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.     

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.     

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.     

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.     

Evidence for a critical role for the cytoplasmic region of the interleukin 2 (IL-2) receptor gamma chain in IL-2, IL-4, and IL-7 signalling.

The high-affinity interleukin 2 receptor (IL-2R) consists of at least three distinct subunits: the IL-2R alpha chain (IL-2R alpha), beta chain (IL-2R beta), and gamma chain (IL-2R gamma). It has been shown that the cytoplasmic region of IL-2R beta, but not of IL-2R alpha, is essential for IL-2 signalling to the cell interior. In the present study, we examined the functional role of the IL-2R gamma cytoplasmic region in the IL-3-dependent mouse hematopoietic cell line BAF-B03, which expresses the endogenous IL-2R alpha and IL-2R gamma, or its subline F7, which additionally expresses human IL-2R beta cDNA. We show that overexpression of a mutant IL-2R gamma, lacking all but 7 amino acids of its cytoplasmic region, results in the selective inhibition of IL-2-induced c-fos gene activation and cellular proliferation in F7 cells. When two chimeric receptor molecules in which the cytoplasmic regions of IL-2R beta and IL-2R gamma had been swapped with each other (IL-2R beta/gamma and IL-2R gamma/beta) were coexpressed in BAF-B03, the cells responded to IL-2. These results indicate the critical importance of the IL-2-induced functional cooperation of the two cytoplasmic regions. Finally, we provide evidence that the IL-2R gamma cytoplasmic region is also critical for the IL-4 and IL-7-induced growth signal transduction in BAF-B03.     

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.     

Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.     

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)     

Activation of the interleukin 2 receptor: a possible role for tyrosine phosphatases

Engagement of interleukin-2 (IL-2) mediates the heterodimeridation of the common beta chain (beta(c)) and common gamma chain (gamma(c)) of the IL-2 receptor (IL-2R). This is sufficient and necessary for receptor activation and signal transduction. It is generally held that the IL-2R is activated by the trans-activity of the protein tyrosine kinases (PTKs) Jak1 and Jak3 associated with beta(c) and gamma(c) respectively. Transduction of proliferative signals requires Jak3 activity. A Jak3 independent signalling pathway involving p56(lck), generating anti-apoptotic signals, can be observed and requires the PROX domain of gamma(c). p56(lck) can be activated by dephosphorylation of an inhibitory carboxyl terminal phosphorylated tyrosine residue (Y505). We propose that this is mediated by a PROX domain associated protein tyrosine phosphatase (PTP). Activation of p56(lck) alone is insufficient for transduction of proliferative signals and thus works in concert with Jak3 mediated receptor activation. This indicates that both gamma(c) domains are vital for signal transduction.     

Interleukin-2 (IL-2) induces erythroid differentiation and tyrosine phosphorylation in ELM-I-1 cells transfected with a human IL-2 receptor beta chain cDNA.

The molecular mechanism of erythroid differentiation has been still ill-defined. In this study, we introduced a human interleukin-2 receptor (IL-2R) beta chain cDNA into ELM-I-1 cells which differentiated into hemoglobin-positive cells in the presence of erythropoietin (Epo), and established the transformant which expressed IL-2R beta chain. In this transformant, we revealed that IL-2 induced erythroid differentiation and the same pattern of tyrosine phosphorylation as Epo. These data suggest that tyrosine phosphorylation is involved in signal transduction pathway of erythroid differentiation. It is also implicated that the Epo and IL-2 receptor system share a common signal transduction pathway.     

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.     

Internalization and nuclear localization of interleukin 1 are not sufficient for function.

The human fibroblast interleukin 1 (IL-1) receptor is a glycosylated transmembrane protein with a cytoplasmic domain of 213 amino acids. We have constructed a series of deletion mutants of the cytoplasmic region of the IL 1 receptor and have used these mutants to examine its role in ligand binding, internalization, signal transduction, and nuclear localization of IL-1. Mutant receptors lacking most of the cytoplasmic domain are expressed at the cell surface and can bind, internalize, and localize IL-1 at the nucleus, but they do not allow IL-1-mediated induction of interleukin 2 and SV40 promoters. We have localized a critical region for signal transduction to a 50-amino acid segment of the cytoplasmic domain of the receptor. These studies demonstrate that IL-1 internalization and nuclear localization are not sufficient to trigger IL-1 activation of gene expression in T-cells.     

The integrity of the conserved ''WS motif'' common to IL-2 and other cytokine receptors is essential for ligand binding and signal transduction.

Recent studies have identified a new family of cytokine receptors, which is primarily characterized by the conservation of periodically interspersed four cysteine residues and the W-S-X-W-S sequence ('WS motif') within the extracellular domain. However, the role of such conserved structures still remains elusive, in particular that of the WS motif. Interleukin-2 (IL-2) is known to play a critical role in the clonal expansion of antigen-stimulated T lymphocytes, and the IL-2 signal is delivered by one of the receptor components, the IL-2 receptor beta (IL-2R beta) chain. The IL-2R beta chain, unlike the IL-2R alpha chain, belongs to this receptor family. In the present study, we analyzed the function of the WS motif of IL-2R beta (Trp194-Ser195-Pro196-Trp197-Ser198) with the use of site-directed mutagenesis. Our results indicate the critical role of the two Trp residues in the proper folding of the IL-2R beta extracellular domain and point to the general functional importance of the WS motif in the new cytokine receptor family.     

Role of the interleukin 5 receptor system in hematopoiesis: molecular basis for overlapping function of cytokines.

Interleukin 5 (IL-5) is a kind of peptide hormone released from T lymphocytes of mammals infected with microorganisms or parasites. It is an acidic glycoprotein with a molecular mass of 40 to 50 kDa that consists of a homodimer of polypeptides. It controls hematopoiesis so that it increases natural immunity. In the mouse, IL-5 acts on committed B cells to induce differentiation into Ig-producing cells and on common progenitors for CD5+ pre-B cells and CD5+ macrophages to support their survival. The antibodies secreted by CD5+ B cells seem to be responsible for the primary protection against the infection with microorganisms or parasites. It also supports the growth and/or differentiation of eosinophil precursor and mature eosinophils, which can be effective for the removal of parasites in combination with the antibodies against them. Murine IL-5 receptor (IL-5R) consists of two different polypeptide chains; alpha chain and beta chain. The IL-5R alpha chain is 60 kDa protein that binds IL-5 with low affinity. The IL-5R beta chain is a 130 kDa protein which does not bind IL-5 by itself but is necessary to form the high affinity IL-5R. The beta chain was identified by using one of the anti-IL-5R mAb and anti-IL-3R mAb as the IL-3R homologue. This beta chain is also used as the beta chain of GM-CSF receptor. This fact suggests that there is a common signaling mechanism among these cytokines and efficient cooperation among them. At the same time, these findings may explain the overlapping role of these cytokines in the development of granulocytes.     

The structure of the interleukin-15 alpha receptor and its implications for ligand binding

Interleukin (IL)-15 is a member of the small four alpha-helix bundle family of cytokines. IL-15 was discovered by its ability to mimic IL-2-mediated T-cell proliferation. Both cytokines share the beta and gamma receptor chains of the IL-2 receptor for signal transduction. However, in addition, they target specific alpha chain receptors IL-15Ralpha and IL-2Ralpha, respectively. The exceptionally high affinity binding of IL-15 to IL-15Ralpha is mediated by its sushi domain. Here we present the solution structure of the IL-15Ralpha sushi domain solved by NMR spectroscopy and a model of its complex with IL-15. The model shows that, rather than the familiar hydrophobic forces dominating the interaction interface between cytokines and their cognate receptors, the interaction between the IL-15 and IL-15Ralpha complex involves a large network of ionic interactions. This type of interaction explains the exceptionally high affinity of the IL-15.IL-15Ralpha complex, which is essential for the biological effects of this important cytokine and which is not observed in other cytokine/cytokine receptor complexes.     

IL-2R beta agonist P1-30 acts in synergy with IL-2, IL-4, IL-9, and IL-15: biological and molecular effects

From the sequence of human IL-2 we have recently characterized a peptide (p1-30), which is the first IL-2 mimetic described. P1-30 covers the entire alpha helix A of IL-2 and spontaneously folds into a alpha helical homotetramer mimicking the quaternary structure of a hemopoietin. This neocytokine interacts with a previously undescribed dimeric form of the human IL-2 receptor beta-chain likely to form the p1-30 receptor (p1-30R). P1-30 acts as a specific IL-2Rbeta agonist, selectively inducing activation of CD8 and NK lymphocytes. From human PBMC we have also shown that p1-30 induces the activation of lymphokine-activated killer cells and the production of IFN-gamma. Here we demonstrate the ability of p1-30 to act in synergy with IL-2, -4, -9, and -15. These synergistic effects were analyzed at the functional level by using TS1beta, a murine T cell line endogenously expressing the common cytokine gamma gene and transfected with the human IL-2Rbeta gene. At the receptor level, we show that expression of human IL-2Rbeta is absolutely required to obtain synergistic effects, whereas IL-2Ralpha specifically impedes the synergistic effects obtained with IL-2. The results suggest that overexpression of IL-2Ralpha inhibits p1-30R formation in the presence of IL-2. Finally, concerning the molecular effects, although p1-30 alone induces the antiapoptotic molecule bcl-2, we show that it does not influence mRNA expression of c-myc, c-jun, and c-fos oncogenes. In contrast, p1-30 enhances IL-2-driven expression of these oncogenes. Our data suggest that p1-30R (IL-2Rbeta)(2) and intermediate affinity IL-2R (IL-2Rbetagamma), when simultaneously expressed at the cell surface, may induce complementary signal transduction pathways and act in synergy.     

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.     

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.     

Reconstitution of a functional IL-2 receptor by the beta-chain cDNA. A newly acquired receptor transduces negative signal

The high-affinity IL-2R results from the noncovalent association between at least two subunits; alpha (p55) and beta (p70), both of which are capable of binding IL-2 with a low and intermediate affinity, respectively. Although the alpha-chain itself has been shown to be nonfunctional, suggestions have been made that the beta-chain mediates an IL-2 signal. To directly study the role of the beta-chain in the signal transduction, we transfected with the cDNA encoding the IL-2R beta-chain a human T lymphotropic virus-I-transformed T cell line, MT-1 originally expressing low-affinity alpha-chain alone, and established a stable transformant (designated MT-beta 7) which expressed both alpha- and beta-chains simultaneously. We showed 1) MT-beta 7 manifested the high-affinity IL-2 binding, which was completely disrupted by the anti-beta chain mAb (Mik-beta 1), 2) the 125I-IL-2 crosslinking patterns of MT-beta 7 were indistinguishable from those of cells expressing the native high-affinity IL-2R, 3) MT-beta 7, but not parental MT-1, internalized the bound IL-2 and responded to IL-2 with a negative signal, i.e., inhibition of the de novo DNA synthesis. These results clearly demonstrate that the beta-chain not only participates in forming the high-affinity IL-2R with the alpha-chain but also is directly involved in the IL-2 signal transduction.