Interleukin 1 alpha and interleukin 1 beta bind to the same receptor on T cells

Pure, E. coli-derived recombinant murine interleukin 1 alpha (IL 1 alpha) was labeled with 125I and used for receptor binding studies. The 125I-IL 1 binds to murine EL-4 thymoma cells in a specific and saturable manner. Scatchard plot analysis for binding studies carried out at 4 degrees C reveals a single type of high affinity binding site with an apparent dissociation constant of approximately 2.6 X 10(-10) M and the presence of approximately 1200 binding sites per cell. The rate of association of the 125I-IL 1 with EL-4 cells is slow, requiring more than 3 h to reach apparent steady state at 4 degrees C. Cell-bound 125I-IL 1 cannot be dissociated from EL-4 cells upon removal of unbound 125I-IL 1 and incubation of the cells at 4 degrees C in the presence or absence of unlabeled IL 1. Unlabeled recombinant murine IL 1 competes for 125I-IL 1 binding in a dose-dependent manner, whereas interferon-alpha A, interleukin 2 (IL 2), epidermal growth factor, and nerve growth factor have no effect. The 125I-IL 1 binding site is sensitive to trypsin, suggesting that it is localized on the cell surface. We have also examined the ability of purified recombinant human IL 1 alpha and IL 1 beta to compete for binding of the radiolabeled murine IL 1 to its receptor and to stimulate IL 2 production by EL-4 cells. Previous reports have shown that human IL 1 alpha is approximately 60% homologous in amino acid sequence with murine IL 1, but that human IL 1 beta is only about 25% homologous with either murine IL 1 or human IL 1 alpha. Despite these marked differences, however, we report here that both human IL 1 proteins are able to recognize the same binding site as mouse IL 1. In addition, murine as well as both human IL 1 proteins stimulate IL 2 production by EL-4 cells.     

Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line

The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied. YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1. YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M. Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells. In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80%. The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells. The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells. Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C. Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C. Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C. However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta. This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detergent solubilization of the interleukin 1 receptor

Interleukin 1 (IL 1) receptors were solubilized from membranes prepared from murine EL-4 thymoma cells with the zwitterionic detergent 3[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Binding of IL 1 to the solubilized receptor was detected by a polyethylene glycol (PEG) precipitation procedure. Concentrations of CHAPS from 4 to 8 mM were effective in solubilizing the IL 1 receptor. At 10 mM CHAPS, there was some loss in binding activity, whereas 2 mM CHAPS was completely ineffective in solubilizing the receptor. Detergent concentrations of 4 mM were routinely used. The solubilized receptor retains the ability to bind 125I-IL 1 in a specific and saturable manner. Scatchard analysis reveals a single type of high affinity binding site having an apparent dissociation constant (KD) of approximately 1.2 X 10(-10) M. Nearly identical KD values are observed for membrane fractions. There are approximately 400 to 500 fmol receptor/mg protein in the detergent extract, corresponding to a two- to threefold enrichment in the Bmax observed for membranes. There is no loss in receptor activity as determined by complete recovery of the total number of binding sites from membranes after solubilization. Binding kinetics show that apparent steady state for the solubilized receptor is reached after 60 min at 37 degrees C. The binding of 125I-IL 1 is essentially irreversible because relatively little bound ligand can be dissociated from the receptor on the addition of excess unlabeled IL 1 at 37 degrees C. Both human IL 1 alpha and IL 1 beta compete for binding of 125I-IL 1 to the soluble receptor, confirming that IL 1 alpha and IL 1 beta bind to the same receptor. Other recombinant proteins, including interferon-alpha A, interferon-gamma, and interleukin 2 have no inhibitory effect.     

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.     

Properties of a specific interleukin 1 (IL 1) receptor on human Epstein Barr virus-transformed B lymphocytes: identity of the receptor for IL 1-alpha and IL 1-beta

The properties of specific human interleukin 1 (IL 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human IL 1-beta from a myelomonocytic cell line (THP-1) was labeled with 125I by the Bolton-Hunter method without detectable loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of 125I-IL 1-beta. Maximal binding was reached within 20 min at 4 degrees C. Scatchard plot analysis of the binding of 125I-IL 1-beta to VDS-O cells yielded a Kd (dissociation constant) of 2.4 to 5.9 X 10(-10) M with 110 to 220 binding (receptor) sites/cell. The binding of 125I-IL 1-beta to VDS-O cells was also inhibited by F(ab)'2 fragments of anti-human IL 1 and recombinant human IL 1-alpha, as well as by unlabeled human IL 1-beta but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that IL 1-alpha and IL 1-beta bind specifically to the same receptor. The m.w. of IL 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC) gel filtration analysis of receptor extracted from membrane enriched fraction by a non-ionic detergent (CHAPS). The isoelectric point of solubilized human IL 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of IL 1 receptor on human EBV-B cells additionally supports the hypothesis that IL 1 may be an autocrine signal for these cells.     

The interleukin 1 receptor. Dynamics of interleukin 1 binding and internalization in T cells and fibroblasts

In this study, we have demonstrated that a murine T cell lymphoma, EL 4, and a murine fibroblast cell line, Swiss 3T3, possess a single class of high affinity interleukin 1 (IL 1) receptors that exist in a dynamic state of equilibrium that is influenced by IL 1. In the absence of IL 1, the IL 1 receptor appears to turnover with a t1/2 of approximately 11 hr. However, when cells are incubated in the presence of IL 1, the IL 1 receptor undergoes extensive ligand-induced down-regulation. IL 1 itself is internalized at 37 degrees C; 50% of the surface-bound IL 1 is internalized in 60 to 120 min. IL 1 does not undergo degradation for at least 6 hr after internalization. By using electron microscopy and autoradiography, we observed several important features of the internalization process. When cells having bound 125I-IL 1 at 4 degrees C were shifted to 37 degrees C, IL 1 moved from the cell membrane to the cytoplasm where it was found in proximity to nuclei or within lysosomes. IL 1 appeared to progressively accumulate in nuclei. Six hours after shifting cells to 37 degrees C, 30 to 35% of the internalized 125I-IL 1 is associated with the cell nucleus. The accumulation of relatively high levels of IL 1 in the nucleus raises the interesting possibility that IL 1 may not only interact in a highly specific manner with cell surface receptors, but also with potentially important nuclear receptors.     

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.     

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.     

Murine interleukin-1 receptor: differences in binding properties between fibroblastic and thymoma cells and evidence for a two-chain receptor model

The concentration of porcine interleukin-1 beta (pIL1 beta) required to elicit half-maximal IL2 production from NOB-1, a subline of murine thymoma EL4, was 100-fold greater than for p1L alpha. In contrast, similar doses of each type of IL1 stimulated increased lactate production by Balb/C 3T3 fibroblasts. Receptor-bound 125I-IL 1 alpha was displaced with equal efficiency by both unlabelled forms from 3T3 cells, but a 20-fold lower affinity for p1L1 beta was observed using NOB-1. Crosslinking experiments suggested that the IL1 receptors on each line consisted of two polypeptides of 80 and 100 kDa. The results provide the first evidence for a multiple-component IL1 receptor within which IL1 alpha and IL1 beta may bind at different loci, and suggest the receptors may have evolved differently in the two lines.     

Localization of putative gonadotrophin releasing hormone receptor protein in the anterior pituitary

Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 +/- 0.07, 0.78 +/- 0.04, 0.31 +/- 0.03 and 0.27 +/- 0.03 fmol micrograms-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10(-9) M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p greater than 0.05) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p less than 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p less than 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of high affinity receptor for interleukin-1 (IL-1) on cultured porcine thyroid cells

Using 125I-interleukin-1 beta (125I-IL-1 beta) as a ligand, a specific receptor of high affinity dissociation constant (1.1 +/- 0.2 x 10(-10) M) with binding sites (350 +/- 40/cell) for interleukin-1 beta (IL-1 beta) has been demonstrated on cultured porcine thyroid cells. IL-1 alpha almost equally cross-reacted with the receptor (Kd = 1.2 +/- 0.3 x 10(-10) M and 350 +/- 50 binding sites/cell). TSH, IL-2 and other peptide hormones did not inhibit the binding of 125I-IL-1 beta to thyroid cells. Crosslinking study revealed a major band (approximately 95 kD) with a corrected molecular mass of approximately 78 kD. Moreover, both IL-1 beta and IL-1 alpha stimulated prostaglandin E2 production of cultured porcine thyroid cells, although the potency of IL-1 alpha was slightly greater than that of IL-1 beta. These results suggest that IL-1 may be involved in the regulation of thyroid cell function.     

TCGF(IL 2)-receptor inducing factor(s). II. Possible role of ATL-derived factor (ADF) on constitutive IL 2 receptor expression of HTLV-I(+) T cell lines

Interleukin 2 (IL 2) receptor (IL 2-R) is constitutively expressed on T cell lines established from the patients with adult T cell leukemia (ATL), which is a human T cell leukemia lymphoma virus (HTLV-1)(+) T4(+)-leukemia endemic in Japan, the United States, and other countries. Many of these cell lines continuously produce an acidic lymphokine, ATL-derived factor (ADF), which preferentially induces the synthesis and expression of IL 2-R on a sensitive HTLV-1(-) non-T cell line (YT). The induced IL 2-R was characterized by the binding of 125I-IL 2 and flow cytometry by using fluoresceinated anti-human IL 2-R monoclonal antibodies (anti-Tac). Scatchard analysis with 125I-IL 2 showed ADF induced high-affinity receptor sites on YT cells. To test the possibility that ADF produced by HTLV-1(+) T cells is involved in the abnormal expression of IL 2-R, we studied the effect of ADF on an HTLV-1(+) IL 2-dependent T cell line (ED) in which the beta-chain gene of the T cell antigen receptor (T beta) was rearranged. Unlike IL 2-independent HTLV-1(+) cell lines that constitutively expressed Il 2-R, the IL 2-R expression on ED cells declined in the absence of crude IL 2 or recombinant IL 2. When either ADF or recombinant IL 2 was added to the culture of ED cells, there was a dose-dependent enhancement of IL 2-R expression in 24 hr. ADF and IL 2 showed a synergism in the IL 2-R induction, and both factors were needed to induce the maximal receptor expression in these T cells. The lack of IL 2 production by ADF-treated YT, as well as ED cell line suggested IL 2 may not be involved in the IL 2-R induction by ADF. Northern blot hybridization with human IL 2-R cDNA probe showed the increase of IL 2-R mRNA in YT cells after ADF-treatment. ADF also enhanced IL 2-R expression of a rat T cell line transformed by HTLV-1(TARS-1), as demonstrated with anti-rat IL 2 receptor monoclonal antibodies (ART-18). An ADF-like IL 2-R-inducing factor was also detected in the conditioned medium of two HTLV-1(+) rat T cell lines (TARL-2 and TART-1), which constitutively expressed a higher number of Il 2-R than TARS-1 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Visualization and characterization of interleukin 1 receptors in brain

Interleukin 1 (IL 1) is a polypeptide hormone produced by macrophages, keratinocytes, and brain glial cells which acts as a soluble mediator in immunological and inflammatory reactions. Although its best known effect on the central nervous system is its ability to cause fever, it has been found to influence cell growth, food intake, and slow-wave sleep. We have developed a binding assay for 125I-labeled recombinant murine IL 1 and show it to be highly specific. Additionally, affinity cross-linking studies indicate that the rat brain IL 1 receptor has a m.w. of approximately 80,000, which is similar to the previously described recognition molecule on T cells and fibroblasts. Using autoradiographic techniques, we visualized the distribution of 125I-IL 1 binding in sections of fresh frozen rat brain. IL 1 receptors were found to be widespread throughout the brain, forming a distinctive pattern of distribution. Areas especially dense in receptors were typically neuron-rich sites of the brain such as granule cell layer of the dentate gyrus, the pyramidal cell layer of the hippocampus, and the granule cell layer of the cerebellum as well as in the hypothalamus. The pattern of IL 1 receptor distribution indicates the presence of receptors on neuron cell bodies and the localization to numerous discrete brain areas other than those hypothalamic sites involved in temperature regulation, suggesting a broader role for IL 1 in brain functioning than previously recognized. IL 1, derived from local or systemic sources, may function in the brain to coordinate behavioral and neuroendocrine activities with immunological and inflammatory reactions throughout the body.     

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.     

Intracellular distribution of endothelin-1 receptors in rat liver cells.

We studied the binding of (125I)-endothelin-1 as well as that of the vasopressin analogue (125I)-[8-phenylpropionyl]-LVP to purified plasma membranes, Golgi cisternae and cell nuclei from rat liver. Cell organelles were isolated by differential centrifugation and discontinuous sucrose gradients. Endothelin-1 exhibited specific binding to plasma membranes, Golgi cisternae and nuclei, while the binding of (125I)-[8-phenylpropionyl]-LVP was restricted to the plasma membranes. The number of receptors (Bmax) and the binding constants (Kd) were determined by Scatchard analysis of competition binding studies. In all cases only one class of Et-1 binding sites could be detected. The presence of Et-1 receptors on the Golgi complex either indicates that the receptor is glycosylated within the cisternae or alternatively, there exists a recycling pathway. The unexpected finding of Et-1 receptors on highly purified nuclei suggests that this peptide may exert part of its biological functions intracellularly via the nucleus.     

Recombinant murine and human IL 1 alpha bind to human endothelial cells with an equal affinity, but have an unequal ability to induce endothelial cell adherence of lymphocytes

Consistent with the reports of others, we have demonstrated that human peripheral blood lymphocytes adhere to cultured human umbilical vein-derived endothelial cells (EC) in vitro. In our studies adherence was increased twofold to threefold by a 6-hr preincubation of the EC with IL 1. Recombinant human IL 1 alpha induced a maximal adherence response at less than 1 U per 2 X 10(4) EC. In contrast, recombinant murine IL 1 alpha was found to be 250- to 1250-fold less active in the adherence assay, based on units of IL 1 activity defined by the murine thymocyte proliferation assay. Moreover, when EC were preincubated with excess murine IL 1, no inhibition of the adherence-inducing effect of human IL 1 was noted. To characterize further this dichotomy of biological potency of murine and human IL 1 on the adherence assay, IL 1 binding studies were initiated. Recombinant human and murine IL 1 alpha were equally effective in inhibiting the binding of 125I-labeled human and murine IL 1, based on both micrograms of protein and units of IL 1 activity. The results of this study demonstrate that although human and murine IL 1 bind with equal affinity to receptors on human EC, human IL 1 is significantly more potent at inducing the increased EC adhesiveness for lymphocytes. The implications of these results for endothelial cell IL 1 receptor function are discussed.     

Functional endothelin receptors are present on nuclei in cardiac ventricular myocytes

Endothelins are thought to act through two specific, plasmalemmal G protein-coupled receptor subtypes, ETAR and ETBR. However, in subfractionated cardiac membranes, ETAR immunoreactivity was detected only in the plasma membrane whereas ETBR immunoreactivity was detected predominantly in membranes of intracellular origin. Confocal microscopy demonstrated the presence of intracellular ETAR and ETBR in ventricular myocytes. ETAR were primarily on plasma membrane (surface membranes and transverse-tubules) and to a lesser extent on the nucleus while ETBR localized primarily to the nuclei. Western blot analysis of nuclei isolated from the heart indicated the presence of endothelin receptors: both ETAR and ETBR copurified with nucleoporin 62, whereas markers of endoplasmic reticulum and Golgi membranes were depleted. Radioligand binding studies revealed that isolated nuclei contain specific [125I]ET-1 binding sites. Specific [125I]ET-1 binding was reduced by 70-80% using the ETAR-selective antagonist BQ610 and 20-30% using the ETBR-specific antagonist BQ788. IRL-1620, an ETBR-specific agonist, also reduced [125I]ET-1 binding. Furthermore, ET-1 and IRL-1620 altered the incorporation of 32P into nuclear proteins and caused a transient increase in nuclear Ca2+ concentration. Hence, cardiac nuclei possess both ETAR and ETBR subtypes, which are functional with respect to ligand binding and are coupled to signaling mechanisms within the nuclear membrane.     

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.     

Heterogeneity in interleukin (IL)-1 receptors expressed on human B cell lines. Differences in the molecular properties of IL-1 alpha and IL-1 beta binding sites

IL-1 alpha and IL-1 beta although distantly related at the primary sequence level, bind to the same Mr 80,000 IL-1 receptor on various cell types. Several lines of evidence indicate, however, that the IL-1 receptor on B cells and T cells differ. By binding experiments with 125I-IL-1, marked heterogeneity in IL-1 receptor binding was observed in 13 of 24 B cell lines studied. This was classified into three categories: (I) in nine cell lines, 125I-IL-1 alpha binding revealed high (kD = 10(-10) M) and low affinity (kD = 10(-8) M) IL-1 alpha receptors, whereas 125I-IL-1 beta binding showed one class only with intermediate affinity (kD = 10(-9) M); (II) in three cell lines selective binding with 125I-IL-1 beta was observed; (III) in one cell line only, 125IL-1 alpha and 125I-IL-1 beta bind to a single class of IL-1 receptors as has been described for most cell types. Cross-linking with 125I-IL-1 alpha or 125I-IL-1 beta demonstrated their specific binding to Mr 80,000 and to Mr 68,000 in cell lines in categories I and III, whereas for those in category II, binding to the IL-1 receptor was confined to 125I-IL-1 beta. The expression of two subsets of IL-1 alpha receptors but only one class of IL-1 beta receptors was further confirmed in kinetic studies. Internalization at 37 degrees C demonstrated that only 19% of IL-1 beta was internalized and that binding with IL-1 alpha was entirely cell surface. Flow cytometry studies showed that IL-1 alpha and IL-1 beta do not influence B cell surface antigen expression, suggesting that the ability of IL-1 to influence B cell proliferation is not mediated via direct binding to the IL-1 receptor only.     

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography

Comparison of [125I]epibatidine and 5-[125I]iodo-3-(2-azetidinylmethoxy)pyridine ([125I]A-85380) autoradiography showed evidence for nicotinic receptor heterogeneity. To identify the receptor subtypes, we performed [125I]epibatidine autoradiography in the presence of cytisine or A-85380. By comparing these results with binding data from human embryonic kidney (HEK) 293 cells stably transfected with different combinations of rat nicotinic receptor subunits, we were able to quantify three distinct populations of [125I]epibatidine binding sites with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 receptors. Although the predominant subtype in rat brain was alpha4beta2, non-alpha4beta2 binding sites were prominent in many regions. In the habenulo-peduncular system, cerebellum, substantia gelatinosa, and many medullary nuclei, alpha3beta4-like binding accounted for more than 40% of [125I]epibatidine binding, and nearly all binding in superior cervical ganglion and pineal gland. Other regions enriched in alpha3beta4-like binding included locus ceruleus, dorsal tegmentum, subiculum and anteroventral thalamic nucleus. Regions enriched in alpha3beta2-like binding included the habenulo-peduncular system, many visual system structures, certain geniculate nuclei, and dopaminergic regions. The combination of autoradiography using a broad spectrum radioligand in the presence of selective competitors, and data from binding to defined receptor subtypes in expression systems, allowed us to quantify the relative populations of these three subtypes.