Three residues in the common beta chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and interleukin-5 (IL-5) is essential for high affinity ligand-binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM-CSF, IL-3 and IL-5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL-2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site-directed mutagenesis and binding assays with radiolabelled GM-CSF, IL-3 and IL-5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365-Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM-CSF and IL-5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL-3 binding to high affinity by beta c. To identify likely contact points in GM-CSF involved in binding to the 365-368 beta c region we used the GM-CSF mutant eco E21R which is unable to interact with wild-type beta c whilst retaining full GM-CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild-type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM-CSF and IL-5 can be dissociated from IL-3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM-CSF.     

Expression cloning of the human IL-3 receptor cDNA reveals a shared beta subunit for the human IL-3 and GM-CSF receptors.

A cDNA for a human interleukin-3 (hIL-3) binding protein has been isolated by a novel expression cloning strategy: a cDNA library was coexpressed with the cDNA for the beta subunit of human granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (hGMR beta) in COS7 cells and screened by binding of 125I-labeled IL-3. The cloned cDNA (DUK-1) encodes a mature protein of 70 kd, which belongs to the cytokine receptor family and which alone binds hIL-3 with extremely low affinity (Kd = 120 +/- 60 nM). A high affinity IL-3-binding site (Kd = 140 +/- 30 pM) was reconstituted by coexpressing the DUK-1 protein and hGMR beta, indicating that hIL-3R and hGMR share the beta subunit. Therefore, we designated DUK-1 as the alpha subunit of the hIL-3R. As in human hematopoietic cells, hIL-3 and hGM-CSF complete for binding in fibroblasts expressing the cDNAs for hIL-3R alpha, GMR alpha, and the common beta subunit, indicating that different alpha subunits compete for a common beta subunit.     

Identification of three adjacent amino acids of interleukin-2 receptor beta chain which control the affinity and the specificity of the interaction with interleukin-2.

The beta chain of the interleukin-2 (IL-2) receptor (IL-2R beta) and the interleukin-3 (IL-3) binding protein AIC2A are members of the family of cytokine receptors, which also includes the receptors for growth hormone (GHR) and prolactin. A four amino acid sequence of AIC2A has recently been shown to be critical for IL-3 binding. We analyze here the function of the analogous sequence of human IL-2R beta and identify three amino acids, Ser132, His133 and Tyr134, which play a critical role in IL-2 binding. We show that some mutant IL-2 proteins with substitutions of a critical Asp residue in the N-terminal alpha-helix bind the mutant IL-2R beta receptor with a higher affinity than the wild-type receptor. This suggests that the critical Asp34 in the ligand and the sequence Ser-His-Tyr (positions 132-134) in the receptor interact directly. On the double barrel beta-stranded structural model of cytokine receptors, the residues important for ligand binding in IL-2R beta, AIC2A and GHR map to strikingly similar locations within a barrel, with the interesting difference that it is the N-terminal barrel for GHR and the C-terminal barrel for IL-2R beta and AIC2A.     

Identification of the second subunit of the murine interleukin-5 receptor: interleukin-3 receptor-like protein, AIC2B is a component of the high affinity interleukin-5 receptor.

Murine interleukin-5 (IL-5) binds to its receptor with high and low affinity. It has been shown that the high affinity IL-5 receptor (IL-5-R) is composed of at least two membrane protein subunits and is responsible for IL-5-mediated signal transduction. One subunit of the high affinity IL-5-R is a 60 kDa membrane protein (p60 IL-5-R) whose cDNA was isolated using the anti-IL-5-R monoclonal antibody (mAb), H7. This subunit alone binds IL-5 with low affinity. The second subunit does not bind IL-5 by itself, and is expressed not only on IL-5-dependent cell lines but also on an IL-3-dependent cell line, FDC-P1. Expression of the p60 IL-5-R cDNA in FDC-P1 cells, which do not bind IL-5, reconstituted the high affinity IL-5-R. We have characterized the second subunit of the IL-5-R by using another anti-IL-5-R mAb, R52.120, and the anti-IL-3-R mAb, anti-Aic-2. The anti-Aic-2 mAb down-regulated binding of IL-5 to an IL-5-dependent cell line, Y16. Both R52.120 and anti-Aic-2 mAbs recognized membrane proteins of 130-140 kDa expressed on FDC-P1 and Y16 cells. The R52.120 mAb recognized both murine IL-3-R (AIC2A) and its homologue (AIC2B) expressed on L cells transfected with suitable cDNAs. The high affinity IL-5-R was reconstituted on an L cell transfectant co-expressing AIC2B and p60 IL-5-R, whereas only the low affinity IL-5-R was detected on a transfectant co-expressing AIC2A and p60 IL-5-R.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of mouse interleukin 3 (IL-3) binding protein (AIC2A). Amino acid residues critical for IL-3 binding.

Despite the high degree of sequence homology between two mouse proteins AIC2A and AIC2B (91% at the amino acid level), only the AIC2A protein binds interleukin 3 (IL-3). Soluble AIC2A protein bound IL-3 with affinity similar to the membrane-bound AIC2A protein, indicating that binding of IL-3 to AIC2A was mediated by the external domain alone. The extracellular domain of the AIC2A protein has two repeats of the common motif shared by members of the cytokine receptor family. Neither one of these repeats alone bound IL-3. Hybrids of AIC2A and AIC2B revealed that the first domain of the cytokine receptor motif could be replaced with the AIC2B sequence without an affinity change, suggesting the importance of the second domain. By changing individual amino acid residues of AIC2A in the second domain which differ from those of AIC2B, we identified several amino acid residues critical for IL-3 binding. All these residues are located at the putative hinge region within the second domain.     

Subunit structure of the high and low affinity human interleukin-15 receptors

Radio-iodinated cytokines and monoclonal antibodies directed at the IL-2R beta- and gamma-chains were used to analyze the structure of the cell-surface IL-15 and IL-2 receptors expressed by the human lymphoma cell clone YT-2C2. YT-2C2 cells are IL-2R alpha negative and express IL-2R gamma (15,000 molecules/cell) in excess of IL-2R beta (11,000 molecules/cell). Accordingly, they display a number of beta/gamma complexes of intermediate affinity for IL-2 and IL-15 which is equivalent to the number of beta-chains. Both cytokines compete for binding to this beta/gamma complex. There are about 800 high affinity IL-15 receptors, suggesting the presence of a similar number of IL-15R alpha-chains. Within the common intermediate affinity beta/gamma-complex, the anti-beta-chain A41 mAb defines an epitope which is similarly engaged in IL-2 and IL-15 binding, whereas the anti-beta-chain 284 mAb defines an epitope which does not display similar interaction with either cytokines. Thus, although IL-2 and IL-15 compete for binding to this beta/gamma-complex, they do not use similar binding areas. Cross-linking and immunoprecipitation experiments have shown that the high affinity IL-15 receptors comprises IL-2R beta/gamma, in association with IL-15R alpha and that the three chains can be efficiently cross-linked to IL-15 and co-immunoprecipitated. Contrary to the intermediate affinity situation, high affinity IL-15 binding and subunit cross-linking were not affected by excess amounts of IL-2, A41 or 284 mAb, suggesting that when engaged in the IL-15 high affinity complex, the beta- and gamma-chains adopt different conformations, at least with respect to IL-15 binding. Finally, we provide evidence for the participation of a novel 35 kDa component within the high affinity structure. This component is immunoprecipitated with anti-IL-2R gamma mAb but not with anti-IL-2R beta mAb and might correspond to a truncated form of IL-2R gamma-chain.     

Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding.

The human interleukin-3 receptor (IL-3R) is a heterodimer that comprises an IL-3 specific alpha chain (IL-3R alpha) and a common beta chain (beta C) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. These receptors belong to the cytokine receptor superfamily, but they are structurally and functionally more related to each other and thus make up a distinct subfamily. Although activation of the normal receptor occurs only in the presence of ligand, the underlying mechanisms are not known. We show here that human IL-3 induces heterodimerization of IL-3R alpha and beta c and that disulfide linkage of these chains is involved in receptor activation but not high-affinity binding. Monoclonal antibodies (MAb) to IL-3R alpha and beta c were developed which immunoprecipitated, in the absence of IL-3, the respective chains from cells labelled with 125I on the cell surface. However, in the presence of IL-3, each MAb immunoprecipitated both IL-3R alpha and beta c. IL-3-induced receptor dimers were disulfide and nondisulfide linked and were dependent on IL-3 interacting with both IL-3R alpha and beta c. In the presence of IL-3 and under nonreducing conditions, MAb to either IL-3R alpha or beta c immunoprecipitated complexes with apparent molecular weights of 215,000 and 245,000 and IL-3R alpha and beta c monomers. Preincubation with iodoacetamide prevented the formation of the two high-molecular-weight complexes without affecting noncovalent dimer formation or high-affinity IL-3 binding. Two-dimensional gel electrophoresis and Western blotting (immunoblotting) demonstrated the presence of both IL-3R alpha and beta c in the disulfide-linked complexes. IL-3 could also be coimmunoprecipitated with anti-IL-3R alpha or anti-beta c MAB, but it was not covalently attached to the receptor. Following IL-3 stimulation, only the disulfide-linked heterodimers exhibited reactivity with antiphosphotyrosine antibodies, with beta c but not IL-3R alpha being the phosphorylated species. A model of IL-3R activation is proposed which may be also applicable to the related GM-CSF and IL-5 receptors.     

The amino-terminal helix of GM-CSF and IL-5 governs high affinity binding to their receptors.

Transduction of the biological effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5) requires the interaction of each cytokine with at least two cell surface receptor components, one of which is shared between these two cytokines. A strategy is presented that allowed us to identify receptor binding determinants in GM-CSF and IL-5. Mixed species (human and mouse) receptors were used to locate unique receptor binding domains on a series of human-mouse hybrid GM-CSF and IL-5 cytokines. Results show that the interaction of these two cytokines with the shared subunit of their high affinity receptor complexes is governed by a very small part of their peptide chains. The presence of a few key residues in the amino-terminal alpha-helix of each ligand is sufficient to confer specificity to the interaction. Comparison with other cytokines suggests that the amino-terminal helix of many of these proteins may contain the recognition element for the formation of high affinity binding sites with the alpha subunit of their multi-component receptors.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.     

Granulocyte-macrophage colony-stimulating factor signals for increased glucose transport via phosphatidylinositol 3-kinase- and hydrogen peroxide-dependent mechanisms

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates cellular glucose uptake by decreasing the apparent K(m) for substrate transport through facilitative glucose transporters on the plasma membrane. Little is known about this signal transduction pathway and the role of the alpha subunit of the GM-CSF receptor (alpha GMR) in modulating transporter activity. We examined the function of phosphatidylinositol 3-kinase (PI 3-kinase) in GM-CSF-stimulated glucose uptake and found that PI 3-kinase inhibitors, wortmannin and LY294002, completely blocked the GM-CSF-dependent increase of glucose uptake in Xenopus oocytes expressing the low affinity alpha GMR and in human cells expressing the high affinity alpha beta GMR complex. We identified a Src homology 3 domain-binding motif in alpha GMR at residues 358-361 as a potential interaction site for the PI 3-kinase regulatory subunit, p85. Physical evidence for p85 binding to alpha GMR was obtained by co-immunoprecipitation with antibodies to alpha GMR and p85, and an alpha GMR mutant with alteration of the Src homology 3 binding domain lost the ability to bind p85. Experiments with a construct eliminating most of the intracellular portion of alpha GMR showed a 50% reduction in GM-CSF-stimulated glucose uptake with residual activity blocked by wortmannin. Searching for a proximally generated diffusible factor capable of activating PI 3-kinase, we identified hydrogen peroxide (H(2)O(2)), generated by ligand or antibody binding to alpha GMR, as the initiating factor. Catalase treatment abrogated GM-CSF- or anti-alpha GMR antibody-stimulated glucose uptake in alpha GMR-expressing oocytes, and H(2)O(2) activated PI 3-kinase and led to some stimulation of glucose uptake in uninjected oocytes. Human myeloid cell lines and primary explant human lymphocytes expressing high affinity GM-CSF receptors responded to alpha GMR antibody with increased glucose uptake. These results identify the early events in the stimulation of glucose uptake by GM-CSF as involving local H(2)O(2) generation and requiring PI 3-kinase activation. Our findings also provide a mechanistic explanation for signaling through the isolated alpha subunit of the GM-CSF receptor.     

Coupling of osteopontin and its cell surface receptor CD44 to the cell survival response elicited by interleukin-3 or granulocyte-macrophage colony-stimulating factor

The receptors for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) share a common beta subunit, the distal cytoplasmic domain of which is essential for the promotion of cell survival by these two cytokines. Genes whose expression is specifically induced by signaling through the distal cytoplasmic domain of this receptor beta subunit were screened by a subtraction cloning approach in derivatives of a mouse pro-B-cell line. One gene thus identified was shown to encode a protein highly homologous (with only 7 amino acid substitutions) to murine osteopontin (OPN), a secreted adhesion protein. Conditioned medium from cells expressing wild-type OPN, but not that from cells expressing a deletion mutant lacking residues 79 to 140, increased the viability of a non-OPN-producing cell line in the presence of human GM-CSF. Antibody blocking experiments revealed that OPN produced as a result of IL-3 or GM-CSF signaling was secreted into the medium and, through binding to its cell surface receptor, CD44, contributed to the survival-promoting activities of these two cytokines. Furthermore, coupling of the OPN-CD44 pathway to the survival response to IL-3 was also demonstrated in primary IL-3-dependent mouse bone marrow cells. These results thus show that induction of an extracellular adhesion protein and consequent activation of its cell surface receptor are important for the antiapoptotic activities of IL-3 and GM-CSF.     

Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation.

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 5 (IL-5) are composed of two distinct subunits, alpha and beta c. The alpha subunits are specific for each cytokine, whereas the beta subunit (beta c) is shared by the three receptors and is an essential component of signal transduction. We have made a series of mutant beta c cDNAs that delete various regions of the cytoplasmic domain and examined the function of these mutants by coexpressing them with the alpha subunit of the human GM-CSF receptor (hGMR) in an IL-3-dependent mouse pro-B cell line BaF3. Two domains in the membrane-proximal portion of beta c were found to be important for transducing the hGM-CSF-mediated growth signals: one domain between Arg456 and Phe487 appears to be essential for proliferation, and the second domain between Val518 and Asp544 enhances the response to GM-CSF, but is not absolutely required for proliferation. The region between Val518 and Leu626 was responsible for major tyrosine phosphorylation of 95 and 60 kDa proteins. Thus, beta c-mediated major tyrosine phosphorylation of these proteins was apparently separated from proliferation. However, the beta 517 mutant lacking residues downstream of Val518 transmitted a herbimycin-sensitive proliferation signal, suggesting that beta 517 still activates a tyrosine kinase(s). We also evaluated the role of the cytoplasmic domain of the GMR alpha subunit and the results suggest that it is involved in the hGM-CSF-mediated signal transduction, but is not essential.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-2 regulation of tyrosine kinase activity is mediated through the p70-75 beta-subunit of the IL-2 receptor

The stimulation of activated human T lymphocytes with IL-2 results in increased tyrosine kinase activity. IL-2 treatment of Tac+ T cells stimulates the rapid phosphorylation of multiple protein substrates at M of 116, 100, 92, 70 to 75, 60, 56, 55, 33, and 32 kDa. Phosphorylation on tyrosine residues was detected by immunoaffinity purification of protein substrates with Sepharose linked antiphosphotyrosine mAb, 1G2. Although phorbol ester stimulated serine phosphorylation of the IL-2R alpha (p55) subunit recognized by alpha TAC mAb, IL-2 did not stimulate any detectable phosphorylation of IL-2R alpha or associated coimmune precipitated proteins. In fact, the tyrosine phosphorylated proteins did not coprecipitate with alpha Tac antibody and similar phosphoproteins were stimulated by IL-2 in IL-2R alpha- human large granular lymphocytes which express only the 70 to 75 kDa IL-2R beta subunit of the high affinity IL-2R. Anti-Tac mAb could inhibit IL-2-stimulated tyrosine phosphorylation in activated T cells, which express both IL-2R subunits that together form the high affinity receptor complex, but not in large granular lymphocytes expressing only the IL-2R beta subunit. The data suggest that IL-2 stimulation of tyrosine kinase activities requires only the IL-2R beta subunit.     

Analysis of signals and functions of the chimeric human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells and transgenic mice

Receptors for GM-CSF, IL-3, and IL-5 are composed of two subunits: alpha, which is specific for each cytokine, and betac, which is shared by all. Although the role of betac in signal transduction has been extensively studied, the role of the alpha subunit has remained to be clarified. To analyze the role of the human (h) GM-CSF receptor alpha subunit, we constructed a chimeric receptor subunit composed of extracellular and transmembrane regions of alpha fused with the cytoplasmic region of betac, designated alpha/beta. In BA/F3 cells, chimeric receptor composed of alpha/beta,beta can transduce signals for mitogen-activated protein kinase cascade activation and proliferation in response to hGM-CSF. Although phosphorylation of Jak1 but not of Jak2 occurred with stimulation of hGM-CSF, the dominant-negative Jak2 but not the dominant-negative Jak1 suppresses c-fos promoter activation. To determine whether the chimeric receptor alpha/beta,beta is functional in vivo, we developed transgenic mice expressing the chimeric receptor alpha/beta,beta. Bone marrow cells from the transgenic mice expressing the alpha/beta,beta receptor form not only GM colonies but also various lineages of colonies in response to GM-CSF. In addition, mast cells were produced when bone marrow cells of the transgenic mouse were cultured with hGM-CSF. Thus, it appears that the cytoplasmic region of the alpha subunit is not required for hGM-CSF promoting activities, even in bone marrow cells.     

IL-4 inhibits the expression of high affinity IL-2 receptors on monoclonal human B cells

In the presence of anti-mu antibodies (anti-microAb), monoclonal B lymphocytes from patients suffering from B type chronic lymphocytic leukemia (B-CLL) can respond to IL-2. In contrast to the effect it exerts on normal B cells, IL-4 does not promote DNA synthesis by B-CLL lymphocytes. Rather this interleukin inhibits the response to IL-2 in all patients' cells that responded to this interleukin. We thus examined whether IL-4 would modulate the number and/or the affinity of IL-2 receptors. A 3-day activation of cells by anti-microAb induced a few hundred high affinity IL-2 receptors (HA-IL-2R) on B-CLL cell surface, as determined by Scatchard analysis. Treatment of cells with IL-4 caused a marked decrease in the number of HA-IL-2R without interfering with the binding ability of IL-2. In contrast with this profound suppressive effect, IL-4 did not down-regulate the expression of each chain, alpha and beta (p55 and p75, respectively), of the HA-IL-2R heterodimer. In fact, the expression of alpha and beta induced by anti-microAb was enhanced by IL-4. Altogether, IL-4 exerts a critical influence on the function and the configuration of HA-IL-2R without inhibiting the expression of two subunits, alpha and beta.     

Expression of low-, intermediate-, and high-affinity IL-2 receptors on B cell lines derived from patients with undifferentiated lymphoma of Burkitt's and non-Burkitt's types

IL-2 receptors on T cells exist in at least three forms which differ in their ligand-binding affinity. The low-affinity IL-2 receptor (IL-2R) consists of the 55-kDa Tac protein (p55 alpha), the intermediate-affinity site corresponds to the 70-kDa molecule (p70 beta), and the high-affinity IL-2R consists of a noncovalent heterodimeric structure involving both p55 alpha and p70 beta. We studied 24 B cell lines (8 EBV-negative and 16 EBV-positive) for IL-2R expression in the presence or absence of the tumor promoter, teleocidin. 125I-IL-2 radioreceptor binding assays and crosslinking studies demonstrated the sole expression of p55 alpha in EBV-negative cell lines only, whereas p55 alpha present in EBV-positive cell lines was always associated with p70 beta to construct high-affinity IL-2R. p70 beta was not detected in any of the EBV-negative cell lines, but was expressed on most of the EBV-positive cell lines (13 of 16). Our data also indicate that the expression of p55 alpha and p70 beta by radiolabeling correlates with their expression in flow cytometry, and that a large excess of p55 alpha is required to construct high-affinity IL-2R. Coexpression of p55 alpha and p70 beta on human B cells contributed to constructing high-affinity IL-2R hybrid complex as shown by (i) rapid association rate contributed by p55 alpha and slow dissociation rate by p70 beta; (ii) teleocidin's ability to induce p55 alpha on cell lines which express p70 beta only, resulting in appearance of high-affinity IL-2R; (iii) blocking p55 alpha by anti-Tac mAb in cell lines which constitutively express high-affinity IL-2R eliminated both high- and low-affinity components. The existence of low, intermediate, and high IL-2R on human B cells bears important future implications for understanding the mechanism of IL-2 signaling and the role of IL-2 in B cell activation, proliferation, and differentiation.     

Molecular cloning of the guinea-pig IL-5 receptor alpha and beta subunits and reconstitution of a high affinity receptor

The functional IL-5 receptor is a heteromeric complex consisting of an alpha and beta subunit. The cloning, sequencing and expression of guinea-pig IL-5Ralpha and beta subunits is described. The guinea-pig IL-5Ralpha subunit cDNA encodes a protein of M(r)47 kDa, which is 72 and 66% homologous to the human and murine orthologs, respectively. Three guinea-pig IL-5Rbeta subunit cDNA clones were isolated, which differ in the N-terminus and are 56-64% homologous to the human and murine IL-5Rbeta subunits. Expressing human IL-5Ralphabeta and guinea-pig IL-5Ralphabeta(1)in the baculovirus-insect cell system resulted in recombinant receptors which bound hIL-5 with high affinity (K(d)=0.19 and 0.11 nM, respectively). Expressing just gpIL-5Ralpha was not sufficient to demonstrate binding. This contrasts with the human receptor, where hIL-5Ralpha alone can bind hIL-5 with high affinity. gpIL-5Ralphabeta(1)bound both hIL-5 and mIL-5 with comparable affinity (K(i)=0.10 and 0.06 nM), similar to that seen with hIL-5Ralphabeta. Thus, both the heteromeric hIL-5R and gpIL-5Ralphabeta(1)can bind multiple IL-5 orthologs with high affinity whereas the murine IL-5R is selective for the murine ligand.     

Characterization of the heterodimeric complex of human IL-2 receptor alpha.beta chains reconstituted in a mouse fibroblast cell line, L929

The receptor for IL-2 has been known to exist in three forms on the basis of their affinities to IL-2: high, intermediate, and low affinity forms. Two IL-2R components have been identified as IL-2R alpha (p55, Tac Ag) and IL-2R beta (p70-75) chains, both bind IL-2 with low and intermediate affinities, respectively. Recently, we cloned human IL-2R beta chain cDNA and demonstrated that the cDNA product binds IL-2 with intermediate affinity and forms high affinity IL-2R with coexpressed IL-2R alpha chain in a human T cell line, Jurkat. In this study, we report the establishment of the mouse fibroblast transformants expressing either the IL-2R beta chain alone or both the IL-2R alpha and IL-2R beta chains. In contrast to lymphoid cells, significant IL-2 binding was not detected in the transformants expressing the IL-2R beta chain alone at IL-2 concentrations (50 pM to 10 nM) generally utilized. Nonetheless, the transformants expressing both IL-2R alpha and IL-2R beta chains displayed two forms of the IL-2R with high and low affinities to IL-2. However, neither IL-2 internalization nor signal transduction via the high affinity IL-2R complex were observed in the L929 transformants. Those findings suggest that the interaction of the IL-2R beta chain with the IL-2R alpha chain occurs in the absence of additional lymphoid specific component(s) to form high affinity IL-2R, but that this interaction is insufficient for IL-2 internalization and signal transduction just as observed in lymphoid cells. The experimental approach described here may allow further dissection of the molecular architecture of the IL-2R complex in the ligand binding, internalization, and signal transduction.     

Interleukin-1 receptor antagonist binds to the type II interleukin-1 receptor on B cells and neutrophils.

We have examined the binding of human and rodent interleukin-1 receptor antagonist (IL-1ra) to the type II IL-1 receptor on the human B cell line, Raji, on the mouse pre-B cell line, 70Z/3, and on human polymorphonuclear leukocytes (PMNs). Human IL-1ra binds to the receptors on the human B cells with an affinity (KD = 15 +/- 3 nM) equal to that of IL-1 alpha and only 15-fold lower than that of IL-1 beta and, likewise, binds to human PMNs with an affinity (KD = 8 +/- 4 nM) 15-fold lower than that of IL-1 beta. Mouse and rat IL-1ra bind to these two human cell types with an affinity similar to that of the human protein. Human IL-1ra binds very weakly to the type II receptor on the mouse pre-B cells with an affinity (KD = 1.4 +/- 0.2 microM) about 1500-fold lower than human IL-1 beta. Mouse and rat IL-1ra also bind to the mouse pre-B cells with low affinity. The weak binding of the three IL-1ra proteins to these mouse cells appears to be more a consequence of the cell type rather than species specificity. There may be a population of cells for which the actions of IL-1 cannot be effectively opposed by IL-1ra, although this group does not include mature B cells and PMNs.