Identification and distribution of two forms of the interleukin 1 receptor

Using affinity crosslinking techniques, we have biochemically characterized the interleukin-1 (IL1) receptor and investigated its distribution on a range of murine and human cell lines. We show that two forms of IL1 receptor can be identified on the basis of specific crosslinking with 125I-IL1 alpha and 125I-IL1 beta. The two receptor forms have an approximate molecular mass of approximately 80 and approximately 60 kDa, and were found on both murine and human cells. Their relative distribution shows no clear cell lineage restriction and does not correlate with preferential binding of IL1 alpha or IL1 beta. Some cells, such as the T helper cell line D10.G4.1, express both forms of the receptor. Iodine 125-IL1 was crosslinked to the two receptor forms and a partial peptide map analysis of the two receptor/ligand complexes was performed. Comigration of the major partial peptide fragments suggests that the approximately 80 and approximately 60 kDa forms of the receptor may be differentially processed forms of the same protein. Treatment of the approximately 60 kDa IL1 receptor on Raji cells with N-glycanase reduced its molecular mass by 12 kDa, showing that this lower molecular mass form is a glycoprotein; glycosylation differences alone probably do not account for the difference in mass between the two forms.     

The hepatic interleukin-6 receptor. Down-regulation of the interleukin-6 binding subunit (gp80) by its ligand.

Interleukin-6 (IL6) exerts its action via a cell surface receptor composed of an 80 kDa IL6-binding protein (gp80) and a 130 kDa polypeptide involved in signal transduction (gp130). We studied the role of gp80 in binding, internalization and down-regulation of the hepatic IL6-receptor (IL6R) by its ligand in human hepatoma cells (HepG2). Comparison of transfected HepG2 cells overexpressing gp80 with parental cells indicate that gp80 is responsible for low affinity binding (Kd = 500 pM) of IL6. Furthermore, gp80 is rate-limiting in internalization and degradation of IL6. Internalization resulted in a rapid down-regulation (t1/2 approximately 15-30 min) of IL6-binding sites at the cell surface. More than 80% of the internalized [125I]rhIL6 was degraded. The reappearance of IL6-binding sites at the cell surface required greater than 8 h and was sensitive to cycloheximide, suggesting that gp80 is not recycled after internalization. The down-regulation of the hepatic IL6R by its ligand might play an important role as a protection against overstimulation.     

A soluble binding protein specific for interleukin 1 beta is produced by activated mononuclear cells

Soluble interleukin 1 (IL 1) binding proteins were identified by gel filtration and covalent cross-linking of 125I IL 1 in normal human serum and inflammatory exudate. High molecular weight 125I IL 1 protein complexes occurred with both IL 1 alpha and IL 1 beta, however, high molecular weight binding appeared to be non-specific. One specific IL 1 beta binding protein was observed to elute at approximately 100 kDa on gel filtration when bound to 125I IL 1 beta. This complex migrated as a broad band at 60 kDa when covalently cross-linked and analyzed by SDS-PAGE. The protein did not bind 125I IL 1 alpha and 125I IL 1 beta binding was only displaceable by excess cold IL-1 beta. The production of the specific IL 1 beta binding protein was assessed in a number of cell populations. Unstimulated peripheral blood mononuclear cells (PBMNC) did not produce the binding protein, but stimulation with phytohemagglutinin (PHA) caused production within 24 hr and binding protein levels remained elevated for up to 7 days. Stimulation with lipopolysaccharide (LPS) and IL 1 alpha did not consistently induce synthesis of the binding protein. Ligand-binding studies were performed to compare solubilized EL 4 NOB.1 cell membrane IL 1 receptor (sIL 1R) with semi-purified IL 1 beta binding protein from pooled synovial fluid. The sIL 1R preparation bound ligand with an affinity of 168 pM while the IL 1 beta binding protein bound 125I IL 1 beta with an affinity of 370 pM. This protein may function as an important carrier molecule for IL 1 beta and determine its distribution and kinetics in vivo.     

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm-activating peptide I (SAP-I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP-I analogue [GGGY(¹²⁵I)-SAP-I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP-I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(¹²⁵I)-SAP-I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino-terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.     

Molecular identification of the human tumor necrosis factor receptor on interleukin-2-stimulated peripheral blood lymphocytes

The human tumor necrosis factor (TNF) receptor on interleukin (IL)-2-stimulated lymphocytes was characterized by binding and crosslinking techniques. The TNF receptor on IL-2-activated lymphocytes has an affinity of approximately 50 pM. Conventional crosslinking studies with the DSS analog bis(sulfosuccinimidyl) suberate demonstrated a ligand-receptor complex molecular weight of 106-108 kDa. Lectin precipitation experiments indicated that the receptor is a glycoprotein with an affinity for lectin isolated from Ricinus communis. Affinity crosslinking studies with the iodinateable, cleavable crosslinker sulfosuccinimidyl 2-(p-azido-salicylamido) ethyl 1,3'-dithiopropionate demonstrated that the TNF receptor, by itself, in the absence of bound ligand, has a molecular weight of approximately 90 kDa. Furthermore, these results indicate that the crosslinked TNF:TNF-receptor complexes observed at 104-108 kDa are composed of receptor and monomeric TNF.     

Interleukin 1 induces NF-kappa B through its type I but not its type II receptor in lymphocytes.

It is not known whether one or both of the interleukin 1 (IL1) receptors mediates the induction of the DNA-binding protein NF-kappa B. Nuclear extracts of the murine lines EL4.NOB.1 and 70Z/3, which bear the type I (80 kDa) and type II (67 kDa) IL1 receptor, respectively, were analyzed by an electrophoretic mobility shift assay. A 265-base pair sequence of the human serum amyloid A gene or a synthetic oligonucleotide each containing the NF-kappa B site were used as the DNA probes. IL1 induction of NF-kappa B was rapid (optimal at 15-30 min) and transient in both cell types. The IL1 receptor antagonist (IL1ra), which binds strongly to the type I receptor, inhibited the NF-kappa B response in both cell lines. IL1ra did not bind to the type II receptor on 70Z/3 cells as judged by competition for binding with 125I-IL1 alpha. When 125I-IL1ra binding to 70Z/3 cells was measured, a small number (10/cell) of high affinity sites (Kd = 5 x 10(-12) M) were detected. These were likely to have been type I receptor because an antibody to this inhibited the NF-kappa B induction in 70Z/3 cells (as well as EL4). Potential signal transduction mechanisms involving protein kinase C or oxygen radicals were studied. Phorbol 12-myristate 13-acetate induced NF-kappa B with a similar time course to IL1 in 70Z/3 but only after 4 h in EL4.IL1 was unaffected by a protein kinase C inhibitor (staurosporine). H2O2 did not mimic IL1, and IL1 was not inhibited by an antioxidant. The type I receptor mediates the induction of NF-kappa B in response to IL1 via a signaling mechanism that still remains to be identified.     

Purification and characterization of human recombinant precursor interleukin 1 beta

We have purified the 31-kDa precursor of human interleukin 1 beta (proIL1 beta) from recombinant Escherichia coli expressing the protein. The recombinant precursor was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, spectroscopy, Western blot, and for biological and receptor binding activity. The protein migrates at the expected molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical gel filtration columns. The specific activity of the recombinant precursor is less than 10(2) units/mg in the EL4 thymoma assay compared with 5 x 10(8) units/mg for the recombinant 17-kDa mature protein. The inactivity of the precursor is attributable to the inability of the protein to bind the IL1 receptor on EL4 cells as shown by receptor competition studies using 125I-labeled 17-kDa IL1 beta. Inactivity of the IL1 beta precursor is not due to degradation of the protein in either the bioactivity or receptor binding assays. The inactive IL1 beta precursor is converted to an active form following proteolysis with chymotrypsin which generates a carboxyl-terminal fragment of 17 kDa that is 6 orders of magnitude more active than the starting IL1 beta precursor. Removal of the first 114 amino acids from proIL1 beta generates a fully active molecule. In contrast, removal of the first 77 amino acids by treatment with trypsin only partially restores activity. The resultant 22-kDa protein exhibits a 600-fold increase in both biological and receptor binding activity, demonstrating a direct correlation between the ability of sequences within the pro-region to inhibit biological activity and inhibit binding to the IL1 receptor. Far-UV circular dichroism spectroscopy indicates that proIL1 beta is similar in secondary structure to mature IL1 beta; both proteins are nonhelical beta sheet proteins.     

Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein.

Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173-178-residue stretch of the human 37LRP sequence, has been identified as the laminin-1-binding site. Peptides from 37LRP of species that contain this palindrome-bind laminin-1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15-residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin-1-binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin-1-binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin-1-binding affinity of the peptides influences their inhibitory activity.     

Involvement of a specific guanine nucleotide binding protein in receptor immunoglobulin stimulated inositol phospholipid hydrolysis

The role of a specific guanine nucleotide binding (G protein) protein in coupling murine B lymphocyte receptor immunoglobulin to inositol phospholipid hydrolysis was investigated. Using an in vitro system with isolated membranes, we have observed specific enhancement of GTP binding subsequent to ligand-induced receptor crosslinking. Induced increases were inhibited by pretreatment with pertussis toxin which catalyzed ADP-ribosylation of a 43 kDa substrate. Involvement of this G protein with receptor immunoglobulin-induced inositol phospholipid hydrolysis was evidenced by the ability of pertussis toxin to block this response. This report, then, indicates that the B lymphocyte antigen receptor belongs to a family of receptors which are linked to inositol phospholipid hydrolysis through a G protein.     

Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.

We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.     

Pathways of human T lymphocyte development and activation

The T lymphocyte receptor for antigen, which operates in conjunction with gene products of the major histocompatibility complex (MHC), is a molecular complex comprised of five polypeptide chains. Both the 49 kDa alpha and 43 kDa beta chains are immunoglobulin-like and thus contain variable domains responsible for ligand binding. In contrast, the 20–25 kDa T3 gamma, delta and epsilon chains are monomorphic structures presumably involved in transmembrane signalling. The alpha and beta subunits are disulfide bonded to each other and held in noncovalent association with the T3 chains. T3-Ti receptor crosslinking leads to conformational modification of a second T lineage specific molecule, termed the 50 kDa T11 structure which in turn leads to protein kinase C activation, elevation in intracytoplasmic free calcium and Na⁺/H⁺ antiport stimulation.     

Binding of interleukin 2 to its 75-kDa intermediate affinity receptor is sufficient to activate Na+/H+ exchange

High affinity interleukin 2 (IL-2) binding sites are composed of two IL-2-binding molecules: one of 55 kDa, commonly called TaC, and another of 75 kDa. In the absence of the other IL-2-binding molecule, the 55-kDa molecule binds IL-2 with a relatively low affinity and the 75 kDa molecule binds IL-2 with an intermediate affinity. One of the earliest events following interaction of IL-2 with its receptor on the surface of cells is an increase in intracellular pH due to activation of the Na+/H+ antiport. In contrast to IL-2-induced proliferation of human IL-2-sensitive T cells, interaction of IL-2 with a low affinity binding site was sufficient to activate the Na+/H+ antiport. By determining the effect of IL-2 on cytosolic pH in cells that express one of the two IL-2-binding molecules in the absence of the other IL-2-binding molecule, we have demonstrated that interaction of IL-2 with the 75 kDa IL-2-binding molecule is sufficient to activate the Na+/H+ antiport and thus induce cytosolic alkalinization. This indicates that the 75-kDa IL-2-binding molecule, in the absence of the 55-kDa IL-2-binding molecule, forms a functional receptor that can transduce an activation signal across the cell membrane.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

Purification of a 130-kDa T cell glycoprotein that binds human interleukin 4 with high affinity

The interleukin 4 (IL-4) receptor was purified from the gibbon T cell line MLA 144. These cells were found to express high numbers of human IL-4-binding proteins (5000-6000 sites/cell) with an affinity constant (Kd) similar to that measured in human cell lines (Kd = 40-70 pM). Affinity cross-linking of 125I-IL-4 to human cell lines and MLA 144 cells demonstrated the labeling of three proteins of approximately 130, 75, and 65 kDa. Human IL-4-binding sites were solubilized from MLA 144 cells using Triton X-100 and then purified by carboxymethyl chromatography, which removed 50% of the protein without loss of IL-4-binding activity. Then sequential affinity purification over wheat germ agglutinin and a single IL-4 Affi-Gel 10 column resulted in a final 8000-fold purification of the IL-4 receptor. When analyzed on a silver-stained sodium dodecyl sulfate-polyacrylamide gel, the purified receptor migrated as a single molecular species of 130 +/- 5 kDa. Identification of the 130-kDa protein as the IL-4 receptor was demonstrated by cross-linking experiments and specific binding of 125I-IL-4 to nitrocellulose membranes after electrophoretic transfer of the purified receptor on sodium dodecyl sulfate-polyacrylamide gel.     

A 64 kDa protein is a candidate for a thyrotropin-releasing hormone receptor in prolactin-producing rat pituitary tumor cells (GH4C1 cells)

A thyrotropin-releasing hormone (TRH) binding protein of 64 kDa has been identified by covalently crosslinking [3H]TRH to GH4C1 cells by ultraviolet illumination. The crosslinkage of [3H]TRH is UV-dose dependent and is inhibited by an excess of unlabeled TRH. A 64 kDa protein is also detected on immunoblots using an antiserum raised against GH4C1 cell surface epitopes. In a closely related cell line (GH12C1) which does not bind [3H]TRH, the 64 kDa protein cannot be demonstrated by [3H]TRH crosslinking nor by immunoblotting. These findings indicate that the 64 kDa protein is a candidate for a TRH-receptor protein in GH4C1 cells.     

Identification of an interleukin-1 beta binding protein in human plasma

A covalent cross-linking technique was used to bind iodinated interleukin-1 (IL1) alpha and beta to plasma proteins. One specific IL1 beta binding protein was observed, that when cross-linked to ¹²⁵I-ILl beta migrated to approximately 60 kDa on SDS-PAGE. The protein did not bind IL1 alpha. The 43 -kDa protein was partially purified using a wheat germ agglutinin affinity column. The isolated factor again specifically bound IL1 beta, and appeared to consist of single chain glycoprotein. The protein was heat stable and had a rapid association time with IL1 beta. This protein may be an important carrier molecule for IL1 beta in vivo.     

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.     

HepG2 cells predominantly express the type II interleukin 1 receptor (biochemical and molecular characterization of the IL-1 receptor).

In this study we have characterized the cell surface interleukin 1 (IL-1) receptor in HepG2 hepatoma cells. We found that HepG2 cells bind both IL-1 alpha and beta with high affinity, KDs of 136 and 180 pM and receptor densities of 16,000 and 8500 binding sites/cell respectively. The binding sites appeared to be predominantly type II since phorbol ester treatment of the cells, which selectively downregulates type II IL-1 receptors, reduced binding by 68% while treatment of the cells with an inhibitory monoclonal antibody specific for the type I receptor had no significant effect on IL-1 binding. Competition studies with a modified IL-1 beta analog (Glu4) also revealed binding kinetics more consistent with binding to type II receptors than to type I. Crosslinking and ligand blotting with human 125I-IL-1 demonstrated the presence of two bands, a 78 kDa band typical of crosslinking to type II (p60) receptor, and a 98 kDa band, typical of crosslinking to the type I (p80) receptor. Low level expression of the type I receptor was consistent with molecular biological studies employing polymerase chain reaction (PCR) amplification which indicated that mRNA for the type I receptor was produced by the HepG2 cells. Functional receptors were demonstrated by the induction of IL-8 by IL-1 stimulated cells.     

Two Plasmodium falciparum merozoite proteins binding to erythrocyte band 3 form a direct complex

Erythrocyte invasion by malaria parasites requires multiple protein interactions. Our earlier studies showed that erythrocyte band 3 is an invasion receptor binding Plasmodium falciparum merozoite surface protein 1 and 9 (MSP1, MSP9) existing as a co-ligand complex. In this study, we have used biochemical approaches to identify the binding sites within MSP1 and MSP9 involved in the co-ligand complex formation. A major MSP9-binding site is located within the 19kDa C-terminal domain of MSP1 (MSP1(19)). Two specific regions of MSP9 defined as Delta1a and Delta2 interacted with native MSP1(19). The 42 kDa domain of MSP1 (MSP1(42)) bearing MSP1(19) in the C-terminus bound directly to both MSP9/Delta1a and Delta2. Thus, the regions of MSP1 and MSP9 interacting with the erythrocyte band 3 receptor are also responsible for assembling the co-ligand complex. Our evidence suggests a ternary complex is formed between MSP1, MSP9, and band 3 during erythrocyte invasion by P. falciparum.     

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.