box 2 (amino acids 809 -820). The Box 3 motif is present with two copies (amino acids 917-920 and 945-948) in the OSMR␤ carboxyl-terminal region. The biological assays utilized in this report, cell proliferation, which requires functional Box 1 and Box 2 sequences, and haptoglobin synthesis, which requires all three functional domains (57), indicate that OSMR␤ is fully competent to signal through all three domains. Given the presence of clearly homologous signaling domains between the gp130 family signal transducing molecules and specifically LIFR and OSMR␤, many overlapping signal transducing elements and functions would be expected to be activated.
Given that cytoplasmic domains able to activate the Jak/ STAT signal transduction pathway are present in both OSM receptor forms, differences in biological activities between OSM and LIF must be ascribed to less well characterized signaling events specific to different receptor complexes. The presence of a second signaling receptor specific for OSM allows for overlapping of LIF and OSM signals through the LIF/OSM receptor and for an OSM-specific set of signals through the OSM-specific receptor.

RT-PCR analysis of OSM receptor components from various cell sources
Blots of radiolabeled PCR-generated products were exposed to Phosphorimager screens, and the resulting image was scanned. Counts incorporated in individual bands were normalized for the amount of cDNA (DHFR control) and for amplification efficiency of each template. An arbitrary scale indicates the relative amounts of specific mRNAs from various sources. The ratio of type I and type II OSM receptors and their distribution on various cells is a potential mechanism for regulating the differentiation of signals mediated by OSM and LIF. Current analysis of the distribution of OSM receptors on a limited array of cell types shows a relatively even and broad distribution of type I and type II receptors on many normal neural and fibroblast type cell lines, yet the ratio of OSM receptor types varies significantly in many of the tumor and carcinoma cell lines, indicating a potentially different role for LIF and OSM in the regulation of these cell types. FIG. 8. Homology among members of the gp130 family of receptors. The complete amino acid sequence for each receptor was compared using the ALIGN program which computes the optimal alignment of two sequences and calculates the percent amino acid identity.