Analysis of a soluble mutant des-methionine interleukin-2 receptor alpha chain (Tac protein) produced by transfected mammalian cells

By using recombinant DNA technology the cytoplasmic and trans-membrane domain of the human interleukin-2 receptor alpha chain (IL-2R alpha, Tac) and of a mutant protein lacking methionine-residues 18, 25, 44, 88, 92, 126, 149, 167, 205, and 209 (des-Met IL-2R alpha) encoded by a chemically and enzymatically synthesized gene, were deleted. This leads to secretory expression of soluble wild-type and des-Met mutant Tac protein of 42-45 kDa after transfection of BHK-21 cells. Transfectants secreted up to 1.6 micrograms soluble wild-type IL-2R alpha protein/10(6) cells in 24 h into the culture medium. LTK- cell lines, expressing a large number of wild-type and des-Met mutant low-affinity IL-2R alpha of 50-55 kDa on their surface, shed a truncated form of the Tac protein of about 40 kDa into the culture medium. In contrast to wild-type IL-2R alpha, shedding of mutant Tac protein is strongly reduced. This phenomenon might be the result of higher protein stability of the mutant receptor which may also explain the about 10 times higher surface expression of des-Met IL-2R alpha in LTK- cells. There are no significant differences in the biosynthesis and post-translational modification of mutant or wild-type Tac proteins either in transfected LTK- or BHK-21 cells as analysed by pulse/chase labeling experiments.     

Molecular cloning and expression of the murine interleukin-5 receptor

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.     

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.     

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 soluble interleukin-1 binding protein in cell-free supernatants. Evidence for soluble interleukin-1 receptor

This study describes the identification and characterization of a soluble interleukin-1 (IL-1) binding protein in the conditioned media from Raji human B-lymphoma cells. The soluble IL-1 binding material was isolated by IL-1 affinity chromatography, and treatment with trypsin decreased its ability to bind to IL-1 demonstrating its protein nature. The soluble IL-1 binding protein was specific for IL-1 and was able to discriminate between Il-1 alpha and IL-1 beta in a manner analogous to the membrane-bound Raji IL-1 receptor. The specificity of the IL-1 binding protein was further established in two ways. 1) Cell-free supernatants from Raji "receptor-negative" cells did not contain any IL-1 binding protein, thus ruling out nonspecific interactions between IL-1 and a serum or other protein present in the conditioned medium; and 2) the soluble binding protein inhibited IL-1 binding to Raji cells in a dose-dependent manner. Scatchard analysis of IL-1 beta binding showed the dissociation constant (KD) to be 5.1 nM for the soluble IL-1 binding protein compared with 0.8 nM for the membrane-bound IL-1 receptor. Gel chromatography of the soluble binding protein yielded a major peak of IL-1 binding activity with a molecular mass of 35-45 kDa. The characteristics of the soluble IL-1 binding protein described above are consistent with those of the extracellular binding domain of the membrane-bound Raji IL-1 receptor.     

A biochemical and kinetic analysis of the interleukin-1 receptor. Evidence for differences in molecular properties of IL-1 receptors

This study describes the biochemical characterization and kinetic analysis of the interleukin-1 (IL-1) receptor in Raji human B-lymphoma and EL4 murine T-lymphoma cells. The internalization of 125I-IL-1 was studied in both cell types by an acid extraction technique which removes surface bound ligand. At 37 degrees C, binding to Raji IL-1 receptors was almost entirely cell surface (91%). EL4 cells, in contrast, internalized 59% of ligand at this temperature and this was almost totally inhibited by sodium azide. Receptor binding studies showed that the B-cells had a lower binding affinity but much higher receptor density per cell (KD = 2.1 nM, Ro = 7709) than the T-cells (KD = 0.4 nM, Ro = 241). The receptor binding affinity of two IL-1 analogs, Glu-4 and clone 18, was determined in competitive binding studies. In the B-cells the analogs had binding affinities of 25 and 90%, respectively, whereas in the T-cells the affinities were 0.2 and 200%, respectively. Chemical cross-linking studies showed that the IL-1 receptor in B-cells had a lower molecular weight than that in T-cells (68 kDa compared to 80 kDa). In summary these studies demonstrate that structural differences exist between IL-1 receptors in Raji and EL4 cells.     

Suppression of interleukin-2 and interleukin-2 receptor expression in Jurkat cells stably expressing the human immunodeficiency virus Tat protein.

The Jurkat T cell line was stably transfected with an Epstein-Barr virus-based episomal replicon designed to express high levels of the HIV-1 Tat protein. After selection in hygromycin B, high-level Tat activity was detected in 3 of 18 transfected cell lines. After stimulation with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), Tat transfectants with high Tat expression showed diminished expression of interleukin-2 (IL-2) and the interleukin-2 receptor alpha chain (IL-2R) when compared to untransfected Jurkat cells or Jurkat cell lines transfected with the parent control plasmid. Sublines derived from the high-level Tat transfectants with reduced Tat activity showed normalization of PHA/PMA-induced IL-2 expression. Northern analysis showed diminished expression of IL-2 and IL-2R mRNA in the stimulated Tat transfectants. Inhibition of IL-2 and IL-2R expression by the HIV-1 Tat protein may contribute to the immune suppression that characterizes HIV-1 infection.     

Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs.     

Biochemical and functional analysis of soluble human interleukin-2 receptor produced in rodent cells. Solid-phase reconstitution of a receptor-ligand binding reaction

The binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) on human T-cells is a key regulatory event which is absolutely required for T-cell-mediated immune responses. To understand further this binding event, we modified the human IL-2R gene to encode a secreted form of IL-2R. Secreted IL-2R was then expressed at very high levels (approximately 11 micrograms/10(6) cells/48 h) in rodent cells using gene-linked co-amplification. The soluble forms of IL-2R were shown to retain IL-2 affinity shown by cell-surface IL-2R (Kd approximately 18 nM) and were purified to homogeneity using IL-2 affinity chromatography. Purified, recombinant IL-2R and biotinylated IL-2 were used to establish a solid-phase receptor binding assay. Binding of IL-2-biotin was demonstrated to be dose-dependent at concentrations ranging from 10 to 1000 ng/ml, and the specificity of receptor-ligand binding was demonstrated by competition with non-biotinylated IL-2 and with anti-receptor antibodies known to block IL-2 binding in vivo. This immunosorbent receptor assay offers a simple and rapid method for studying the binding of IL-2 to human IL-2R.     

Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.     

Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution

We created a novel mutated form of human interleukin-13 (IL-13) in which a positively charged arginine (R) at position 112 was substituted to a negatively charged aspartic acid (D). This mutant, termed IL-13R112D, was expressed in Escherichia coli and purified to near homogeneity. IL-13R112D was found to be a potent IL-13 agonist with 5-10-fold improved binding affinity to IL-13 receptors compared with wild-type IL-13 (wtIL-13). The conclusion of IL-13 agonist activity was drawn on the basis of approximately 10-fold improved activity over wtIL-13 in several assays: (a) inhibition of CD14 expression in primary monocytes; (b) proliferation of TF-1 and B9 cell lines; and (c) activation of STAT6 in Epstein-Barr virus-immortalized B cells, primary monocytes, and THP-1 monocytic cell line. Furthermore, mutant IL-13R112D neutralized the cytotoxic activity of a chimeric fusion protein composed of wtIL-13 and a Pseudomonas exotoxin A (IL-13-PE38) approximately 10 times better than wtIL-13. Based on these results, it was concluded that IL-13R112D interacts with much stronger affinity than wtIL-13 on all cell types tested and that Arg-112 plays an important role in the interaction with its receptors (IL-13R). Thus, these results suggest that IL-13R112D may be a useful ligand for the study of IL-13 interaction with its receptors or, alternatively, in designing specific targeted agents for IL-13R-positive malignancies.     

Biochemical evidence for a third chain of the interleukin-2 receptor

Two receptor proteins that specifically bind interleukin-2 (IL-2) have been identified previously. The L (Tac or alpha) chain can bind IL-2 with a Kd value of 10 nM (low affinity). Although the H (beta) chain expressed on lymphocytes can bind IL-2 with a Kd value of 1 nM (intermediate affinity), transfected fibroblasts expressing the H chain cannot bind IL-2, suggesting the involvement of other lymphocyte-specific factors for the function of the H chain. To obtain direct evidence for the presence of a third component of the IL-2 receptor, we examined the IL-2 binding activity of detergent-solubilized cell membrane preparations. We found that lysates of transfected Cos7 cells expressing H chains can bind IL-2 when mixed with lysates from lymphocytes that cannot bind IL-2. Chemical cross-linking of 125I-IL-2-bound lysate mixture and subsequent immunoprecipitation with a noncompetitive anti-H chain antibody gave rise to two 125I-IL-2-bound proteins, a 56-kDa protein (p56) and the H chain, although neither the H chain nor p56 alone is able to bind IL-2. These results indicate that p56 is the IL-2 receptor third chain that is required for IL-2 binding to the H chain. A similar lysate mixing experiment also showed that p56 is involved in IL-2 binding to the high affinity IL-2 receptor by forming the quaternary complex of IL-2, p56, L chain, and H chain.     

Kinetic analysis of ligand binding to interleukin-2 receptor complexes created on an optical biosensor surface.

The interleukin-2 receptor (IL-2R) is composed of at least three cell surface subunits, IL-2R alpha, IL-2R beta, and IL-2R gamma c. On activated T-cells, the alpha- and beta-subunits exist as a preformed heterodimer that simultaneously captures the IL-2 ligand as the initial event in formation of the signaling complex. We used BIAcore to compare the binding of IL-2 to biosensor surfaces containing either the alpha-subunit, the beta-subunit, or both subunits together. The receptor ectodomains were immobilized in an oriented fashion on the dextran matrix through unique solvent-exposed thiols. Equilibrium analysis of the binding data established IL-2 dissociation constants for the individual alpha- and beta-subunits of 37 and 480 nM, respectively. Surfaces with both subunits immobilized, however, contained a receptor site of much higher affinity, suggesting the ligand was bound in a ternary complex with the alpha- and beta-subunits, similar to that reported for the pseudo-high-affinity receptor on cells. Because the binding responses had the additional complexity of being mass transport limited, obtaining accurate estimates for the kinetic rate constants required global fitting of the data sets from multiple surface densities of the receptors. A detailed kinetic analysis indicated that the higher-affinity binding sites detected on surfaces containing both alpha- and beta-subunits resulted from capture of IL-2 by a preformed complex of these subunits. Therefore, the biosensor analysis closely mimicked the recognition properties reported for these subunits on the cell surface, providing a convenient and powerful tool to assess the structure-function relationships of this and other multiple subunit receptor systems.     

Receptor binding and internalization of mouse interleukin-2 derivatives that are partial agonists.

Mouse interleukin-2 (mIL-2) mutant proteins with subunit-specific receptor binding defects have been previously described. Some of these mutant proteins are unable to trigger a maximum proliferative response of T cells. In this study, mIL-2 and mIL-2 mutant proteins were labeled with 32P, and their association and dissociation kinetics with the high affinity IL-2 receptor (IL-2R) were investigated. A mIL-2 mutant protein with a partial defect in binding to the low affinity component of IL-2R had a slower on-rate than mIL-2. On the other hand, a mIL-2 antagonist with a binding defect to the intermediate affinity component of IL-2R had a normal on-rate, whereas its off-rate at 37 degrees C was faster than mIL-2. This fast off-rate at physiological temperature interfered with mIL-2 internalization. When three mIL-2 partial agonists, each inducing a different maximal response, were examined, no difference was found between their dissociation rates or their internalization properties. The significance of these findings for the function of each receptor subunit in the IL-2R complex, as well as for the mechanism of activation of the receptor, is discussed.     

The cell surface expression level of the human interleukin-5 receptor alpha subunit determines the agonistic/antagonistic balance of the human interleukin-5 E13Q mutein.

The human interleukin-5 (IL-5) receptor consists of an alpha-chain that specifically binds the ligand with intermediate affinity, and a beta c-chain, that associates with the IL-5/IL-5R alpha complex, leading to a high-affinity, signal transducing receptor complex. Structure-function studies showed that modification of the putative beta c-chain binding site in IL-5 (E13Q mutein) converted the molecule into an antagonist. However, analysis of the effect of this mutant IL-5 on COS-1 cells transfected with both receptor subunits, did not show reduced interaction with the beta c subunit [Tavernier, J., Tuypens, T., Verhee, A., Plaetinck, G., Devos, R., Van der Heyden, J., Guisez, Y. & Oefner, C. (1995) Proc. Natl Acad. Sci. USA 89, 7041-7045]. To gain more insight into the mechanism of IL-5 antagonism by E13Q, we tested its biological activity on two FDC-P1 subclones that express clearly different numbers of alpha-subunits yet an almost constant number of murine beta c-subunits. Here we show that E13Q has a biological activity comparable to wild-type IL-5 only when a high number of alpha-chains is present on the cells. Confirming the critical role of the IL5R alpha cell-surface expression level, treatment with suboptimal doses of a neutralising anti-IL-5R alpha antibody results in reduced activity of the mutant but not of wild-type IL-5.     

Biological characterization of human interleukin-2 mutant proteins. Structure-activity relationship studies

Several human interleukin-2 (IL-2) mutant proteins have been produced previously by site-directed mutagenesis and found to have different capacities to induce T-cell proliferative activity. In this study, the abilities of these IL-2 mutant proteins to activate natural killer cells and to induce interferon-gamma production have been evaluated, and the binding of these proteins to IL-2 receptors analyzed. Natural killer cell activation and interferon-gamma induction assays showed that the relative activities of IL-2 mutant proteins were consistent with their relative activities in T-cell proliferation assay. Receptor-binding studies showed that the activities of most proteins correlated well with their respective affinities for high-affinity IL-2 receptors on CTLL-2 cells. Interestingly, although the mutant protein with deletion of cysteine 125 (des-Cys125) was biologically less active than the protein with substitution of alanine for cysteine 105 (Ala105), both proteins exhibited similar affinity. Des-Cys125, like IL-2 and Ala105, also caused down-regulation of high-affinity IL-2 receptors. Binding studies on MLA-144, a cell line expressing mainly intermediate-affinity IL-2 receptors (IL-2R beta), however, showed that des-Cys125 had much lower affinity than Ala105. These results suggest that binding of IL-2 and mutant proteins to the IL-2R beta component of the high-affinity receptor is essential for the induction of biological effects.     

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.