Structure-function relationships for the IL-2 receptor system. V. Structure-activity analysis of modified and truncated forms of the Tac receptor protein: site-specific mutagenesis of cysteine residues

IL-2R on activated lymphocytes contain the Tac protein. As part of an effort to characterize this molecule, we examined the structure-activity relationship for each of its 12 Cys residues. A preliminary map of intramolecular disulfide bonding was derived by analysis of cystine-linked enzymatic fragments of the Tac protein. The results indicated that disulfide bonds linked Cys-3 with Cys-147, Cys-131 with Cys-163, and Cys-28,30 with Cys-59,61. The contribution of the Cys residues to an active protein conformation was tested by site-specific mutagenesis, followed by expression of the modified molecules in murine L cells. The results indicated that Cys-192 and -225 could be replaced without affecting ligand binding. In contrast, modification of any of the other 10 Cys residues, either singly or in combinations corresponding to the predicted disulfide bonds, greatly reduced the ability of the corresponding protein to bind IL-2 or either of two mAb (anti-Tac and 7G7/B6) which recognize the Tac protein. Each of the latter mutations also interfered with the molecule's post-translational modification and cell-surface expression. Consistent with these findings, transfection of the L cells with vectors containing truncated Tac cDNA inserts resulted in secretion of Tac fragments capable of ligand binding when the polypeptide chains terminated after Cys-163 (the 10th Cys residue in the full length molecule), but resulted in inactive fragments of Tac which were poorly secreted when they terminated before Cys-163. These findings emphasize the remarkable sensitivity of the active conformation of the Tac molecule to each of the postulated intramolecular disulfide bonds.     

Sequence requirements for ligand binding and cell surface expression of the Tac antigen, a human interleukin-2 receptor

Discrete peptide domains within the primary sequence of cell-surface receptor glycoproteins are believed to regulate not only their function but also their targeting to the cell membrane. To identify sequence elements required for intracellular transport and ligand binding by the human Tac interleukin-2 (IL-2) receptor, we prepared expression plasmids encoding a series of artificially mutated or naturally occurring variants of the Tac cDNA. In particular, we sought to further delineate the functional role of the sequences contributed by each of the eight exons that together encode the Tac protein. Deletion of exons 5 through 8 of the receptor had no detectable effect on IL-2 binding or intracellular transport of the Tac protein, and resulted in secreted forms of this IL-2-binding protein. Removal of sequences corresponding to all of exon 4 ablated IL-2 binding activity yet still permitted transport to the cell surface. In contrast, partial deletion of exon 4 sequences resulted in proteins that not only lacked IL-2 binding activity but also were sequestered within the endoplasmic reticulum. Removal of one or both of the N-linked glycosylation sites present in the Tac protein did not impair receptor transport or ligand binding. These results demonstrate that exon 4 of the Tac gene encodes amino acid residues that play an important role in regulating both the intracellular transport and function of this IL-2 receptor.     

Structure-function relationships for the IL 2-receptor system. II. Localization of an IL 2 binding site on high and low affinity receptors

The ligand-binding component of high and low affinity IL 2 receptors is a 55,000 m.w. glycoprotein termed Tac. Correlating the structure and function of this molecule should provide insight into the mechanism of IL 2-initiated signal transduction and the structural basis for high and low affinity receptor forms. As a first step in this process, various approaches were used to localize the IL 2 binding region of the Tac molecule. Antibodies prepared to synthetic fragments of Tac were tested for their ability to interfere with IL 2 binding and bioactivity. The results delineated segments in the C-terminal portion of the molecule which appeared to be distal to the ligand binding site. In a more direct approach, radioiodinated IL 2 was cross-linked to high and low affinity receptors, and the resulting complexes were subjected to mild tryptic digestion. Consistent with the antibody data, the IL 2 remained covalently associated with an N-terminal tryptic fragment which apparently consisted of residues 1-83 of the Tac protein. These results suggest that the N-terminal region of the Tac molecule contains important contact sites for ligand-receptor interaction.     

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.     

Expression of functional IL 2 receptors by lipopolysaccharide and interferon-gamma stimulated human monocytes

Human peripheral blood monocytes were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) alone or in combination. Stimulated but not resting monocytes displayed the Tac peptide of the interleukin 2 (IL 2) receptor within 24 hr as measured by immunofluorescence staining and [3H] Tac binding. The total number of anti-Tac binding sites on co-stimulated monocytes was 13,700. By using scatchard analysis with radiolabeled IL 2, the activated cells were shown to express low numbers (below 100 sites/cell) of high affinity binding sites with a KD of approximately 15 pM. LPS and IFN-gamma were additive in augmenting the number of IL 2 and anti-Tac binding sites. By using an ELISA assay specific for the soluble released form of the Tac peptide we identified 112 U/ml of IL 2 receptors in the supernatant of monocytes stimulated for 24 hr with IFN-gamma, 233 U/ml after stimulation with LPS, and 519 U/ml after the addition of both stimulating agents. Both the membrane form (55,000 daltons), as well as the soluble form (45,000 to 50,000 daltons) of the Tac, IL 2 receptor, peptide from monocytes were shown by immunoprecipitation and gel electrophoresis to be similar size to the comparable forms of these receptors derived from activated T cells. In addition, monocytes stimulated for 8 hr contained mRNA specifically hybridizing to a cDNA probe coding for the Tac peptide. Finally, activated monocytes responded to the addition of recombinant IL 2 by an increase in H2O2 production that was measured by using fluorescent indicator 2,7-dichlorofluorescein. This response as well as the observed induction of monocytic IL 2 receptors by LPS may point to a functional role for this receptor during monocyte/macrophage responses to microbial infections.     

The murine IL 2 receptor. I. Monoclonal antibodies that define distinct functional epitopes on activated T cells and react with activated B cells

The properties of three distinct rat monoclonal antibodies, designated 3C7, 7D4, and 2E4, to the murine IL 2 receptor have been compared in binding, biochemical, and functional assays. 3C7 appears to define an epitope near or identical to the IL 2-binding site of the receptor, because 3C7 inhibited the binding of radiolabeled IL 2 to CTL-L cells and because unlabeled IL 2 inhibited the binding of FITC-3C7 to CTL-L cells. 7D4 and 2E4 had no effect on IL 2 binding. Competitive antibody-binding studies confirmed that the epitope seen by 3C7 was distinct from the epitope(s) seen by 7D4 and 2E4. Sequential immunoprecipitation studies demonstrated that all three antibodies were reactive with the same molecular species, and that each precipitated identical components of 20,000 to 25,000 daltons, 50,000 to 60,000 daltons, and 100,000 to 120,000 daltons from the surface of CTL-L. FACS studies demonstrated a quantitatively and qualitatively identical cell distribution for the antigen defined by each antibody. They failed to stain more than 95% of resting lymphocytes, but were strongly reactive with Con A T blasts and substantially less reactive with LPS B blasts. Unlabeled IL 2 was also able to inhibit the binding of FITC-3C7 to LPS B cell blasts, suggesting the presence of IL 2-binding sites on activated B cells. Each antibody inhibited IL 2-driven proliferation of HT2 or CTL-L cells. 3C7 and 7D4 were more potent inhibitors of proliferation than was 2E4, and the combined use of 3C7 and 7D4 resulted in greater levels of inhibition of proliferation than that shown from the use of either antibody alone. Collectively, the results support the hypothesis that these antibodies detect two distinct functional regions of the IL 2 receptor.     

TCGF (IL 2)-receptor inducing factor(s). I. Regulation of IL 2 receptor on a natural killer-like cell line (YT cells)

A continuous cell line (YT cells) with inducible receptor for T cell growth factor (TCGF)/interleukin 2 (IL 2) was established from a 15-yr-old boy with acute lymphoblastic lymphoma and thymoma. YT cells were tetraploid, having 4q+ chromosomal markers, and proliferated continuously in vitro without conditioned medium (CM) or IL 2. They were weakly positive for OKT9, OKT11, and Tac antigen (Ag), a determinant closely associated with the receptor for IL 2 (IL 2-R), and were negative for OKT1, OKT3, OKT4, and OKT8 Ag. YT cells also expressed HNK-1 Ag and Fc receptors for IgG, which are expressed on natural killer (NK) cells. They retained a killing activity against human cell lines, including K562 (myeloid), T, and B cell lines. Unlike Tac Ag/IL 2-R(+) cell lines derived from adult T cell leukemia (ATL), YT cells were negative for HTLV, as proved by Southern blotting with cDNA for viral DNA. The expression of Tac Ag was markedly enhanced in 18 hr, when YT cells were incubated with CM from PHA-stimulated peripheral blood leukocytes (PBL) or spleen cells, as determined by immunofluorescence by using flow cytometry and binding assay with 125I-anti-Tac antibody (Ab). The binding study with 125I-labeled recombinant IL 2 showed 3.2 X 10(4) IL 2 receptor sites on YT cells precultured with CM. PHA-P and Con A neither agglutinate nor enhance the expression of IL 2-R/Tac antigen on these non-T cell line cells. Furthermore, neither recombinant IL 2 nor gamma-interferon could induce IL 2-R on YT cells, suggesting the presence of a unique IL 2-R inducing factor in PBL or spleen CM. Unlike Tac Ag on HTLV(+), ATL-derived cell lines (Hut-102, MT-1, ATL-2), the expression of Tac Ag on YT cells was down-regulated by anti-Tac Ab. The induction of Tac Ag/IL 2-R on YT cells seemed specific, because the enhancement of Tac Ag expression was not associated with that of Ia Ag and T9/transferrin receptor.     

Structure-function relationships for the IL 2-receptor system. IV. Analysis of the sequence and ligand-binding properties of soluble Tac protein

The Tac protein is one of at least two glycoproteins known to bind the growth and differentiation factor interleukin 2 (IL 2). In addition to its location on the cell surface, where it plays a part in high and low affinity IL 2 receptors, Tac is released from activated lymphocytes in a soluble form. We observed this release both for Tac protein labeled biosynthetically and for Tac protein labeled by surface iodination of intact cells. Competitive binding studies indicated that the soluble Tac protein retained an ability to bind IL 2 with a low affinity (Kd of 11.1 nM). In addition, structural analysis revealed that the polypeptide chain began at position 1 and ended at or just before Cys-192 of the full-length molecule. Thus, the protein was missing its normal transmembrane and intracytoplasmic segments, accounting for its solubility and cellular release. The apparent lack of modification in the amino acid sequence and the termination at Cys-192 are inconsistent with a mechanism of cellular release dependent only on alternate mRNA splicing. Instead, the results suggest that proteolysis may accompany the release of soluble Tac protein from cells expressing IL 2 receptors.     

IL 2 receptor induction on human T lymphocytes: role for IL 2 and monocytes

In this report we studied the requirements for the activation and proliferation of highly purified human T lymphocytes. Purified T cells incubated for 3 days with PHA neither proliferate nor express IL 2 receptors as detected by FACS analysis with the use of anti-Tac antibodies. However, purified T cells incubated with Con A or anti-T3 moAb do not proliferate, albeit 30 to 35% T cells express Tac epitopes. The addition of IL 2, either natural purified or recombinant, resulted in both the appearance of Tac antigen and the proliferation of PHA-activated T cells. Much to our surprise, IL 2 did not induce proliferation of Tac-positive T cells activated by Con A or soluble anti-T3 unless monocytes were added to the cultures. These data suggested that two classes of IL 2 receptors might exist on T cells, one of which was not functionally involved in T cell proliferation. In keeping with this interpretation, we have been able to demonstrate, using a radiolabeled IL 2 binding assay, that anti-T3 moAb induced almost exclusively IL 2 receptors of low affinity (Kd = 30 to 70 X 10(-9) M) and that additional signals, provided by monocytes, are required for the acquisition of high affinity receptors. IL 2 itself can induce high affinity receptors on PHA-stimulated T cells but not on cells activated by Con A or anti-T3. In this latter case the physical presence of monocytes is required and cannot be substituted by IL 1, thus indicating a previously unreported role for monocytes. It is postulated that the contact of monocytes with T cells induces a switch from an inactive low affinity conformation of the IL 2 receptor to a functional high affinity one.     

Interleukin 2 (IL 2) up-regulates its own receptor on a subset of human unprimed peripheral blood lymphocytes and triggers their proliferation

Several reports indicate that human peripheral blood lymphocytes (PBL) seeded in culture with purified or recombinant interleukin 2 (IL 2) immediately after separation from the blood display a substantial level of proliferation at day 5 or 6, even in the absence of any activating signal. The spontaneously IL 2 proliferating cells are large lymphocytes, and they co-purify on a Percoll gradient in the large granular lymphocytes (third (LGL) fraction) together with the natural killer (NK) activity. When LGL were separated into NKH1 (an NK-specific surface marker)-positive and NKH1-negative cells by fluorescence-activated cell sorting (FACS), proliferating cells were mainly found in the NKH1-negative fraction. On the contrary, when cells from Percoll fraction 3 were separated into OKT3-negative and positive cells, the majority of the proliferating cell was found in the OKT3-positive cells. These results indicate that spontaneously IL 2 proliferating (SIP) cells most probably belong to the T cell lineage, but are distinct from NK cells. Surprisingly, cells from this Percoll fraction examined immediately after separation from the blood do not express detectable amounts of IL 2 receptors as assessed by three different techniques: binding of [3H]IL 2, binding of [125I]anti-Tac antibodies, and FACS analysis with the use of anti-Tac antibodies. However, after 18 hr of culture in IL 2-supplemented medium, 5 to 7% of these cells became Tac-positive by FACS analysis. Additional analysis of IL 2 receptor induced in culture with IL 2 was performed by [125I]anti-TAC binding and by [3H]IL 2 binding. Scatchard analysis of [3H]IL 2 binding, in the range of concentrations leading to the detection of high-affinity binding sites, showed an affinity constant similar to that of conventional phytohemagglutinin blasts. The role of IL 2/IL 2 receptor interaction in the proliferation process was confirmed by the fact that proliferation, in contrast with NK activation, was clearly inhibited by anti-Tac antibodies. When LGL activated with IL 2 for 60 hr were sorted into Tac+ and Tac- cells, equal levels of NK activity was found in the two fractions. Proliferation, however, was only observed in the Tac+ population. We interpret these results to indicate that SIP cells are preactivated cells circulating in the blood. They are large cells and represent a very small proportion of circulating lymphocytes (0.3%). They express a subliminal amount of IL 2 receptor. Cultivated in the presence of IL 2, IL 2 receptor expression is enhanced to a detectable level, and the SIP cells begin to proliferate. These SIP cells could be activated T cells present in every normal individual.     

Association of protein kinase C activation with IL 2 receptor expression

Tac antigen (as a measure of the IL 2 receptor) acquisition and regulation by IL 2, an antigen-receptor agonist (anti-T3), phorbol esters, and phytohemagglutinin (PHA) were studied. Phorbol esters stimulated de novo acquisition of Tac antigen, which was associated with the subcellular redistribution of protein kinase C (PK-C) from cytosol to particulate membranes of human T lymphocytes. PHA and anti-T3 (alpha-T3) antibody also stimulated a transient redistribution and activation of PK-C that reached a maximum within 20 min after stimulation. Both phorbol esters and alpha-T3 could increase Tac expression and stimulate PK-C translocation on 5 and 12 day activated T cells that were at the G0/G1 stage of the cell cycle due to IL 2 deprivation. Tac antigen-specific mRNA was seen in the nucleus within 2 hr after stimulation. In contrast, IL 2 alone could only increase Tac expression and stimulate PK-C translocation on day 5 but not day 12 activated T cells. IL 2 synergizes with alpha-T3 and phorbol ester for the regulation of Tac expression. Although IL 2 increased expression of Tac, the majority if not all of these receptors possessed low affinity for IL 2. These data suggest that the activation of PK-C is a common transmembrane signal shared by IL 2 and antigen stimulation. The results also imply that PK-C activation is necessary for the regulation of Tac antigen expression.     

Stimulation of immunoglobulin secretion in human B lymphocytes as a direct effect of high concentrations of IL 2

In certain human IgM and IgG cell lines, immunoglobulin (Ig) secretion is highly stimulated by a B cell inducing factor (BIF) that is free of interleukin 2 (IL 2). BIF also induces Ig secretion in purified peripheral blood B cell populations that have been mitogenically stimulated by Staphylococcus aureus bacteria. Low concentrations of IL 2 (less than 20 U/ml) are not active in these systems. We now show that IL 2 at concentrations above 100 U/ml can induce Ig secretion in these blood B cells and B cell lines. Both conventional IL 2, purified from the human JURKAT and gibbon MLA-144 cell lines, and recombinant IL 2 are active. Very high concentrations approaching 10(4) U/ml are optimal for Ig secretion. Antibody to the T cell IL 2 receptor, anti-Tac, did not inhibit stimulation of the IgM cell line SKW6.4 by IL 2, and no Tac antigen was detected on the cells. The 9B11 monoclonal anti-IL 2 antibody that neutralizes T cell growth activity also abrogates stimulation of Ig secretion by conventional and recombinant IL 2 in the SKW6.4 cell line. However, the 1H11 monoclonal anti-(conventional thr3-glycosylated IL 2), which does not neutralize T cell growth activity, does inhibit induction of Ig secretion by the corresponding IL 2 in the B cell line. These results suggest that IL 2 stimulates B cells via a low-affinity interaction with a receptor different from the Tac receptor identified on T cells, and that the active site on the IL 2 molecule for B cells differs from that for T cell targets. If IL 2 promotes Ig secretion by binding with a low affinity to the B cell BIF receptor, IL 2 and BIF could be homologous proteins.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

An exon 5-deleted mRNA encodes a functional interleukin 2 receptor alpha-subunit.

Two polypeptides are involved in interleukin 2 binding: a low-affinity receptor of 55 kD (IL2-R alpha) and an intermediate affinity component of 75 kD (IL2-R beta). We describe the cloning by the Polymerase Chain Reaction of the coding region of IL2-R alpha from a human T-cell lymphoma cell line. One clone presented a 72-bp deletion that precisely corresponds to exon 5. The deleted form and the normal IL2-R alpha cDNA were expressed CHO cells. Stable transfected cellular clones were compared for their immunoreactivity to monoclonal antibodies directed against IL2-R alpha and for their ability to bind radiolabeled IL2. The presence or absence of the protein region encoded by exon 5 did not modify the IL2-binding capacity of the receptor.     

Identification of a major microfibril-associated glycoprotein-1-binding domain in fibrillin-2

Using yeast two-hybrid, ligand blotting, and solid phase binding assays, we have shown that microfibril-associated glycoprotein-1 (MAGP-1) interacts with the 8-cysteine motif of fibrillin-2 encoded by exon 24. Binding to this sequence was demonstrated for full-length MAGP-1 as well as for the MAGP-1 matrix-binding domain encoded by exons 7 and 8. The matrix-binding domain, but not the full-length protein, also bound to regions of fibrillin-2 defined by exons 16 and 17, exon 20, and exons 23 and 24. Interestingly, no binding was detected to sequences near the N or C terminus where MAGP-1 and MAGP-2, respectively, were shown to interact with fibrillin-1. The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of fibrillin-2. Exon 24 in fibrillin lies in the region of the molecule where mutations produce the most severe phenotypes associated with Marfan syndrome (fibrillin-1) and congenital contractural arachnodactyly (fibrillin-2). It is possible that these mutations alter the ability of fibrillin to bind MAGP-1, which may contribute to the severity of the disease.     

Limited proteolysis of recombinant human soluble interleukin-2 receptor. Identification of an interleukin-2 binding core

Limited proteolysis of a recombinant, soluble form of the Tac protein, a human interleukin-2 receptor (rIL-2R), was performed using trypsin, Staphylococcus aureus V8 protease and proteinase K to study the structural requirements of interleukin-2 receptor (IL-2R) for interleukin-2 (IL-2) binding. Sensitive proteolytic sites were found to be clustered in the regions of the polypeptide encoded by exons 3, 5, and 6, with a few semi-sensitive sites located within the two homologous domains encoded by exons 2 and 4. A number of nicked and truncated rIL-2R species generated by proteolysis were assayed for IL-2 binding using recombinant IL-2 (rIL-2) affinity gel and then structurally characterized. The results demonstrated that only the species that consist of the regions encoded by exons 2 and 4, joined by five disulfide bonds, are capable of binding IL-2 and that the presence of semi-sensitive cleavage sites within the two homologous domains had no apparent effect on IL-2 binding. These results suggest that the pattern of the sensitive cleavage sites in rIL-2R is closely related to the structural requirements for IL-2 binding. Based on the experimental results, a highly symmetrical core structure of IL-2R with a total of 135 amino acid residues was identified. This is the smallest protein moiety so far known to be capable of binding IL-2.     

Effect of recombinant IL 2 and gamma-IFN on proliferation and differentiation of human B cells

The effects of IL 2 and gamma-IFN on the activation of human B cells was studied with recombinant IL 2 and gamma-IFN. BCDF-responsive B lymphoblastoid cell lines and highly purified human B cells were employed as target B cells. IL 2 or gamma-IFN did not induce any IgG or IgM secretion in the B cell lines CESS and SKW6-CL4, in which IgG and IgM were inducible with conventional T cell factor(s). IL 2 alone did not induce the optimum production of Ig, but did induce proliferation in the SAC-stimulated B cell population. No Leu-1-, Leu-4-, or Leu-7-positive cells were detected in B cell populations that had been stimulated with SAC for 3 days. FACS analysis showed that a portion of the SAC-stimulated B cells (30%) were in the G2 or M stages by IL 2 stimulation. The addition of gamma-IFN together with IL 2 induced IgM and IgG secretion in SAC-stimulated B cells that was comparable with that induced by a conventional T cell factor(s). IL 2 induced proliferation not only in SAC-stimulated B cells but also in an anti-mu-stimulated B cell population. Stimulation of T cell populations with anti-mu and IL 2 did not induce significant proliferation, suggesting the direct effect of IL 2 on B cells. Double staining of anti-mu-stimulated B cells with anti-Ig and anti-Tac antibodies demonstrated that anti-mu stimulation induced an increased expression of Tac antigen on surface Ig-positive B cells. All of these results strongly supported the notion that IL 2 was one of the growth factors for B cells, and gamma-IFN was one of the differentiation factors for B cells.     

Structure-function relationships for the IL 2-receptor system. I. Localization of a receptor binding site on IL 2

Interleukin 2 (IL 2) modulates the growth and differentiation of a variety of lymphocyte subclasses through its interaction with a specific cell surface receptor. Although both IL 2 and its receptor have been characterized extensively, the location of interaction sites on the two molecules is unknown. Synthetic peptides based on the IL 2 sequence were used to determine the epitopes seen by a number of antibodies reactive with IL 2, some of which inhibited the receptor binding of the factor and the proliferative response of target cells. The results indicated that inhibitory antibodies bound at either of two spatially distinct sites defined by amino acids 8-27 and 33-54. By inference, these segments may also encode distinct contact sites for receptor association. Alternatively, a single contact site may be located between the antibody epitopes in the three-dimensional configuration of the molecule. Although the data did not directly address the role of the C-terminal portion of IL 2, this preliminary localization of receptor contact sites within the N-terminal half of the molecule should prove useful in correlating structure and function during crystallographic analysis of the factor.     

The third molecule associated with interleukin 2 receptor alpha and beta chain.

It is known that the affinity cross-linking study of the human high-affinity Interleukin 2 (IL-2) receptor reveals triplet bands consisting of 70 kDa alpha chain(Tac)-IL-2 and the 90/80 kDa doublet. We found the cell lines lacking the lower band of the doublet in spite of the expression of both alpha and beta chains. No IL-2 binding was detectable in the presence of anti-Tac antibody in these cells. Immunoprecipitation from the cell extract of [125 I] IL-2-cross-linked T cells with anti-beta chain polyclonal IgG detected the upper band, but not lower band of the doublet. These data suggest that the lower band of the doublet represents an unknown IL-2-binding protein (p65) distinct from the beta chain and this molecule may be involved in the intermediate-affinity IL-2 binding together with the beta chain.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.