Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.     

Highly concentrated urine-purified Tac peptide fails to inhibit IL-2-dependent cell proliferation in vitro.

Tac peptide, i.e., the p55 chain of the human interleukin-2 receptor (IL-2R) complex, is detectable as a soluble from (sIL-2R) in normal sera and, at increased levels, in patients with different diseases. Since several immunological abnormalities are observed in most conditions associated with an increase in sIL-2R levels, a down-regulatory effect on IL-2-dependent functions has been postulated as a consequence of binding and functional block of IL-2 by the excess of sIL-2R. To test this hypothesis, we purified sIL-2R from the urine of a patient with hairy cell leukemia and investigated the possible inhibitory effect of this peptide on the in vitro IL-2-induced cell proliferation. The urine-purified molecule was detectable by the specific immunoassay utilized to measure the serum Tac peptide and was constructed by a single polypeptide of about 50 kDa which was able to bind IL-2. Experiments performed with the IL-2-dependent murine CTLL-2 cell line and with PHA-stimulated human peripheral blood mononuclear cells showed that the purified sIL-2R at concentrations up to about 300 nM was unable to block IL-2-dependent cell proliferation. According to these data, which can be explained by the low affinity for IL-2 of the p55 IL-2R chain, it seems unlikely that in vivo the soluble Tac peptide can exert a down regulatory effect on IL-2-induced phenomena through a functional block of IL-2.     

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-γ (IFN-γ). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.     

IL-2-dependent proliferation of murine T cells requires expression of both the p55 and p70 subunits of the IL-2 receptor

An IL-4-dependent T cell clone (LD8) was isolated from the murine IL-2-dependent cytotoxic T cell line C30.1. This clone has lost the capacity to proliferate in response to IL-2 after long-term culture in IL-4. LD8 cells express the p70, but not the p55, subunit of the IL-2R on their cell surface. The number of p70 IL-2R molecules on LD8 cells is comparable with the number of high-affinity IL-2R on the parental C30.1 cell line. LD8 cells can efficiently internalize IL-2 through the p70 IL-2R subunit. Following stimulation by IL-2, LD8 cells up-regulate p70 IL-2R mRNA, but do not express p55 IL-2R mRNA. IL-2-dependent proliferation of LD8 cells was reconstituted after introduction and expression of a human p55 IL-2R cDNA. To further investigate the role of p70 IL-2R, we have measured IL-2-induced proliferation of C30.1 cells in the presence of three anti-p55 IL-2R mAb (5A2, PC61, and 7D4) that recognize different epitopes. Under the experimental conditions used, the combination of anti-p55 IL-2R mAb prevents the formation of high-affinity IL-2R, but does not affect the binding of IL-2 to p70 IL-2R or IL-2 internalization. However, these three mAb inhibit proliferation of C30.1 cells even in the presence of IL-2 concentrations sufficient to saturate p70 IL-2R. Together these results demonstrate that p70 IL-2R alone is not sufficient to transmit IL-2-induced growth signals and that formation of p55-p70 IL-2R complex is required for IL-2-dependent proliferation of murine T cells.     

Why are multiple chains required for the interleukin 2 receptor?

The interleukin 2 receptor (IL-2R) is composed of at least two proteins, that is, a 55 kDa L chain (p55, α chain) and a 75 kDa H chain (p75, β chain). The high-affinity binding of IL-2 results in the formation of the ternary complex consisting of IL-2, the L chain and the H chain. Kinetic studies on the IL-2 binding to the high-affinity IL-2R have shown that the association of IL-2 to the L chain is the first step of the ternary complex formation and that expression of a larger number of L chains accelerates the association of IL-2 to the high-affinity IL-2R in agreement with the stepwise binding/affinity conversion model. This conclusion was supported by experiments using several monoclonal antibodies directed to either H or L chain and murine T cell lines which was transfected by the human L chain cDNA. Temperature-sensitive IL-2 binding to the high-affinity receptor is also consistent with the above conclusion. Signal transduction by the IL-2R appears to involve the activation of tyrosine protein kinase. IL-2 signal transduction seems to require the H chain and another yet unidentified molecule, which might have the kinase activity.     

A tyrosine kinase physically associates with the beta-subunit of the human IL-2 receptor

Cell surface expression of the high affinity IL-2R regulates, in part, the proliferative response occurring in Ag- or mitogen-activated T cells. The functional high affinity IL-2R is composed of at least two distinct ligand-binding components, IL-2R alpha (Tac, p55) and IL-2R beta (p70/75). The IL-2R beta polypeptide appears to be essential for growth signal transduction, whereas the IL-2R alpha protein participates in the regulation of receptor affinity. We have prepared and characterized two mAb, DU-1 and DU-2, that specifically react with IL-2R beta. In vitro kinase assays performed with DU-2 immunoprecipitates, but not anti-IL-2R alpha or control antibody immunoprecipitates, have revealed co-precipitation of a tyrosine kinase enzymatic activity that mediates phosphorylation of IL-2R beta. Because both IL-2R alpha and IL-2R beta lack tyrosine kinase enzymatic domains, these findings strongly suggest that noncovalent association of a tyrosine kinase with the high affinity IL-2R complex. Deletion mutants of the intracellular region of IL-2R beta, lacking either a previously described "critical domain" between amino acids 267 and 322 or the carboxyl-terminal 198 residues (IL-2R beta 88), lacked the ability to co-precipitate this tyrosine kinase activity, as measured by phosphorylation of IL-2R beta in vitro. Both of these mutants also failed to transduce growth-promoting signals in response to IL-2 in vivo. Analysis of the IL-2R beta 88 mutant receptor suggested that a second protein kinase mediating phosphorylation on serine and threonine residues physically interacts with the carboxyl terminus of IL-2R beta. This kinase may be necessary but, alone, appears to be insufficient to support a full IL-2-induced proliferative response. These studies highlight the physical association of protein kinases with the cytoplasmic domain of IL-2R beta and their likely role in IL-2-induced growth signaling mediated through the multimeric high affinity IL-2R complex.     

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.     

Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.     

Detailed analysis of the IL-5-IL-5R alpha interaction: characterization of crucial residues on the ligand and the receptor.

The receptor for interleukin-5 (IL-5) is composed of two different subunits. The IL-5 receptor alpha (IL-5R alpha) is required for ligand-specific binding while association with the beta-chain results in increased binding affinity. Murine IL-5 (mIL-5) has similar activity on human and murine cells, whereas human IL-5 (hIL-5) has marginal activity on murine cells. We found that the combined substitution of K84 and N108 on hIL-5 by their respective murine counterpart yields a molecule which is as potent as mIL-5 for growth stimulation of a murine cell line. Since the unidirectional species specificity is due only to the interaction with the IL-5R alpha subunit, we have used chimeric IL-5R alpha molecules to define regions of hIL-5R alpha involved in species-specific hIL-5 ligand binding. We found that this property is largely determined by the NH2-terminal module of hIL-5R alpha, and detailed analysis defined D56 and to a lesser extent E58 as important for binding. Moreover, two additional residues, D55 and Y57, were identified by alanine scanning mutagenesis within the same region. Based on the observed homology between the NH2-terminal module and the membrane proximal (WSXWS-containing) module of hIL-5R alpha we located this stretch of four amino acid residues (D55, D56, Y57 and E58) in the loop region that connects the C and D beta-strands on the proposed tertiary structure of the NH2-terminal module.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lymphokine-regulated differential expression of mRNA for p75kDa-IL-2R and p55kDa-IL-2R in a cloned B lymphoma line (BLC1-CL-3 cells)

IL-5 renders BCL1-CL-3 (CL-3) cells responsive to IL-2 by increasing the number of high affinity IL-2R, whereas IL-4 prohibits such action of IL-5 to prepare CL-3 cells responsive to IL-2. Here we have found that genes for p75kDa-IL-2R and p55kDa-IL-2R are differentially regulated by IL-4 and IL-5. Nonstimulated CL-3 cells constitutively express mRNA for p75kDa-IL-2R and p55kDa-IL-2R. IL-5 stimulation principally augments the expression of p75kDa-IL-2R mRNA (4- to 8-fold), although modestly increasing the expression of p55kDa-IL-2R mRNA. Kinetic studies have revealed a maximal increase in p75kDa-IL-2R mRNA expression at 12 h and a decline thereafter, substantiating our previous kinetic study of the expression of high affinity IL-2R after the IL-5 stimulation. By contrast, IL-4 stimulation modestly increases the expression of p75kDa-IL-2R mRNA, whereas markedly reducing the expression of p55kDa-IL-2R mRNA, irrespective of whether CL-3 cells were stimulated with IL-4 alone or together with IL-5 and IL-2. Moreover, addition of IL-4 into the culture containing IL-5 and IL-2 causes striking reduction in the level of J-chain mRNA, which otherwise is markedly induced by stimulation with IL-5 and IL-2. These results clearly illustrate the differential regulation of p75kDa- and p55kDa-IL-2R-gene expression by IL-5 and IL-4, and reinforce our notion that increased expression of high affinity IL-2R induced by IL-5 is responsible for the IL-2 competent state, and decreased expression of p55kDa-IL-2R by IL-4 is responsible for IL-2 unresponsive state.     

The IL-4 receptor: biochemical characterization of IL-4-binding molecules in a T cell line expressing large numbers of receptors

Cross-linking of 125I-IL-4 to the surface of cells expressing IL-4R yields as the major IL-4-binding molecules, polypeptide chains with inferred m.w. of approximately 70,000 (p70) and approximately 120,000 to 140,000 (p120-p140). The demonstration that the functional product of the IL-4R cDNA clone has m.w. of approximately 140,000 and that no p70 product is detected in transfected COS-7 cells has led to an uncertainty regarding the nature of p70. To study this issue, we examined the relationship of the IL-4-binding molecules p120 and p70 and, in parallel, attempted to immunoprecipitate p70 from surface and internally labeled cells using IL-4 and two anti-IL-4R antibodies (M1 and M2), bound to Affigel 10, as ligands. Cross-linked complexes containing 125I-IL-4 and p70 or p120 were isolated and digested with chymotrypsin or with V8 protease. Three distinct IL-4-binding peptides could be compared; these were indistinguishable for cross-linked p70 and p120, strongly implying that p70 and p120 were structurally related. Furthermore, immunoprecipitates made with IL-4 or anti-IL-4R-Affigel did not contain p70. This led us to conclude that p70 is a breakdown product of p120. A second IL-4-binding molecule of 40,000 Da (p40) expressing the M1 and M2 epitopes of the IL-4R was detected and appears to be the product of an mRNA coding for the soluble form of the receptor. mRNA for p40 was detected in both the T cell line CT.4R and the mast cell line CFTL.12 using polymerase chain reaction primers unique to this species of message. Pulse-chase studies of IL-4R in [35S] methionine-labeled cells indicates that p40 is derived from a 42,000-Da precursor that is detectable at the end of the pulse period, and thus, further argue that p40 is an independently translated molecule and not a degradation product of p120. Although p40 has been previously shown to be a soluble, truncated form of the receptor, we failed to observe secretion of p40 into the medium by internally labeled CT.4R cells.     

Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.     

Diversity of the human allergen-specific T cell repertoire associated with distinct skin test reactions: delayed-type hypersensitivity-associated major epitopes induce Th1- and Th2-dominated responses.

Distinct immune responses in humans to the Trichophyton rubrum Ag, Tri r 2, are associated with different patterns of T cell epitope recognition based on in vitro proliferation to peptides derived from this 29-kDa protein. Specifically, the amino-terminal immunodominant epitope, peptide 5 (P5), stimulates strong T cell proliferative responses in subjects with delayed (DTH), but not immediate (IH) hypersensitivity skin tests. Evidence of a role for cytokines or changes in epitope recognition over time was examined in responses to Trichophyton using primary PBMC cultures established from seven IH and seven DTH subjects. Responses stimulated by Tri r 2 were dominated by the Th1 cytokine IFN-gamma (IFN-gamma:IL-5 > or = 4:1) in five DTH subjects, even in the presence of Th2-dominated responses (IFN-gamma:IL-5 < or = 3:1) to a subset of major epitopes. Paradoxically, P5 induced IL-5 and IL-10 production in DTH, but not IH subjects (p = 0.003 (IL-5), p = 0.024 (IL-10)), with no significant difference in IFN-gamma levels between the two groups. In cultures from IH responders, no IL-5 was measurable after stimulation with P6 and P7 (as well as P5); this region of the molecule was shown previously to stimulate markedly reduced T cell proliferation in these individuals. Repeat proliferation assays confirmed no change in the pattern of peptide recognition after > or =20 mo in IH or DTH subjects. We conclude that T cell repertoires associated with distinct immune responses to Tri r 2 can be distinguished based on Th2 cytokine induction by DTH-associated major epitopes localizing to the amino-terminal region of the molecule.     

Synthesis and immunochemical properties of peptides corresponding to sequences 59-72 and 25-36 of human interleukin-2

Synthesis of peptides corresponding to the 59-72 and 25-36 sequences of human IL-2 is reported. The former peptide, which had been shown to be immunogenic in the protein molecule, was prepared to obtain antipeptide antibodies for isolation and purification of the recombinant IL-2. We located the epitope at the C-terminus of this peptide. In accordance with the IL-2 secondary structure and hydrophilicity profile analysis, the 25-36 fragment was chosen as the potential epitope. The peptides were synthesized by conventional methods in solution, conjugated with a protein carrier, and polyclonal rabbit antisera were obtained. Antibodies against both peptides were shown to be specific to human IL-2 in ELISA. Besides, monoclonal antibodies to IL-2 recognized in ELISA the 59-72 peptide, suggesting the epitope located in this region to be main one in the protein molecule.     

Expression of the human IL-2 receptor on lymphocytes involves rapid turnover of its p55 alpha-subunit (Tac)

Upon mitogenic stimulation, both mRNA encoding the p55 alpha-subunit (Tac) of the human IL-2R alpha and IL-2R alpha protein are induced and expressed in tonsil lymphocyte populations over several days. Using a quantitative dot-blot immunoassay for the IL-2R alpha subunit, a rapid disappearance of this polypeptide from cells is demonstrated in the presence of the translation inhibitor, cycloheximide. The half-life of IL-2R alpha subunit protein is 2 to 3 h. This decline in cell-associated IL-2R alpha subunit is matched by a rapid decline in IL-2R alpha on the cell surface and is not accompanied by any increase in soluble IL-2R alpha protein. Long term expression of the IL-2R on the cell surface is thus the result of continual synthesis and rapid breakdown of IL-2R alpha chains in the cell. Steady state expression of the IL-2R after an immune stimulus hence depends upon continuous expression of the IL-2R alpha subunit gene. Rapid turnover of the unstable alpha-subunit on the cell surface provides a novel mechanism for sensitive control of functional IL-2R expression.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.     

Ligand-stimulated tyrosine phosphorylation of the IL-2 receptor beta chain and receptor-associated proteins.

Interleukin-2 (IL-2) stimulates the rapid phosphorylation on tyrosine of several specific cellular proteins. However, the high-affinity human IL-2 receptor, composed of an alpha (p55) and beta (p70/75) subunit, does not contain a cytoplasmic tyrosine kinase domain. In this study, we investigated the identities of the proteins phosphorylated on tyrosine in response to IL-2 stimulation to examine possible pathways of signal transduction. By the use of immunoblotting with anti-phosphotyrosine antibodies, we demonstrate that IL-2 augments tyrosine phosphorylation of the IL-2 receptor beta chain in human cell lines expressing either high-affinity (alpha/beta) receptors or only the beta chain. In IL-2-dependent mouse T cell lines, a 100,000-Da protein was phosphorylated on tyrosine in response to IL-2 and is proposed to be the mouse IL-2 receptor beta chain. Two other cellular proteins, pp55 and pp105 in human or pp55 and pp115 in mouse cell lines, were phosphorylated on tyrosine in response to IL-2 and coimmunoprecipitated with the high-affinity IL-2 receptor after chemical crosslinking of IL-2-stimulated cells. Thus, the IL-2 receptor may associate with additional subunits or with cellular proteins involved in signal transduction.     

An associated molecule, p64, with IL-2 receptor beta chain. Its possible involvement in the formation of the functional intermediate-affinity IL-2 receptor complex.

We identified previously a membrane molecule, p64, which co-precipitates with the IL-2R beta-chain in human T cells. We have now investigated the biologic significance of p64 in the formation of the functional IL-2R complex with cell lines transfected with cDNA of IL-2R alpha- and/or beta-chains. Two functional parameters associated with IL-2R, IL-2 binding ability and association of p64 with the beta-chain, were examined. Two subclones, MOLT beta-11 and MOLT beta-12, of an IL-2R beta cDNA-transfected MOLT4 clone expressed similar numbers of IL-2R beta molecules on cell surfaces and bound to IL-2 with intermediate affinity. However, the numbers of IL-2 binding sites were significantly lower than those of IL-2R beta molecules and considerably different between the two subclones. The amount of p64 co-precipitated with IL-2R beta was proportional to numbers of the IL-2 binding sites in the two subclones. In addition, neither p64 co-precipitation nor IL-2 binding was detected in HeLa and COS7 cells transfected with IL-2R beta, and no p64 precipitation was seen even in those transfectants with both IL-2R alpha and beta cDNAs, which bind to IL-2 with high affinity but are not able to transduce intracellular signals. These results suggest the possibility that p64 associates with IL-2R beta and has an important role in formation of the functional IL-2R complex.