Evidence for additional subunits associated to the mouse interleukin 2 receptor p55/p75 complex.

Previous studies have indicated that high-affinity interleukin 2 receptors (IL-2R) are comprised of at least two distinct noncovalently associated subunits of Mr 55,000 (p55) and Mr 75,000 (p75). To biochemically characterize p75, we have directly isolated IL-2R using affinity precipitations with immobilized IL-2. We now report that at least some mouse p75 appears to exist as a disulfide-linked heterodimer with a subunit of Mr 22,000 (p22). These findings suggest that functional high-affinity mouse IL-2R may be comprised of at least three distinct subunits. The possibility of an even more complex structure is suggested by the coprecipitation of a protein of Mr 40,000-45,000 (p40) that may represent an additional IL-2R-associated protein.     

Demonstration of a non-Tac peptide that binds interleukin 2: a potential participant in a multichain interleukin 2 receptor complex.

The interleukin 2 (IL-2) receptor system plays a key role in the T-cell immune response. Although IL-2 binding was reported to be restricted to the Tac peptide, we have identified an IL-2 binding peptide that does not react with anti-human IL-2 receptor monoclonal antibodies, including anti-Tac on MLA 144, a gibbon ape T-cell line. The MLA 144 cell line expressed 6800 IL-2 binding sites per cell with a low (Kd = 14 nM) affinity for human recombinant IL-2. Using cross-linking methodology, we demonstrated that the IL-2 binding peptide on MLA 144 is larger (Mr 75,000) than the Tac peptide, which has a Mr of 55,000. An IL-2 binding peptide of similar size (Mr 75,000) was also identified in addition to the Tac peptide (Mr 54,000-57,000) on Hut 102, a human T-cell lymphotrophic virus I-induced T-cell leukemia line, and phytohemagglutinin-activated normal human and gibbon ape lymphoblasts. Anti-Tac antibody did not block the binding of 125I-labeled IL-2 to MLA 144 cells. However, this antibody abolished the binding of 125I-labeled IL-2 not only to the Tac peptide on Hut 102 cells and normal lymphoblasts but also to the Mr 75,000 IL-2 binding peptide, suggesting that this latter peptide is associated with the Tac peptide to form the high-affinity IL-2 receptor complex.     

Localization in human interleukin 2 of the binding site to the alpha chain (p55) of the interleukin 2 receptor.

Human interleukin 2 (IL-2) analogs with defined amino acid substitutions were used to identify specific residues that interact with the 55-kDa subunit (p55) or alpha chain of the human IL-2 receptor. Analog proteins containing specific substitutions for Lys-35, Arg-38, Phe-42, or Lys-43 were inactive in competitive binding assays for p55. All of these analogs retained substantial competitive binding to the intermediate-affinity p70 subunit (beta chain) of the receptor complex. The analogs varied in ability to interact with the high-affinity p55/p70 receptor. Despite the lack of binding to p55, all analogs exhibited significant biological activity, as assayed on the murine CTLL cell line. The dissociation constants of Arg-38 and Phe-42 analogs for p70 were consistent with intermediate-affinity binding; the Kd values were not significantly affected by the presence of p55 in binding to the high-affinity IL-2 receptor complex. These results confirm the importance of the B alpha-helix in IL-2 as the locus for p55-receptor binding and support a revised model of IL-2-IL-2 receptor interaction.     

Association of intercellular adhesion molecule 1 with the multichain high-affinity interleukin 2 receptor.

Previously, using flow cytometric resonance energy transfer and lateral diffusion measurements, we demonstrated that a 95-kDa protein identified by two monoclonal antibodies (OKT27 and OKT27b) interacts physically with the 55-kDa alpha protein of the high-affinity interleukin 2 (IL-2) receptor. In the present study, this 95-kDa protein (p95) was purified and amino acid sequence data were obtained that showed strong homology to the human intercellular adhesion molecule 1 (ICAM-1). The identity of the p95 protein with ICAM-1 was confirmed by sequential immunoprecipitations using OKT27 and an antibody, WEHI-CAM-1, that is directed toward ICAM-1. We confirmed the physical proximity of p95/ICAM-1 to the IL-2 receptor alpha subunit by demonstrating that radiolabeled IL-2 could be cross-linked to this protein expressed on activated T cells. In functional studies, the antibodies OKT27 and OKT27b inhibited T-cell proliferative responses to OKT3, to soluble antigen, and to heterologous cells (mixed lymphocyte reaction). However, these antibodies did not inhibit IL-2-induced proliferation of an IL-2-dependent T-cell line. Taken together with our previous observations, the present studies suggest that ICAM-1 is in proximity and interacts physically with the high-affinity IL-2 receptor. The association of ICAM-1 with the IL-2 receptor may facilitate the paracrine IL-2-mediated stimulation of T cells expressing IL-2 receptors by augmenting homotypic T-T-cell interaction, by receptor-directed focusing of IL-2 release by helper T cells, and by focusing IL-2 receptors of the physically linked cells to the site of lymphocyte function-associated antigen 1-ICAM-1-IL-2 receptor interaction.     

Identification of a direct interaction between interleukin 2 and the p64 interleukin 2 receptor gamma chain.

The interleukin 2 receptor (IL-2R) consists of at least two subunits, alpha and beta, both of which can bind interleukin 2 (IL-2). Recent studies have demonstrated the existence of a third subunit, a 64-kDa molecule termed IL-2R gamma chain, and have suggested that gamma chain functions to regulate the rate of IL-2 dissociation from the receptor. In the present report we have addressed whether the gamma chain modulates IL-2R affinity by contributing contact sites for IL-2 binding. Using reagents that allow the IL-2R complex to be immunoprecipitated through the IL-2 molecule itself, we demonstrate the existence of a stable IL-2-IL-2R gamma-chain complex. These studies thus establish that the IL-2R gamma chain directly contributes to the IL-2-binding site, consistent with the hypothesis that gamma chain influences IL-2R affinity through its direct interaction with IL-2.     

Identification of specific residues of human interleukin 2 that affect binding to the 70-kDa subunit (p70) of the interleukin 2 receptor.

Analogs of interleukin 2 containing defined amino acid substitutions and deletions were assayed for bioactivity and for competitive binding to the high-affinity human interleukin 2 receptor complex and its two component subunits, a 55-kDa subunit (p55 or TAC) and a 70-kDa subunit (p70). Substitution of Asp20 or deletion of Phe124 resulted in inactive analog proteins that were unable to interact with the high-affinity p55/p70 complex or the intermediate-affinity p70 subunit of the interleukin 2 receptor. These analogs, however, retained the capacity to compete for binding to the low-affinity p55 subunit. The presence of the carboxylic acid in the side chain of Asp20 was necessary for effective binding to the p70 protein. In contrast, substitution of Trp121 and Leu17 created analogs that were inactive in the bioassay and all three binding assays. The effects of these mutations on protein conformation were assessed by circular dichroism. These results demonstrate that specific residues in the NH2 and COOH termini of interleukin 2 are crucial for its structure and activity.     

Properties of a high-affinity DNA binding site for estrogen receptor.

It has been shown previously that a short sequence from the 5' regulatory region of the Xenopus laevis vitellogenin gene A2, when appropriately placed, can confer estrogen responsiveness to another gene. Using the Xenopus sequence and similar sequences from the 5' regulatory regions of other estrogen-responsive genes, we derived a consensus sequence 38 nucleotides long. The sequence contains an inverted repeat (5' C-A-G-G-T-C-A-G-A-G-T-G-A-C-C-T-G 3') and an A/T-rich region. Plasmids carrying a single copy of the sequence bound 3-fold-more partially purified estrogen receptor (ER) than did control plasmids when assayed by gel filtration. Maximum specificity for ER binding occurs at 100-150 mM ionic strength and pH 7.5-8.0. Plasmids carrying multiple copies of the sequence bound correspondingly more ER. The dissociation constant for ER bound to the sequence is 0.5 nM. This value is lower by a factor of about 400 than the dissociation constant for ER bound to an equivalent length of plasmid DNA. Portions of the consensus sequence were evaluated for binding efficiency. Plasmids containing the inverted repeat alone bound ER, though less efficiently than did plasmids containing the entire sequence. The A/T-rich region alone was ineffective in binding ER. Linearization of the plasmid DNA did not enhance specific binding efficiency for ER. This model system represents an effective tool for characterization of ER binding to DNA sequences involved in the regulation of gene expression.     

High-affinity interleukin 2 binding by an oncogenic hybrid interleukin 2-epidermal growth factor receptor molecule.

Both interleukin 2 (IL-2) and epidermal growth factor (EGF) receptors exist in two forms that differ with respect to affinity for their ligand. Only the high-affinity receptors appear to be responsible for the proliferation signal delivered upon binding of the growth factor. Fibroblasts transfected with IL-2 receptor cDNA generate only low-affinity receptors for IL-2, but fusion of membranes from these fibroblasts with T-cell membranes converts some receptors to high affinity, indicating the involvement of a T cell-specific factor in the generation of high-affinity receptors. We have constructed a chimeric cDNA molecule containing the extracellular IL-2-binding domain of the IL-2 receptor cDNA and the transmembrane and intracellular tyrosine kinase domains of the EGF receptor cDNA. When transfected into fibroblasts, this IL-2-EGF receptor cDNA generated high-affinity receptors for IL-2. Moreover, fibroblasts transfected with the chimeric molecule were morphologically transformed and produced rapidly growing tumors in nude mice.     

Mitogenicity and down-regulation of high-affinity interleukin 2 receptor by YTA-1 and YTA-2, monoclonal antibodies that recognize 75-kDa molecules on human large granular lymphocytes.

A large number of interleukin 2 receptors lacking the Tac epitope (IL-2R/p75) were found to be constitutively expressed on the human large granular lymphocyte/natural killer cell line YT, which bears inducible IL-2R/p55 associated with Tac antigen. Two anti-YT IgG1 monoclonal antibodies, YTA-1 and YTA-2, recognizing different epitopes of the same 75- to 80-kDa molecule, were established. The 75-kDa antigen recognized by these monoclonal antibodies was strongly expressed on the large granular lymphocytes of normal peripheral blood mononuclear cells and on various lymphoid cell lines bearing IL-2R/p75. The YTA-1 and YTA-2 antibodies were mitogenic and were different from other mitogenic monoclonal antibodies such as anti-T3 (CD3), anti-T11 (CD2), and KOLT-2 (CD28). Further, they down-regulated the high-affinity IL-2R of peripheral blood mononuclear cells within 24 hr in culture. The relationship between the YTA-1/2 antigen and the IL-2R system is discussed.     

Induction and upregulation by interleukin 2 of high-affinity interleukin 2 receptors on thymocytes and T cells.

We show that purified recombinant interleukin 2 (rIL-2) alone induces the expression of high- and low-affinity interleukin 2 (IL-2) receptors in vitro on human T cells and thymocytes that have not been activated previously by lectins or other inducing agents. IL-2 receptors are expressed after 24 hr, as determined by the binding of 125I-labeled monoclonal anti-IL-2 receptor antibody 2A3, which binds equally to high- and low-affinity receptors. High-affinity receptors were distinguished from low-affinity receptors by the binding of 125I-labeled IL-2 to T cells and by the proliferative response of thymocytes to IL-2, in concentrations that selectively interact with the high-affinity class of IL-2 receptors. The IL-2-induced proliferation of thymocytes in vitro induced by IL-2 alone is dependent upon the concentration of IL-2 and is inhibited by monoclonal anti-Tac antibody, indicating that the proliferative response is mediated by the binding of IL-2 to the receptors. In addition, we demonstrate that IL-2 augments the number of high-affinity receptors on concanavalin A-activated thymocytes. These results document that IL-2 acts as a hormone that induces the activation of thymocytes and T cells, as evidenced by the de novo induction of biologically active, high-affinity IL-2 receptors. IL-2 also upregulates the expression of high-affinity IL-2 receptors on activated thymocytes. These observations illustrate the biologic importance of the regulatory role of IL-2 in the immune response.     

T-cell hybridoma specific for a cytochrome c peptide: specific antigen binding and interleukin 2 production.

T-cell hybridomas were obtained after fusion of BW 5147 thymoma and long-term cultured T cells specific for cytochrome c peptide 66-80 derivatized with a 2,4-dinitroaminophenyl (DNAP) group. The resulting hybridomas were selected for their capacity to specifically bind to soluble radiolabeled peptide antigen. One T-cell hybrid was positive for antigen binding. This hybrid T cell exhibits surface phenotypic markers of the parent antigen-specific T cells. The binding could be inhibited either by an excess of unlabeled homologous antigen or by cytochrome c peptide 11-25 derivatized with a 2-nitrophenylsulfenyl group. Several other peptide antigens tested failed to inhibit binding of the radioactive peptide. This suggests that a specific amino acid sequence, modified by a DNAP group, is the antigenic structure recognized by the putative T-cell receptor. In addition, direct interaction of DNAP-66-80 peptide with the hybridoma cell line induced production of the T-cell growth factor interleukin 2. Furthermore, supernatants derived from syngeneic macrophages pulsed with the relevant peptide also induced the antigen-specific hybridoma to produce interleukin 2.     

Phenotypic knockout of the high-affinity human interleukin 2 receptor by intracellular single-chain antibodies against the alpha subunit of the receptor.

The experimental manipulation of peptide growth hormones and their cellular receptors is central to understanding the pathways governing cellular signaling and growth control. Previous work has shown that intracellular antibodies targeted to the endoplasmic reticulum (ER) can be used to capture specific proteins as they enter the ER, preventing their transport to the cell surface. Here we have used this technology to inhibit the cell surface expression of the alpha subunit of the high-affinity interleukin 2 receptor (IL-2R alpha). A single-chain variable-region fragment of the anti-Tac monoclonal antibody was constructed with a signal peptide and a C-terminal ER retention signal. Intracellular expression of the single-chain antibody was found to completely abrogate cell surface expression of IL-2R alpha in stimulated Jurkat T cells. IL-2R alpha was detectable within the Jurkat cells as an immature 40-kDa form that was sensitive to endoglycosidase H, consistent with its retention in a pre- or early Golgi compartment. A single-chain antibody lacking the ER retention signal was also able to inhibit cell surface expression of IL-2R alpha although the mechanism appeared to involve rapid degradation of the receptor chain within the ER. These intracellular antibodies will provide a valuable tool for examining the role of IL-2R alpha in T-cell activation, IL-2 signal transduction, and the deregulated growth of leukemic cells which overexpress IL-2R alpha.     

Cooperative binding of insulin-like Peptide 3 to a dimeric relaxin family peptide receptor 2

Insulin-like peptide 3 (INSL3) binds to a G protein-coupled receptor (GPCR) called relaxin family peptide receptor 2 (RXFP2). RXFP2 belongs to the leucine-rich repeat-containing subgroup (LGR) of class A GPCRs. Negative cooperativity has recently been demonstrated in other members of the LGR subgroup. In this work, the kinetics of INSL3 binding to HEK293 cells stably transfected with RXFP2 (HEK293-RXFP2) have been investigated in detail to study whether negative cooperativity occurs and whether this receptor functions as a dimer. Our results show that negative cooperativity is present and that INSL3-RXFP2 binding shows both similarities and differences with insulin binding to the insulin receptor. A dose-response curve for the negative cooperativity of INSL3 binding had a reverse bell shape reminiscent of that seen for the negative cooperativity of insulin binding to its receptor. This suggests that binding of INSL3 may happen in a trans rather than in a cis way in a receptor dimer. Bioluminescence resonance energy transfer (BRET(2)) experiments confirmed that RXFP2 forms constitutive homodimers. Heterodimerization between RXFP2 and RXFP1 was also observed.     

Mechanism of epidermal growth factor receptor autophosphorylation and high-affinity binding.

Epidermal growth factor (EGF) receptor monomers and noncovalently associated dimers were isolated by sucrose density gradient centrifugation, and their respective binding and autophosphorylation activities were determined. We find that monomers are low-affinity receptors and dimers are high-affinity receptors. In the absence of EGF, dimers exhibit a 4-fold higher autophosphorylation activity than do monomers. Addition of EGF increases autophosphorylation on monomers an average of 4.8-fold but has a minimal effect on autophosphorylation of dimers. Furthermore, EGF binding shifts the receptor monomer-dimer equilibrium to the dimer form. We conclude that EGF stimulates in vitro receptor autophosphorylation by inducing kinase-inactive receptor monomers to associate and form receptor dimers, in which conformation the autophosphorylation activity is enhanced.     

Defective expression of p70/75 interleukin 2 receptor in T cells from patients with systemic lupus erythematosus: a possible defect in the process of increased intracellular calcium leading to p70/75 expression

Phytohemagglutinin (PHA) stimulated T cells from patients with systemic lupus erythematosus (SLE) showed hyporesponsiveness to interleukin 2 (IL-2) and expressed less p70/75 IL-2R than healthy controls. Ionomycine (IM, calcium ionophore) which selectively upregulated p70/75 expression, induced less p70/75 in patients with SLE than in healthy controls. However, intracellular calcium levels of T cells from patients with SLE increased as much as those from healthy controls, when T cells were stimulated by IM or PHA. Our results suggest that an impaired expression of p70/75 IL-2R in T cells from patients with SLE is not due to a defective calcium influx but to the events after the rise of calcium levels.     

Delineation of a region in the B2 bradykinin receptor that is essential for high-affinity agonist binding.

We have made mutations in the predicted sixth transmembrane segment of a rat B2 bradykinin receptor and analyzed the variant proteins by expressing them in COS-1 cells. Two amino acid substitutions reduced the affinity of the receptor for bradykinin (Phe261-->Val by 1600-fold; Thr265-->Ala by 700-fold) with comparatively little effect on the affinity for the bradykinin antagonists NPC17731 and D-Arg-[Hyp3,D-Phe7]bradykinin (where Hyp is hydroxyproline). Three other substitutions (Gln262-->Ala, Asp268-->Ala, and Thr269-->Ala) modestly reduced the affinity for bradykinin and for the antagonist D-Arg-[Hyp3,D-Phe7]bradykinin. Even the most dramatically affected mutated receptors were still able to couple, after bradykinin binding, to phosphatidylinositol turnover. The data suggest that bradykinin directly contacts the face of the sixth transmembrane helix formed by the residues Phe261, Gln262, Thr265, Asp268, and Thr269 or that this face of the helix is the site of intraprotein contacts that serve to stabilize the agonist-binding conformation of the receptor.     

Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.

Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors.     

Conversion of low-affinity interleukin 2 receptors to a high-affinity state following fusion of cell membranes

The cellular receptors for interleukin 2 (IL-2) exist in at least two forms, one with a particularly high affinity and a second, more numerous class, with a much lower affinity for IL-2. Indirect evidence suggests that both classes of receptors use the same p55 glycoprotein as their ligand-binding component. L cells transfected with cDNA encoding this protein, however, displayed only low-affinity IL-2 binding. To determine if such receptors could be converted to a high-affinity state, L-cell membranes containing the murine p55 protein were fused with membranes from human T cells displaying high-affinity receptors. The anti-Tac antibody was used to block ligand binding to human p55 on the fusion product. The results showed that a fraction of the murine p55 chains were converted to a dramatically higher affinity following fusion. Fusion of the L-cell membranes with themselves or with membrane preparations from human T-cell lines lacking the IL-2 receptor resulted in little or no affinity modulation. One explanation of the results is that cofactors present in receptor-positive T-cell lines crossed species lines and combined with the murine p55 chain to create "high-affinity" binding sites. Thus, depending upon its environment, the same p55 molecule can apparently form either a low- or high-affinity IL-2 receptor.     

Cloning of the low-affinity murine granulocyte-macrophage colony-stimulating factor receptor and reconstitution of a high-affinity receptor complex.

A cDNA clone (clone 71) that encodes a low-affinity receptor for murine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been isolated by direct expression. This molecule is the homologue of the human GM-CSF receptor alpha subunit, although homology between these molecules is surprisingly low (less than 35% amino acid identity). The cDNA encodes a polypeptide of 387 amino acids, which contains the conserved features of the hematopoietin receptor superfamily. When expressed in COS-7 cells, this clone encodes a protein that binds radiolabeled murine GM-CSF with low affinity. Coexpression of clone 71 with a cDNA corresponding to a low-affinity interleukin 3 (IL-3) receptor (AIC2A) did not alter the affinity of binding of either GM-CSF or IL-3. However, coexpression of clone 71 with the IL-3 receptor-related cDNA AIC2B generated high-affinity binding sites for murine GM-CSF but not murine IL-3. These studies show that clone 71 and AIC2B are capable of forming an alpha beta complex capable of binding murine GM-CSF with high affinity, while AIC2A appears not to be a component of the murine GM-CSF receptor.