Production of soluble form of human TNF-alpha ligand-binding domain type 1 receptor by expression in Drosophila cells

5His-tagged human TNFalpha type I receptor (TNFR1) ligand-binding domain was produced in Drosophila cells under control of metallothionein Cu-inducible promoter and purified by Ni-NTA affinity chromatography to homogeneity. TNFR1 gene fragment was cloned by PCR from CD8+ in vitro cultured T-killer normal linage cDNA. In despite of three disulfide bonds, the recombinant protein was correctly folded which was conformed by TNFalpha ligand binding assay in ELISA variant.     

The WSAWS motif is C-hexosylated in a soluble form of the erythropoietin receptor

The WSXWS motif is a highly conserved structural feature of the type I cytokine receptor family. It has previously been demonstrated that mutations in the (232)WSAWS(236) motif in the erythropoietin receptor (EPOR) can result in strongly inhibited surface expression, due to defective intracellular transport [Hilton, D. J., et al. (1996) J. Biol. Chem. 271, 4699-4708]. Here we report that the first tryptophan in the motif of the recombinant extracellular domain of EPOR (sEPOR) expressed in HEK-EBNA cells carries a C-linked hexosyl residue. The S233A mutation completely abolished secretion of sEPOR, whereas the A234E mutation resulted in enhanced secretion. Comparison of the level of C-hexosylation in the wild-type protein and in the mutant proteins isolated from the conditioned medium and/or the cells suggested that C-hexosylation of the motif did not play a role in the correct intracellular transport of sEPOR.     

Glycosylation of the murine erythropoietin receptor

Murine erythropoietin-responsive Rauscher Red 5-1.5 cells were used to determine the contribution of glycosylation to the size and function of the erythropoietin receptor. The half life of the receptors was determined to be 4 h. The number of receptors was not significantly decreased in cells treated for 48 h with inhibitors of glycosylation (tunicamycin, glucosamine or swainsonine) and their affinity was slightly enhanced in tunicamycin- or glucosamine-treated cells. Erythropoietin was cross-linked with two proteins of 104 and 86 kDa. Their molecular masses were not significantly reduced in cells treated with the glycosylation inhibitors. When immunoprecipitated cross-linked receptors were digested with endoglycosidases, the molecular masses of both proteins were only slightly modified giving values of 100 and 82 kDa. Thus we can conclude that the proteins cross-linked to erythropoietin are very weakly glycosylated.     

Structure of the murine erythropoietin receptor complex. Characterization of the erythropoietin cross-linked proteins.

The structure of the murine erythropoietin receptor was studied using antibodies against the intracellular part of the cloned erythropoietin receptor chain. These antibodies precipitated erythropoietin-receptor complexes from Triton X-100-solubilized cells. When the complexes were cross-linked by disuccinimidyl suberate, the 85- and 100-kDa erythropoietin-cross-linked proteins previously described were immunoprecipitated. However, these proteins were not precipitated when the complexes were denatured and reduced before immunoprecipitation. Using 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide, we observed erythropoietin cross-linking with a protein of 66 kDa in addition to the 100- and 85-kDa proteins. Only the 66-kDa erythropoietin-cross-linked protein was immunoprecipitated by anti-receptor antibodies after denaturation and reduction of the complex. Thus, our results suggest that the 85- and 100-kDa proteins previously evidenced by cross-linking are associated with the cloned chain of the receptor to form a multimeric complex but these proteins seem immunologically unrelated to the cloned chain. We observed that reducing the length of molecules able to cross-link amino groups decreased the efficiency of cross-linking with the 100-kDa protein and only the 85-kDa protein was cross-linked with erythropoietin using 1,5-difluoro-2,4-dinitrobenzene. These results suggest that the 85- and 100-kDa proteins occupate slightly different positions relative to the erythropoietin molecule bound to the receptor.     

Expression cloning of the murine erythropoietin receptor

Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.     

Characterization of murine erythropoietin receptor genes

We have isolated and characterized the murine genomic and complementary DNAs encoding erythropoietin (Epo) receptor from Epo-responsive and unresponsive mouse erythroleukemia cells. Two classes of Epo receptor cDNAs were isolated from Epo-responsive cells. One is a 55,000 Mr membrane-bound Epo receptor, and the other is a 29,000 Mr soluble Epo receptor lacking the transmembrane and cytoplasmic domains. As a result of alternative splicing, two insert sequences containing termination codons are produced, and the encoded polypeptide diverges four amino acids upstream from the transmembrane domain, adding 20 new amino acids before terminating. Amino acid sequence of the Epo receptor cDNA isolated from Epo-responsive cells was identical with that of Epo-unresponsive cells, indicating that Epo-responsiveness does not depend upon the primary structure of the Epo receptor (binding) protein. Analysis of 6.6 x 10(3) base-pairs (kb) genomic DNA segments covering complete Epo receptor gene and promoter regions revealed that potential regulatory elements (NF-E1, GF-1 or Eryf 1) for erythroid-specific and differentiation stage-specific gene expression are located in the promoter and 3' noncoding regions.     

Characterization of erythropoietin receptor of murine erythroid cells

Radioiodinated or biologically tritiated recombinant human erythropoietin was used to characterize receptors for this hormone on the surface of Friend erythroleukemic cells (745A and TSA8) and cells from mouse erythropoietic tissues (liver from fetus and spleen from animals made anemic by injection of Friend virus or phenylhydrazine). Specific binding of erythropoietin to these cells was time-dependent and dose-dependent. Binding studies at 37 degrees C showed that dissociation constants of erythropoietin-receptor complexes were in the range of 100-300 pM. The number of receptors on erythroleukemic cells increased after treatment with dimethylsulfoxide. Covalent binding of 125I-erythropoietin to its receptors with a cross-linking reagent, disuccinimidyl suberate or glutaraldehyde, resulted in the formation of two major radiolabeled products that migrated as 120-kDa and 140-kDa species on sodium dodecyl sulfate/polyacrylamide electrophoresis gels under reducing conditions. Under non-reducing conditions, both 120-kDa and 140-kDa species disappeared and two cross-linked products, a minor product with a molecular mass of 250 kDa and a major product of high molecular mass that kept it from migration into the separating gels, appeared. The relationship of the cross-linked products found under non-reducing conditions with those under reducing conditions remains to be clarified.     

Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.

Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-[[3-[[4-[(p-azido-m-[125I]iodophenyl)azo]benzoyl]amino] propanoyl]oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D'Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands (100- and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 degrees C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.     

Isolation of a cDNA encoding a potential soluble receptor for human erythropoietin.

Analysis of human erythropoietin receptor-encoding cDNAs revealed the possible existence of a soluble receptor lacking the transmembrane and cytoplasmic domains.     

Solubilization and hydrodynamic characteristics of the erythropoietin receptor. Evidence for a multimeric complex

In order to study the erythropoietin receptor in its native state, we solubilized erythropoietin-receptor complexes from spleen cell membranes of mice infected with the anemia strain of Friend virus using mild detergents. Among 11 tested detergents, Triton X-100 and Lubrol PX were the most effective. Triton X-100 was therefore selected for this study. The solubilized complexes appeared to be well representative of the total membrane receptor population as indicated by cross-linking experiments and affinity measurements. The hydrodynamic characteristics of the complexes were determined by gel filtration chromatography and ultracentrifugation through sucrose gradients prepared with H2O or D2O. Although erythropoietin-receptor-detergent complexes exhibited some heterogeneity, we determined the following minimal hydrodynamic values: sedimentation coefficient (s20,w): 11.7 +/- 0.8 S, Stokes radius: 7.7 +/- 0.2 nm, partial specific volume: 0.774 +/- 0.017 ml/g, giving a molecular mass of 458 +/- 66 kDa. The contribution of the detergent was estimated to be 28% from the measured partial specific volume, giving an estimated molecular mass of 330 +/- 48 kDa for the erythropoietin-receptor complex. The minimal molecular mass value was significantly greater than those obtained by polyacrylamide gel electrophoresis under denaturing conditions, strongly suggesting that the erythropoietin receptors were present as multimeric complexes. The nature of these complexes is discussed. Beside this major component our results revealed the presence of higher-molecular-mass erythropoietin binding components. We also demonstrated that erythropoietin-receptor complexes could be precipitated with anti-erythropoietin antibodies. This property should greatly improve the purification of erythropoietin receptors.     

Epidermal growth factor (EGF) induces oligomerization of soluble, extracellular, ligand-binding domain of EGF receptor. A low resolution projection structure of the ligand-binding domain.

Ligand-induced oligomerization is a universal phenomenon among growth factor receptors. Although the mechanism involved is yet to be defined, much evidence indicates that receptor oligomerization plays a crucial role in receptor activation and signal transduction. Here we show that epidermal growth factor (EGF) is able to stimulate the oligomerization of a recombinant, soluble, extracellular ligand-binding domain of EGF receptor. Covalent cross-linking experiments, analysis by sodium dodecyl sulfate-gel electrophoresis, size exclusion chromatography, and electron microscopy demonstrate that receptor dimers, trimers and larger multimers are formed in response to EGF. This establishes that receptor oligomerization is an intrinsic property of the extracellular ligand-binding domain of EGF receptor. Ligand-induced conformational change in the extracellular domain will stimulate receptor-receptor interactions. This may bring about the allosteric change involved in signal transduction from the extracellular domain across the plasma membrane, resulting in the activation of the cytoplasmic kinase domain. Electron microscopic images of individual extracellular ligand-binding domains appear as clusters of four similarly-sized stain-excluding areas arranged around a central, relatively less stain-excluded area. This suggests that the extracellular ligand-binding domain is structurally composed of four separate domains.     

Normal platelets possess the soluble form of IL-6 receptor

Interleukin 6 is a multifunctional cytokine that exerts its biological activity through binding to an 80 Kd specific receptor (IL-6Ralpha) and a 130 Kd signal-transducing unit (gp130). A 55 Kd soluble IL-6R (IL-6sR) has also been described which, after binding to IL-6 is also able to activate gp130. The presence of IL-6Ralpha was described in some megakaryoblastic cell lines but is still controversial in normal megakaryocytes. In this study we demonstrate the presence of intraplatelet IL-6sR by Western blot through the appearance of a 55 Kd protein and the finding of detectable amounts of IL-6sR in the platelet content by ELISA technique. Besides, we showed IL-6sR release during platelet activation induced by thrombin and a complex of ADP and epinephrine. IL-6Ralpha on platelet membrane could not be found neither by Western blot nor by flow cytometry. The IL-6sR released during platelet activation and complexed to IL-6 could act on cell types such as endothelial cells that do not possess IL-6Ralpha through binding to gp130.Besides, since we could not find IL-6R on platelet membrane, the potentiating effect of IL-6 on platelet function could be explained through binding of IL-6sR/IL-6 complex to platelet membrane gp130.     

Cloning of the human and murine interleukin-7 receptors: demonstration of a soluble form and homology to a new receptor superfamily

cDNA clones encoding the human and murine interleukin-7 (IL-7) receptor were isolated and expressed in COS-7 cells. Binding of radiolabeled IL-7 to the recombinant IL-7 receptors produced curvilinear Scatchard plots containing high and low affinity classes. These binding properties, as well as the molecular size of the cloned receptor, were comparable to the native forms of the IL-7 receptor. In addition, several cDNA clones were isolated that encode a secreted form of the human IL-7 receptor capable of binding IL-7 in solution. Analysis of the sequence of the IL-7 receptor revealed significant homology in the extracellular domain to several recently cloned cytokine receptors, demonstrating that the IL-7 receptor is a member of a new receptor superfamily.     

Preparation of soluble murine IL-6 receptor and anti-murine IL-6 receptor antibodies

Starting with a previously isolated cDNA clone encoding murine IL-6R, a stable transformed Chinese hamster ovary cell line constitutively expressing soluble murine IL-6R (smIL-6R) has been established. The smIL-6R was purified to homogeneity by sequential filtration and chromatography of culture medium. The smIL-6R augmented the sensitivity of M1 cells to IL-6 in their growth inhibition in a dose-response manner. Rat hybridomas producing mAb specific to murine IL-6R were also established. One of the clones, RS13, produced IgG2a isotype that was capable of inhibiting IL-6 activity. ELISA for the quantitation of smIL-6R was established, which could detect smIL-6R in a quantity as low as 1 ng/ml.     

An activating mutation in the murine erythropoietin receptor induces erythroleukemia in mice: a cytokine receptor superfamily oncogene.

A point mutation at codon 129 of the murine erythropoietin receptor (cEpoR) results in constitutive activation. We have generated a recombinant spleen focus-forming retrovirus in which the env gene is replaced by the cEpoR cDNA. Mice infected with this virus (but not by viruses expressing the wild-type EpoR) develop erythrocytosis and splenomegaly. From the spleen of infected animals we have isolated clonal, growth factor-independent, proerythroblast cell lines that express cEpoR, do not express the putative oncogene spi-1, and have rearranged and inactivated expression of the p53 suppressor oncogene. These cells induce erythroleukemia upon injection into mice. This demonstrates that oncogenic point mutations exist in a member of the cytokine receptor superfamily. The activated erythropoietin receptor does not transform cultured fibroblasts, suggesting why oncogenic mutations in other members of this receptor superfamily have not been detected.     

A soluble form of the interleukin 4 receptor in biological fluids

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.     

Isolation of an mRNA encoding a soluble form of the human interleukin-6 receptor.

Soluble forms of the interleukin (IL)-2, IL-4 and IL-7 receptors which lack the transmembrane domain have been described. IL-6 is a growth factor important in the final differentiation of B-cells into plasma cells and in the pathogenesis of multiple myeloma. To determine whether the receptor for IL-6 may exist as a soluble molecule, RNA was analysed from the transformed B-cell lines U266, CESS and Daudi, from bone marrow from two myeloma patients, and from normal leukocytes. Using polymerase chain reaction, oligonucleotide primers which flank the transmembrane domain were selected to generate a 339 bp fragment. All samples produced equivalent amounts of the expected 339 bp fragment plus a smaller 245 bp fragment except Daudi which exhibited virtual absence of both. Sequence analysis of the smaller fragments from each of the five samples demonstrated the deletion of the entire transmembrane region from codons 356 (G-TG) to 387 (AG-G). The boundaries of this deletion were identical in all cases. Partial sequence analysis of the ligand-binding domain for U266 demonstrated identical sequences for the membrane-bound and soluble forms of the IL-6 receptor cDNAs. In summary, an mRNA which encodes a soluble form of the IL-6 receptor is expressed in both normal and myeloma cells.     

Three soluble form messages of murine CD46 are produced through alternative mRNA splicing.

Murine CD46 (mCD46) is a type 1 membrane protein expressed predominantly in testicular germ cells, the distribution profile of which is in contrast to that of human CD46 showing a ubiquitous tissue distribution. We have identified an additional message of mCD46 that encodes a putative secretory form [Nomura et al. (1999) Immunogenetics 50, 245-254]. Here, we cloned three cDNAs encoding putative soluble CD46 from murine testis. These soluble form messages were yielded on insertion of unidentified nucleotide sequences, 77, 179, and 73 ntds, into the junctions between the SCR3 and SCR4 (variant 2), ST(c) and UK (variant 3), and SCR4 and ST(c) (variant 1) domains, respectively, the last one corresponding to the reported soluble form. The exons corresponding to these three inserts were identified in the murine CD46 genome, indicating that the alternative splicing of mRNA participates in the generation of these various CD46 messages. In normal mouse sera and cell lines, however, virtually no soluble CD46 was detected on immunoblotting. On Northern blotting analysis with specific probes, on the other hand, variant 1 was found to be predominantly expressed in the liver and heart. In addition, all variant messages were detected on PCR in all organs examined. When a rabbit cell line, RK13 cells, was transfected with cDNA of variant 1, protein synthesis was detected on immunoblotting. Although the mCD46 protein production was inefficient, this variant 1 exhibited factor I-cofactor activity as to inhibition of the complement cascade. Since the mCD46 protein was reported to be markedly up-regulated on infection of murine cells with mCMV, the soluble mCD46 proteins may act as a complement regulator that controls the systemic complement system under the conditions of a viral infection.     

Production and characterization of a soluble, active form of Tva, the subgroup A avian sarcoma and leukosis virus receptor

The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.