Structural and functional similarity between Yersinia pestis capsular protein Caf1 and human interleukin-1 beta

A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.     

Analysis of interleukin 5 receptors on murine eosinophils: a comparison with receptors on B13 cells

The interleukin 5 receptor (IL-5R) on murine eosinophils and a mouse B cell line (B13) was investigated using iodinated murine IL-5 produced in the baculovirus system. Electrophoretic analysis of this recombinant protein identified a range of bands Mr 26,000 to 32,000 resulting from differential glycosylation. The specific activity and binding kinetics of the iodinated IL-5 (125I-IL-5) were essentially identical to unlabeled material. Both high-affinity (Kd approximately 50 pM) and low-affinity (Kd approximately 1 nM) receptor populations were identified on murine eosinophils. Approximately 50 high-affinity receptors and 10,000 low-affinity receptors were present. This was compared with approximately 2,000 high-affinity (Kd approximately 80 pM) and about 8,000 low-affinity (Kd approximately 3 nM) sites on B13 cells. An antibody that inhibits IL-5 binding to, and proliferation of, B13 cells (R52.120) was also shown to inhibit eosinophil proliferation, suggesting that eosinophils and B cells bear the equivalent IL-5 binding proteins.     

Binding, internalization, and degradation of basic fibroblast growth factor in human microvascular endothelial cells

The binding, internalization, and degradation of basic fibroblast growth factor (bFGF) in human omental microvascular endothelial cells (HOME cells) were investigated. Binding studies of bFGF in human endothelial cells have not yet been reported. Basic FGF bound to HOME cells (KD of 42.0 +/- 3.8 pM and 70,526 +/- 6121 binding sites/cell for the high-affinity sites, KD of 0.933 +/- 0.27 nM and 630,252 +/- 172,459 sites/cell for low-affinity binding sites). The number of low-affinity binding sites was found to be variable. Washing the cells with 2 M phosphate-buffered saline removed completely 125I-bFGF bound to low-affinity binding sites but decreased also the high-affinity binding. The majority of the surface-bound 125I-bFGF was removed by washing the cells with acetic acid buffer at pH 3. At 37 degrees C, 30% of the cell-associated 125I-bFGF became resistant to the acidic wash after 90 min, suggesting that this fraction of bound 125I-bFGF was internalized. At this temperature, degradation of the internalized ligand was followed after 1 h by the appearance of three major bands of 15,000, 10,000, and 8,000 Da and was inhibited by chloroquine. These results demonstrated two classes of binding sites for bFGF in HOME cells; the number of high-affinity binding sites being larger than the number reported for bovine capillary endothelial cells. The intracellular processing of bFGF in HOME cells seems to be different from that of heparin binding growth factor-1 in murine lung capillary endothelial cells and of eye-derived growth factor-1 in Chinese hamster fibroblasts.     

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.     

Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement.

Interleukin-4 (IL-4) represents a prototypic lymphokine (for a recent review see Paul, 1991). It promotes differentiation of B-cells and the proliferation of T- and B-cell, and other cell types of the lymphoid system. An antagonist of human IL-4 was discovered during the studies presented here after Tyr124 of the recombinant protein had been substituted by an aspartic acid residue. This IL-4 variant, Y124D, bound with high affinity to the IL-4 receptor (KD = 310 pM), but retained no detectable proliferative activity for T-cells and inhibited IL-4-dependent T-cell proliferation competitively (K(i) = 620 pM). The loss of efficacy in variant Y124D was estimated to be greater than 100-fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B-cells. The substitution of Tyr124 by either phenylalanine, histidine, asparagine, lysine or glycine resulted in partial agonist variants with unaltered receptor binding affinity and relatively small deficiencies in efficacy. These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand of a receptor belonging to the recently identified hematopoietin receptor family. In addition we show for the first time, that a powerful antagonist acting on the IL-4 receptor system can be derived from the IL-4 protein.     

Molecular determinants of the granulocyte-macrophage colony-stimulating factor receptor complex assembly

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is composed of two chains that belong to the superfamily of cytokine receptors typified by the growth hormone receptor. A common structural element found in cytokine receptors is a module of two fibronectin-like domains, each characterized by seven beta-strands denoted A-G and A'-G', respectively. The alpha-chain (GMRalpha) confers low affinity GM-CSF binding (K(d) = 1-5 nM), whereas the beta-chain (beta(c)) does not bind GM-CSF by itself but confers high affinity binding when associated with alpha (K(d) = 40-100 pM). In the present study, we define the molecular determinants required for ligand recognition and for stabilization of the complex through a convergence of several approaches, including the construction of chimeric receptors, the molecular dynamics of our three-dimensional model of the GM.GMR complex, and site-directed mutagenesis. The functional importance of individual residues was then investigated through ligand binding studies at equilibrium and through determination of the kinetic constants of the GM.GMR complex. Critical to this tripartite complex is the establishment of four noncovalent bonds, three that determine the nature of the ligand recognition process involving residues Arg(280) and Tyr(226) of the alpha-chain and residue Tyr(365) of the beta-chain, since mutations of either one of these residues resulted in a significant decrease in the association rate. Finally, residue Tyr(365) of beta(c) serves a dual function in that it cooperates with another residue of beta(c), Tyr(421) to stabilize the complex since mutation of Tyr(365) and Tyr(421) result in a drastic increase in the dissociation rate (Koff). Interestingly, these four residues are located at the B'-C' and F'-G' loops of GMRalpha and of beta(c), thus establishing a functional symmetry within an apparently asymmetrical heterodimeric structure.     

Interaction of recombinant monocyte-derived interleukin 1 receptor antagonist with rheumatoid synovial cells.

Interleukin 1 receptor antagonist (IL-1ra) is a newly described cytokine that is produced by human monocytes cultured on adherent immunoglobulin G (IgG). These studies have characterized the binding of IL-1ra to receptors on human rheumatoid synovial cells in comparison to binding of IL-1 alpha. The human synovial cells bound 35S-IL-1ra with a Kd of 213 pM and a Ki of 134 pM. 125I-IL-1 alpha bound to the synovial cells with similar values, showing a Kd of 205 pM and a Ki of 58 pM. Cross-inhibition studies were performed to examine whether IL-1ra and IL-1 alpha interacted with the same receptors and in an identical fashion. At the highest concentrations of inhibitory proteins, the binding of each ligand was inhibited 100% by the same or opposite ligand. This result indicated that IL-1ra and IL-1 alpha bound to the same receptors and not to overlapping subsets of receptors. In addition, the binding of 35S-IL-1ra was inhibited in an identical fashion by equimolar amounts of IL-1ra or IL-1 alpha. However, twofold or greater amounts of IL-1ra in comparison to IL-1 alpha were required to offer comparable inhibition of binding of 125I-IL-1 alpha. These results suggest that IL-1ra and IL-1 alpha bind with equal avidity to IL-1 receptors but may not bind identically. Additional experiments are necessary to establish whether these two ligands may bind to different regions of the extracellular portion of the IL-1 receptor.     

Expression and in vitro properties of guinea pig IL-5: comparison to human and murine orthologs

Interleukin-5 (IL-5) is a key mediator of eosinophilic inflammation. The biological role of this cytokine in an allergic airway inflammatory response has been widely demonstrated in guinea pigs, yet the interaction of guinea pig IL-5 (gpIL-5) with its receptor has not been studied. Experiments were performed to quantitate the interaction of gpIL-5 with gpIL-5r and to compare this affinity with that of hIL-5 and mIL-5 and their cognate receptors. The cross-species affinity and agonist efficacy were evaluated to see if gpIL-5r had a restricted species reactivity (as is the case with mIL-5r) or did not distinguish between IL-5 orthologs (similar to hIL-5r). gpIL-5 was cloned using mRNA isolated from cells obtained by bronchoalveolar lavage. Recombinant gpIL-5 was expressed in T. ni insect cells and purified from spent media. Binding assays were performed using insect cells expressing hIL-5ralphabeta or gpIL-5ralphabeta1 as previously described (Cytokine, 12:858-866, 2000) or using B13 cells which express mIL-5r. The agonist potency and efficacy properties of each IL-5 ortholog were evaluated by quantitating the proliferative response of human TF-1 cells and murine B13 cells. gpIL-5 bound with high affinity to recombinant gpIL-5r as demonstrated by displacing [125I]hIL-5 (Ki = 160 pM). gpIL-5 also bound to hIL-5r with high affinity (Ki = 750 pM). hIL-5 and mIL-5 showed similar, high-affinity binding profiles to both gpIL-5r and hIL-5r. In contrast, gpIL-5 and hIL-5 did not bind to the mIL-5r as demonstrated by an inability to displace [125I]mIL-5, even at 1000-fold molar excess. These differences in affinity for IL-5r orthologs correlated with bioassay results: human TF-1 cells showed roughly comparable proliferative responses to guinea pig, human and murine IL-5 whereas murine B13 cells showed a strong preference for murine over guinea pig and human IL-5 (EC50 = 1.9, 2200 and 720 pM, respectively). Recombinant gpIL-5 binds to the gpIL-5r with high affinity, similar to that seen with the human ligand-receptor pair. gpIL-5r and hIL-5r do not distinguish between the three IL-5 orthologs whereas mIL-5r has restricted specificity for its cognate ligand.     

Identification of a critical Ig-like domain in IL-18 receptor alpha and characterization of a functional IL-18 receptor complex

Steady state mRNA levels in various human tissues reveal that the proinflammatory cytokine IL-18 is constitutively and ubiquitously expressed. However, limited IL-18R alpha-chain (IL-18Ralpha) expression in tissues may restrict ligand-acting sites and contribute to a specific response for IL-18. To study the IL-18R complex, [(125)I]IL-18 was studied for binding to the cell surface receptors of IL-18-responsive NK and macrophagic KG-1 cells. After cross-linking, [(125)I]IL-18 formed three IL-18R complexes with sizes of approximately 93, 160, and 220 kDa. In KG-1 cells, Scatchard analysis revealed the presence of 135 binding sites/cell, with an apparent dissociation constant (K(d)) of 250 pM; in NK cells, there were 350 binding sites per cell with an apparent K(d) of 146 pM. Each domain of extracellular IL-18Ralpha was cloned and individually expressed in Escherichia coli. An mAb specifically recognized the membrane-proximal third domain; this mAb blocked IL-18-induced IFN-gamma production in NK cells. Furthermore, deletion of the membrane-proximal third domain of IL-18Ralpha prevented the formation of IL-18R ternary complex with IL-18R beta-chain. The present studies demonstrate that the biologically active IL-18R complex requires the membrane-proximal third Ig-like domain in IL-18Ralpha for the formation of IL-18R ternary complex as well as for signal transduction involved in IL-18-induced IFN-gamma in NK cells.     

Characterization and regulation of the expression of scyllatoxin (Leiurotoxin I) receptors in the human neuroblastoma cell line NB-OK 1.

125I-[Tyr2]scyllatoxin allowed to label a single class of high-affinity receptors in membranes from the human neuroblastoma cell line NB-OK 1. The Kd of these receptors was 60 pM for scyllatoxin (Leiurotoxin I) and 20 pM for apamin and the Bmax was low (3.8 fmol/mg membrane protein). K+ increased toxin binding at low concentrations but exerted opposite effects at high concentrations. Ca2+, guanidinium and Na+ exerted only inhibitory effects on binding. Scyllatoxin binding sites were overexpressed 2.5-fold after a 24-h cell pretreatment with 2 mM butyrate. This effect was suppressed by cycloheximide.     

Characterization of bombesin receptors on canine antral gastrin cells

Dispersed canine antral mucosal cells were prepared by sequential steps of collagenase digestion and EDTA treatment. Cell preparations enriched in gastrin cells were made by centrifugal elutriation followed by step density gradient centrifugation. Specific, saturable, and reversible binding of 125I-[Tyr4]-bombesin was found in all preparations. This saturable binding was time, temperature, and cell number dependent. In both velocity (elutriator) and density cell separation experiments, saturable binding of bombesin correlated with the distribution of cells containing gastrin- but not somatostatin-like immunoreactivity. Maximal specific binding to gastrin (G) cell-enriched fractions was reached in 45 min at 37 degrees C and constituted 90% of total binding. Addition of 100 nM nonradioactive bombesin to cells incubated with 50 pM 125I-[Tyr4]-bombesin for 45 min resulted in time-dependent dissociation of specifically bound tracer to about 40% of the maximal equilibrium binding. Analysis of saturable equilibrium binding yielded a best fit to a one-site model of high affinity binding sites with an apparent Kd of 85 +/- 14 pM and a Bmax of 231,000 +/- 71,000 receptors/gastrin cell. Nonradioactive [Tyr4]-bombesin and related analogs inhibited the specific binding of the tracer in a dose-related manner. The rank order of potency, determined at the IC50, of [Tyr4]-bombesin and related analogs for inhibition of specific binding was bombesin greater than [Tyr4]-bombesin = hGRP-27 greater than GRP-10 greater than ranatensin much greater than neuromedin B. Cholecystokinin, somatostatin, substance K, and kassinin each tested at a concentration of 1 microM did not inhibit bombesin binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two distinct affinity binding sites for IL-1 on human cell lines

We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated.     

Specific binding, internalization, and degradation of human recombinant interleukin-3 by cells of the acute myelogenous, leukemia line, KG-1

We have studied the interaction of 35S-labeled recombinant IL-3 with the acute myelogenous leukemia cell line, KG-1. 35S-IL-3 bound to these cells in a time dependent, saturable, and specific manner at 4 degrees C. Scatchard transformation of binding isotherms demonstrated the existence of a small number (200) of binding sites, with an apparent dissociation constant of 70-105 pM. After a temperature shift from 4 degrees C to 37 degrees C, surface-bound 35S-IL-3 was rapidly internalized and processed into a trichloroacetic acid soluble form that was released into the medium. Experiments to address the specificity of the IL-3 binding site revealed that neither human IL-2, M-CSF, erythropoietin, transferrin, bovine insulin, nor murine nerve growth factor compete with IL-3 for binding to KG-1 cells. Both human and gibbon recombinant IL-3 and, surprisingly, human recombinant GM-CSF effectively competed the binding of the labeled IL-3 to these cells at 4 degrees C. The competition by GM-CSF was found to be concentration dependent, but much higher concentrations were required to achieve the levels obtained with IL-3. These results suggest that GM-CSF may also interact with the high-affinity IL-3 binding site on KG-1 cells or, alternatively, that GM-CSF binding to its own receptor may decrease the affinity of the IL-3 receptor for its ligand.     

Identification of IL-1 receptors on human monocytes

The expression and functional analysis of IL-1 beta R on human monocytes were investigated. Binding of 125I-IL-1 to human monocytes was found to be specific and saturable. Scatchard plot analysis revealed a single class of receptors with a binding constant of 600 pM and a receptor density of approximately 100 binding sites per cell. At 37 degrees C 54% of the labeled ligand was internalized over 2 h of incubation. Addition of 0.2% sodium azide to the cells reduced ligand internalization to 9% of total bound. Cross-linking studies revealed that the IL-1R in human monocytes had a Mr of 80 kDa. The addition of IL-1 to monocytes caused changes in membrane Ag expression as assessed by flow cytometric analysis. The results of this study identify IL-1 receptors on monocytes and suggest that IL-1 may act as an effector molecule for monocytes by enhancing expression of Ag correlated with cell differentiation and immune function.     

Specific receptors for endothelin on membranes from human placenta. Characterization and use in a binding assay

High-affinity binding sites for endothelin have been found in a human placenta membrane preparation. 125I-endothelin bound to placenta membranes at 20 degrees C with an association half-time of 30 min, whereas the binding was only slowly reversed with a dissociation half-time of 250 min. In saturation experiments, a single class of high-affinity binding sites was identified with an apparent dissociation constant (KD) of 24 pM and a maximal density of 240 fmol per mg of protein. The binding of 125I-endothelin was half-maximally inhibited by cold endothelin at a concentration (IC50) of 140 pM. In contrast, no inhibition was found at 10(-4) M for a variety of vasoactive peptides such as angiotensin II, vasopressin, neuropeptide Y, substance P, CGRP, bradykinin, leucine enkephalin or dynorphin A. Similarly, the binding was modulated neither by the calcium channel blockers nifedipine, verapamil or diltiazem, nor by the calcium channel agonist Bay k 8644. There was also no effect with the structurally-related bee venom apamin. Using this membrane preparation, endothelin-like activity could be measured in the medium of cultured human endothelial cells by competition binding technique.     

Characterization of murine IL-1 beta. Isolation, expression, and purification

One cDNA clone encoding a truncated murine IL-1 beta (M IL-1 beta) sequence was isolated from a murine macrophage cDNA library. We reconstituted the coding sequence of the 152-residue mature protein and expressed it in Escherichia coli. rM IL-1 beta was purified to homogeneity and characterized by oligonucleotide and NH2-terminal sequence analysis. Purified rM IL-1 beta exhibited biologic activity equivalent to 7.8 x 10(7) units/mg in the murine thymocyte proliferation assay and 9.9 x 10(3) units/mg in the human gingival fibroblast PGE2 production assay, indicative of species specificity. The isoelectric point of rM IL-1 was found to be 8.85. The circular dichroism spectrum revealed that the secondary structure of M IL-1 is indistinguishable from that of the human protein. Receptor binding studies indicated the rM IL-1 bound to murine EL-4.1 thymoma cells in a specific and dose-dependent fashion with an affinity of 32 pM. Competition binding data suggested that murine and human IL-1 compete for a single class of receptor. Antisera were generated in rabbits against both murine and human IL-1. Results of ELISA binding and antisera neutralization assays indicated that there are common antigenic sites between the two IL-1 beta molecules. These domains are of functional importance because they are capable of mediating the neutralization of biologic activity.     

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.     

Interleukin-1 receptors on human thyroid cells and on the rat thyroid cell line FRTL-5.

Cellular binding of interleukin-1 (IL-1) was tested on monolayers of human thyrocytes in secondary culture, on long-term cultures of human thyrocytes, and on the rat thyroid cell line FRTL-5. The human thyrocytes in secondary culture showed specific binding of human 125I-rIL-1 alpha. Scatchard plots of data obtained at 4 degrees C indicated the presence of a single population of receptors with a Kd of 30 to 170 pM and 2,000 to 6,000 receptors per cell. Incubation at room temperature resulted in internalization of the receptor-ligand complex. Parallel experiments were performed with the IL-1 receptor-positive murine T-cell lines EL-4 and NOB-1. The IL-1 receptors on these cells had Kd values one fifth to one tenth those on human thyroid cells in secondary culture. Both rIL-1 alpha and rIL-1 beta inhibited 125I-rIL-1 alpha binding to human thyrocytes and the murine T cells. In contrast to the cells in secondary culture, there was no specific binding of 125I-rIL-1 alpha to long-term cultivated human thyroid cells or to the FRTL-5 cells. We concluded that recently described differences in the response to IL-1 of different thyroid cell culture systems are most likely caused by differences in expression of IL-1 receptors.     

Interleukin-3 binding to the murine betaIL-3 and human betac receptors involves functional epitopes formed by domains 1 and 4 of different protein chains

Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broad range of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human beta-common (hbetac) receptor and the murine IL-3-specific (beta(IL-3)) receptor in IL-3 binding. We show that the domain 1 residues, Tyr(15) and Phe(79), of the hbetac receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of hbetac, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the beta(IL-3) receptor is also a homodimer. Its high sequence homology with hbetac suggests that their structures are homologous, and we identified an analogous binding interface in beta(IL-3) for direct IL-3 binding to the high affinity binding site in hbetac. Tyr(21) (A-B loop), Phe(85), and Asn(87) (E-F loop) of domain 1; Ile(320) of the interdomain loop; and Tyr(348) (B'-C' loop) and Tyr(401) (F'-G' loop) of domain 4 were shown to have critical individual roles and Arg(84) and Tyr(317) major secondary roles in direct murine IL-3 binding to the beta(IL-3)receptor. Most surprising, none of the key residues for direct IL-3 binding were critical for high affinity binding in the presence of the murine IL-3 alpha receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hbetac.