Leukemic B-cell precursors constitutively express functional receptors for human interleukin-1

This study analyzes the expression of functional interleukin-1 (IL-1) receptors on leukemic B-cell precursors (BCPs) from B-cell precursor acute lymphoblastic leukemia (BCP ALL) patients. We first investigated the specific binding of 125I-labeled recombinant IL-1 (125I-rIL-1) (4 x 10(17) cpm/mol) to fresh marrow blasts from 11 BCP ALL patients. In five of 11 cases, the binding of 125I-rIL-1 was significantly blocked by excess cold rIL-1. In these five cases, the cell-bound radioactivity ranged from 146 cpm/10(6) cells to 2,412 cpm/10(6) cells (mean +/- SE = 782 +/- 414 cpm/10(6) cells), indicating that 4 to 60 femtomols (mean +/- SE = 20 +/- 10 femtomols) of 125I-rIL-1 specifically bound per 10(7) cells. The estimated number of 125I-rIL-1 molecules bound per cell ranged from 219 to 3,618 (mean +/- SE = 1173 +/- 621). In all five cases, BCP colony formation was stimulated by 10 ng/mL (570 femtomolar) rIL-1, and the background-subtracted colony numbers ranged from 130 to 298 (mean +/- SE = 226 +/- 31). In contrast, no stimulation was observed in six cases that showed no significant 125I-rIL-1 binding. Hence, there was a high correlation between 125I-rIL-1 binding and IL-1 responsiveness, indicating that functional IL-1 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for leukemic BCPs from two IL-1-responsive BCP ALL cases yielded straight linear regression lines, indicating the existence of a single class of 132 to 154 high affinity IL-1 receptors/cell. The apparent affinity constants (Ka) values ranged from 5.2 x 10(9) mol/L-1 to 1.2 x 10(10) mol/L-1. Notably, the concentrations of IL-1 required for half-maximal receptor occupancy (kd = 83 pmol/L to 190 pmol/L) were approximately three orders of magnitude higher than those needed to elicit a half-maximal proliferative response of leukemic BCPs in colony assays (0.1 to 1.0 ng/mL = 5.7 to 57 femtomolar), indicating that only a small fraction of IL-1 receptors need to be occupied to stimulate leukemic BCPs. Notably, IL-1 unresponsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines (REH, KM-3) did not exhibit any significant IL-1 binding (less than 10 IL-1 binding sites/cell), and two additional IL-1 unresponsive BCP ALL cell lines (NALM-6, HPB-NULL) expressed only 24 to 54 IL-1 binding sites/cell with a Ka of 7.8 to 9.8 x 10(9) mol/L-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

The coexistence of androgen and glucocorticoid receptors in the DDT1 cloned cell line.

The hamster ductus deferens cloned tumor cell line (DDT1) has been shown to contain both androgen and glucocorticoid binding activity. The androgen receptor binding site concentration is 1.07 x 10(-13) mol of testosterone/mg protein, and testosterone (T) binds with a Kd of 4.3 x 10(-10) M. Dihydrotestosterone (DHT) is also bound to the receptor with a Kd of 2.99 x 10(-10) M and the binding site concentration is 1.33 x 10(-13) mol/mg protein. The order of steroid binding affinity is DHT greater than T greater than Estradiol greater than Progesterone. Cortisol, dexamethasone, and triamcinolone acetonide do not inhibit the androgen binding in vivo or in vitro. In a cell free system antiandrogens inhibit the binding of DHT. The DDT1 cells have a separate receptor for cortisol which binds at saturation 3.44 x 10(-13) mol cortisol/mg protein and has a Kd of 4.54 x 10(-9) M. These studies provide evidence that these endocrine target cells contain specific high affinity receptors for more than one type of steroid. The glucocorticoid receptor may be important for maintaining essential undifferentiated functions while the DHT receptor gives the specific characteristics of sex hormone responsive tissues.     

Direct demonstration of three different states of the pancreatic cholecystokinin receptor.

We used rat pancreatic acini as well as COS-7cells transfected with the cloned pancreatic cholecystokinin (CCK) receptor andmeasured the abilities of CCK octapeptide (CCK-8) and L-364,718 (a CCK receptorantagonist) to inhibit binding of 125I-labeled CCK-8 (125I-CCK-8) and[3H]L-364,718. With pancreatic acini 125I-CCK-8 bound to two different states ofthe CCK receptor. The high-affinity state (1% of the receptors) had a Kd forCCK-8 of 985 pM and the low-affinity state (19% of the receptors) had a Kd forCCK-8 of 30 nM. [3H]L-364,718 bound to low-affinity receptors and to apreviously unrecognized very-low-affinity state (80% of the receptors) having aKd for CCK-8 of 13 microM. L-364,718 had the same affinity (Kd 3 nM) for each ofthe three different states of the CCK receptor. Similar measurements usingtransfected COS cells also identified three different states of the CCKreceptor, with the very-low-affinity state being the most abundant. Thus, theability of the CCK receptor to exist in three different states is an intrinsicproperty of the CCK receptor molecule itself.     

Endothelial cells express the interleukin-1 receptor type I.

Interleukin-1 (IL-1) profoundly affects a number of functions of vascular cells. Two distinct IL-1 receptors (IL-1R) are expressed on different cell types: the 80 Kd IL-1RI on T cells and fibroblasts, and the 68 Kd IL-1RII on B cells and myelomonocytic cells. The presence and functionality of IL-1R on vascular cells has been investigated by using polyomatransformed mouse endothelial cell (EC) lines (sEnd.1 and tEnd.1). These cells expressed specific and saturable binding sites for IL-1 (1,273 sites per cell with kd 9.5 x 10(-11) mol/L for sEnd.1, and 771 sites per cell with kd 8.5 x 10(-11) mol/L for tEnd.1, with radioiodinated IL-1 alpha as ligand). Binding of IL-1 was also evident at single cell level by autoradiography. By cross-linking studies, the molecular weight of the IL-1 binding protein on EC was approximately 80 Kd. This was confirmed by the presence in EC of mRNA for the 80 Kd IL-1RI. The IL-1RI on EC was apparently functional, since EC responded to IL-1 with IL-6 mRNA expression and IL-6 bioactivity production. These results were extended to human EC and vascular smooth muscle cells, which were also found to express mRNA for IL-1RI.     

Leukemic B-cell precursors express functional receptors for human interleukin-3

The purpose of this study was to analyze the expression of functional interleukin-3 (IL-3) receptors on leukemic B-cell precursors (BCPs) from 12 BCP acute lymphoblastic leukemia (ALL) patients and five BCP ALL cell lines. The specific binding of biosynthetically labeled 35S-recombinant (r) IL-3 to freshly obtained leukemic marrow blasts was initially investigated. In five of 12 BCP ALL cases, the binding of 35S-rIL-3 was markedly blocked by excess cold rIL-3, and the percentage of inhibitable binding ranged from 53% to 78% (mean +/- SE = 65% +/- 4%). In these cases, the cell-bound radioactivity ranged from 146 cpm/10(7) cells to 1,433 cpm/10(7) cells (mean +/- SE = 627 +/- 250 cpm/10(7) cells), indicating that 1 to 14 femtomole (mean +/- SE = 6 +/- 2 fms) of [35S]rIL-3/10(7) cells were specifically bound (= 60 to 840 molecules per cell). rIL-3 stimulated the proliferative activity of leukemic BCPs in a dose-dependent fashion without inducing differentiation, and the half-maximal stimulatory activity was observed at a concentration of 17 to 34 pmol/L. Fluorescence-activated cell sorter (FACS)-isolated virtually pure populations of CD10+CD19+ leukemic BCPs from two BCP ALL patients, as well as from two of five BCP ALL cell lines, showed a marked proliferative response to highly purified rIL-3, providing formal evidence that the observed IL-3 responses were not mediated by accessory cells. There was a high correlation between [35S]rIL-3 binding and proliferative response in colony assays, indicating that functional IL-3 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for IL-3-responsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines yielded a straight linear regression line, indicating the existence of a single class of 60 to 210 high-affinity IL-3 binding sites/cell. The calculated apparent affinity constant (Ka) values ranged from 3.6 x 10(9) to 5.9 x 10(9) mol/L-1. Hence, the concentration of IL-3 required to produce 50% maximal receptor occupancy (Kd) was in the range of 168 to 280 pmol/L. These concentrations are approximately tenfold higher than those required to induce 50% maximal proliferative response from leukemic BCPs in colony assays, indicating that low receptor occupancy is sufficient for growth stimulation of leukemic BCPs by rIL-3. In comparison, less than 10 to 20 IL-3 molecules/cell were bound to IL-3 unresponsive leukemic BCPs even when the concentration of free-[35S]rIL-3 was as high as 2 nmol/L.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression cloning of a cDNA encoding the murine interleukin 4 receptor based on ligand binding.

Interleukin 4 (IL-4) is a potent mediator of growth and differentiation for various lymphoid and myeloid cells. To isolate a cDNA encoding the murine IL-4 receptor, we have developed an expression cloning method that uses biotinylated ligand as a probe and that may be generally applicable to cloning of receptor genes. COS-7 cells transiently transfected with the cloned full-length cDNA bind murine IL-4 specifically with a Kd = 165 pM. Crosslinking of 125I-labeled IL-4 to COS-7 cells transfected with the cDNA reveals binding to proteins of 120-140 kDa. IL-4-responsive cells also express IL-4-binding proteins of 120-140 kDa but show additional bands at 60-70 kDa; the relationship of the smaller proteins to the larger ones is unclear. The nucleotide sequence indicates that the full-length cDNA encodes 810 amino acids including the signal sequence. While no consensus sequence for protein kinases is present in the cytoplasmic domain, a sequence comparison with the erythropoietin receptor, the IL-6 receptor, and the beta chain of the IL-2 receptor reveals a significant homology in the extracellular domain, indicating that the IL-4 receptor is a member of a cytokine receptor family.     

Identification of a distinct low-affinity receptor for human interleukin-4 on pre-B cells

Biotinylated interleukin-4 (IL-4) was used to examine IL-4 receptor (IL- 4R) expression on a range of human B-cell lines by flow cytometry. Using high concentrations of biotinylated IL-4, we have identified a novel low-affinity IL-4 receptor expressed at high levels on pre-B lines. Expression of this low-affinity receptor did not correlate with detected mRNA levels for the previously cloned receptor or with reactivity of two anti-human IL-4R monoclonal antibodies (MoAb). Radiolabeled IL-4 cross-linking studies using pre-B lines showed a doublet of 65 to 75 Kd in contrast to the 110- to 130-Kd molecule detected on cells expressing the cloned IL-4R. A soluble IL-4 binding protein (IL-4bp) was purified from the supernatants of three pre-B lines expressing the low-affinity receptor on their surface. IL-4bp could block both IL-4-mediated CD23 induction on tonsil B cells and IL- 4-induced inhibition of proliferation of the pre-B line JM1. Partial N- terminal amino acid sequence was obtained from purified IL-4bp that confirmed this protein to be novel. A 12 amino acid peptide based on the IL-4bp sequence was used to produce a polyclonal antiserum that was reactive with purified IL-4bp, and also bound to the surface of pre-B cells but not to murine CTLL cells transfected with the human IL-4R. Blocking MoAb against the previously characterized high-affinity receptor inhibited IL-4-mediated proliferation of hIL-4R+ CTLL cells but had no effect on IL-4-induced inhibition of JM1 cell proliferation, and only partially inhibited IL-4-mediated CD23 and sIgM induction and proliferation of tonsil B cells. The data presented here provide evidence for a novel cell-surface expressed low-affinity IL-4R that also exists as a biologically active soluble IL-4 binding protein.     

Functional opioid receptor sites in human placentas

We characterized the presence of opioid peptide receptor sites in plasma membranes and cells from human midterm and term placentas. Incubations with [3H]ethylketo-cyclazocine (EKC) at increasing doses revealed the presence of high affinity, low capacity, opioid peptide receptor-specific binding of the kappa-type. Scatchard analysis of the binding data showed, in the plasma membranes, linear plots at both stages of pregnancy with similar mean equilibrium association constants of 1.31 +/- 0.29 (+/- SE) X 10(9) mol/L-1 (n = 4) at midterm and 0.52 +/- 0.63 X 10(9) mol/L-1 at term (n = 4). In placental cells (n = 4) from term gestations, the binding plots were curvilinear; the first component had a Ka of 5.51 +/- 0.50 X 10(9) mol/L-1, and the second component had a Ka of 1.33 +/- 0.81 X 10(8) mol/L-1 (P less than 0.01). When standardized per mg tissue protein, the number of binding sites in plasma membranes increased from 13.8 +/- 9.8 fmol at midterm to 50.0 +/- 18.6 fmol at term (P less than 0.05). For term placental cells, the concentration of binding sites was 81.2 +/- 36.0 fmol for the high affinity sites and 713 +/- 390 fmol for the lower affinity sites. Specificity for the kappa-type of OPR was found based on the inability of mu- or delta-opioid peptides, as well as LHRH and TRH, to compete for [3H]EKC binding. Term placental cells incubated with various doses of opioid peptides had a 50% increase in placental lactogen production. The increase was significantly higher than controls only with kappa-agonists (P less than 0.05), maximal with 10(-9) mol/L EKC, and completely inhibited by 5 X 10(-6) mol/L naloxone. These results expand on previous data demonstrating the presence of opioid peptide receptor in placental plasma membranes and suggest a role for opioid peptides in regulating secretion of placental lactogen by placental cells.     

Human corpus luteum: luteinizing hormone and chorionic gonadotropin receptors during the menstrual cycle

To characterize and determine the concentration of LH/hCG receptors in human corpora lutea of the menstrual cycle, we measured occupied and unoccupied receptors and determined the association (Ka) and dissociation (Kd) constants individually in 23 corpora lutea (CL) and 4 corpora albicantia obtained at the time of tubal ligation from 25 normal cycling women. We found no [125I]hCG binding in any of the corpora albicantia. Scatchard plot analysis for each CL revealed a linear binding plot indicative of a single set of LH/hCG receptors. The mean concentration of unoccupied receptors was 36 +/- 10 (+/- SE) fmol/mg protein in the early luteal phase (days 15-19; n = 5), 64 +/- 11 fmol/mg protein in the midluteal phase (days 20-25; n = 13), and 42 +/- 19 fmol/mg protein in the late luteal phase (days 26-30; n = 5). The concentrations of occupied receptors were 56 +/- 8, 46 +/- 6, and 54 +/- 12 fmol/mg protein in the early, mid-, and late luteal phases, respectively. Total (occupied plus unoccupied) receptor concentrations reached maximum levels of 110 +/- 11 fmol/mg protein in the midluteal phase. Ka increased progressively from 12 +/- 4 X 10(9) mol/L-1 in the early luteal phase to 19 +/- 7 X 10(9) and 21 +/- 8 X 10(9) mol/L-1 in the mid- and late luteal phases. We conclude that in normal CL, 1) total and unoccupied LH/hCG receptor levels parallel progesterone secretion; 2) changes in the binding affinity may be important in sustaining and/or rescuing the CL; and 3) loss of LH/hCG receptors is probably related to luteolysis.     

Nuclear triiodothyronine binding in mononuclear leukocytes in normal subjects and obese patients before and after fasting

We evaluated the maximum binding capacity (MBC) and dissociation constant (Kd) of nuclear T3 receptors (NT3R) in mononuclear cells in normal and obese subjects before and after fasting. Mononuclear cells were isolated by isopycnic centrifugation of 50 ml heparinized blood underlayered with Ficoll-Paque. From 1-5 x 10(6) cells (12-60 microgram DNA) were incubated for 2 h at 37 C in 0.5 ml Ham's F-10 medium with 0.5% bovine serum albumin, 25 mM Hepes buffer (pH 7.4), and six T3 concentrations, each in triplicate (free T3 from 7 x 10(-13) to 5 x 10(-11) and 1.5 x 10(-8) M). Nuclei were isolated from washed cells in sucrose (0.25 M)-Tris (20 mM; pH 7.85)-MgCl2 (1.1 mM) containing 0.5% Triton X-100. Cells took up approximately 5% of the medium T3, and this was not significantly influenced by the T3 concentrations in the medium. About 10% of the cellular T3 was bound to nuclei at 7 x 10(-13) M free T3 in the medium. Nuclear binding in the presence of 1.5 x 10(-8) M free T3 was approximately 20% of that at 7 x 10(-13) M. T4 could compete with T3 for nuclear binding, but it was less than 10% as effective as T3 based on free hormone concentrations. MBC and Kd values of NT3R were the means of two separate determinations on successive days with coefficients of variation of 26% and 28%, respectively. The MBC of NT3R in 9 normal subjects was 2.3 +/- 0.4 (SD) x 10(-15) mol T3/100 microgram DNA, and the apparent Kd was 2.3 +/- 0.5 (SD) x 10(-11) M free T3. For 10 obese subjects, the MBC was 2.5 +/- 0.8 (SD) x 10(-15) mol T3/100 microgram DNA, and the Kd was 2.4 +/- 0.6 (SD) x 10(-11) M free T3. During a 16-day total fast, 7 patients lost 10 +/- 2 kg, and the mean serum T3 decreased by 60%. However, the MBC [2.7 +/- 0.9 vs. 2.4 +/- 0.4 (fasted) x 10(-15) mol/100 microgram DNA] and Kd [2.4 +/- 0.7 vs 2.1 +/- 0.4 (fasted) x 10(-11) M free T3] were not significantly altered. Circulating human mononuclear cells contain limited capacity, high affinity binding sites for T3. We are unable to detect differences in either the MBC or Kd for these sites between lean and obese subjects, nor does fasting alter either of these parameters. If fasting alters NT3R in man, such changes are not detectable in circulating mononuclear cells using these techniques.     

Insulin binding and biological activities in the FRTL-5 rat thyroid cell line

A cloned rat thyroid cell line (FRTL-5) was examined for both insulin binding and responsiveness. The characteristics of insulin binding to thyroid cells were similar to those observed in typical insulin target cells. The 125I-insulin binding was time and temperature dependent and Scatchard analysis suggested the presence of two major binding sites with high and low affinity constant (Kd = 1.4 X 10(-10) mol/L and 1.5 X 10(-9) mol/L, respectively). 125I-insulin was also internalized and degraded in a temperature-dependent manner. IGF1 was weakly effective in completing 125I-insulin binding to FRTL-5 cells (57% inhibition at 333 nmol/L), whereas noninsulin-related peptide hormones were ineffective. Exposure of FRTL-5 cells to insulin stimulated both methyl-aminoisobutyric acid (M-AIB) and 2-deoxy-D-glucose (2DG) transport. These effects were evident at 10(-9) mol/L and maximal at 10(-7) mol/L insulin. Maximal stimulation was three- to four-fold over basal value for both M-AIB and 2DG transport. These data suggest that insulin specifically binds to FRTL-5 cells and regulates different biological functions of this thyroid cell line.     

Binding of oestradiol, progesterone and prolactin in rat lung

Binding of [3H]oestradiol and of [3H]progesterone in the cytosol from lungs of adult male rats was suppressible, dependent on incubation time and on protein concentration and was protein in nature. Suppressible binding of oestradiol consisted of a high affinity site (dissociation constant (Kd), 1 x 10(-9) +/- 0.2 x 10(-9) mol/l; maximum number of binding sites (Bmax), 0.7 +/- 0.2 pmol/mg protein) and a lower affinity site (Kd, 2.4 x 10(-8) +/- 0.6 x 10(-8) mol/l; Bmax, 6.3 +/- 0.4 pmol/mg protein) and showed evidence of positive co-operation. Suppressible binding of progesterone consisted of a single site with a Kd of 6 x 10(-9) +/- 1 x 10(-9) mol/l and Bmax of 44.5 +/- 8 fmol/mg protein. Binding of 125I-labelled ovine prolactin was found in homogenates of fetal lung (20 days of gestation) but not of adult lung (80 days of age). Treatment of adult rats with ovine prolactin was associated with an increase in the number of binding sites of high affinity for 125I-labelled ovine prolactin but these sites showed an altered specificity. This 'up-regulation' of the prolactin binding may provide a mechanism by which prolactin stimulates surfactant production in lung. These results, together with the known effects of these hormones on certain lung functions, provide further evidence that lung is a target organ for oestradiol, progesterone and prolactin.     

Inhibition of luteinizing hormone-human chorionic gonadotropin binding by retinoids in a Leydig cell line.

Treatment of K9 mouse Leydig cells with 3 x 10(-6) M retinol (R) and retinoic acid (RA) resulted in 75% and 65% reduction of 125I-labeled hCG binding respectively, when assayed at 35 degrees C. This effect was dose-dependent and was first detected 12 h after initiation of treatment: it was maximal at 48 h for RA. R and RA had no significant effect on the rate of internalization and degradation of 125I-hCG as measured by disappearance of acid-releasable (i.e. surface-bound) radioactivity from the cells and by the appearance of trichloracetic acid-soluble label in the medium. When exposed to increasing concentrations of hCG for 24 h, both retinoid-treated and control cells 'down-regulated' their gonadotropin receptors with the same dose-dependent pattern. The kinetics of reappearance of the receptors was similar for retinoid-treated and control cells, but for treated cells the maximal number of receptors reinitiated at 24 h never exceeded 40% of the values observed with control cells. Scatchard plot analysis confirmed a decrease in hCG receptor number from approximately 26,000 to approximately 6400 and approximately 3500 sites per cell after R and RA treatment. Kd values for 125I-hCG binding were 2 x 10(-10) M, 7.3 x 10(-11) M and 6.9 x 10(-11) M for control, R- and RA-treated cells respectively. On the basis of our data it is likely that retinoid-induced reduction in 125I-hCG binding to K9 Leydig cells is due to decreased receptor synthesis.     

Increased platelet-fibrinogen affinity in patients with myeloproliferative disorders [see comments]

Patients with myeloproliferative disorders (MPD) are known to have some abnormalities of platelet glycoproteins (Gp). Quantitative changes of the Gp Ib, IIb-IIIa, and/or their glucidic content have been reported. Since the Gp IIb-IIIa complex plays a major role in fibrinogen binding by activated platelets, we measured the platelet fibrinogen affinity in nine patients with polycythemia vera (PV) and one subject with chronic myeloid leukemia (CML) by the aggregometric method of Marguerie. In all patients the Kd of the platelet fibrinogen reaction was significantly decreased as compared to controls, with evidence in two cases with PV of a heterogeneity of platelet-fibrinogen receptor sites. The measurement of 125I-labeled fibrinogen-platelet binding, performed in seven patients (five PV and two CML), showed receptor populations with increased (Kd1 = 0.58 + 0.3 X 10(7) mol/L) and normal affinity (Kd2 = 5.12 + 3.1 X 10(7) mol/L). These results demonstrate a heterogeneity of platelet-fibrinogen receptors in these patients and may explain the thrombotic diathesis of MPD subjects.     

Evidence for specific binding and stimulatory effects of recombinant human erythropoietin on isolated adult rat Leydig cells.

The presence of specific binding of recombinant human erythropoietin and its effect on testosterone production were evaluated in isolated intact adult rat Leydig cells. Maximal specific binding was observed after 135 min incubation at 34 degrees C. Scatchard analysis of the binding data revealed two distinct classes of binding sites for [125I]-recombinant human erythropoietin with dissociation constant of (Kd1) 1.9 x 10(-10) mol/l and (Kd2) 1.37 x 10(-8) mol/l respectively and binding capacity of (Bmax1) 12.3 fmol/10(6) cells and (Bmax2) 42.8 fmol/10(6) cells, respectively. GnRH, hCG, IGF-I and EGF did not induce any modification of recombinant human erythropoietin-specific binding. Recombinant human erythropoietin added to isolated adult rat Leydig cells exerted a stimulatory effect on testosterone production reaching its maximal effect at the dose of 10(-10) mol/l (testosterone production from 14.9 +/- 1.7 to 45.1 +/- 6.2 pmol/10(6) cells/3 h). Addition of anti-recombinant human erythropoietin serum completely blocked the recombinant human erythropoietin-stimulated testosterone production. These results show that purified adult rat Leydig cells possess recombinant human erythropoietin specific binding, and suggest that this glycoprotein directly influences rat Leydig steroidogenesis.     

Modulation of myocardial L-triiodothyronine receptors in normal, hypothyroid, and hyperthyroid rats

To characterize the nuclear receptor believed to mediate the thyroid hormones' actions on the heart, binding of L-[125I]T3 to extracts of myocardial cell nuclei from normal, propylthiouracil, and T4-treated rats has been investigated. Analysis of in vitro iodothyronine binding to this solubilized nuclear preparation revealed multiple saturable, specific binding sites for T3. High affinity binding for T3 (Kd = 4.2 +/- 1.0 X 10(-10) mol/L), and lower affinity (Kd approximately 10(-8) mol/L) binding activity appeared to be independent (Hill plot slope = 1). The mean maximal binding capacity of the high affinity binding site for T3 in euthyroid rats (84 +/- 8 fmol/mg DNA) was increased by approximately 50% in hypothyroidism (120 +/- 6 fmol/mg DNA) and unchanged in hyperthyroidism (88 +/- 25 fmol/mg DNA). The molecular weight of this T3 binding site is estimated to be 50,000-55,000 daltons. The properties of this solubilized binding activity from rat myocardial nuclei are consistent with its putative role as the biologic thyroid hormone receptor. The increase in binding capacity with hypothyroidism suggest regulation by thyroid hormone of its nuclear receptor in myocardium.     

T-cell interleukin 1 receptor cDNA expressed in Chinese hamster ovary cells regulates functional responses to interleukin 1.

We have cloned a cDNA encoding a receptor identical to the native Mr 80,000 glycoprotein that binds interleukin (IL) 1 alpha and -beta in murine T cells. Chinese hamster ovary (CHO) cells transfected with this T-cell IL-1 receptor (IL-1R) [CHO(IL-1R)] cDNA express approximately 100,000 IL-1Rs per cell, compared to the less than 100 receptors present on control CHO cells. For two functional responses to IL-1, prostaglandin synthesis and cytokine secretion, CHO(IL-1R) cells were 1000 times more sensitive to IL-1 alpha than were control CHO cells. Northern blot analysis and antibody precipitation demonstrated that one of the cytokines induced was granulocyte colony-stimulating factor and that mRNA levels for this cytokine were increased in CHO(IL-1R) cells by IL-1 alpha concentrations that had no effect on control cells. To establish the role of the recombinant receptors in signal transduction, an IL-1R cDNA modified by deletion of the predicted cytoplasmic domain was expressed in the CHO cell line termed CHO(IL-1R delta CT). CHO(IL-1R delta CT) cells expressed approximately 100,000 high-affinity IL-1 binding sites per cell, but these cells were less sensitive than control lines to IL-1, as measured by prostaglandin and cytokine release. These results show that the IL-1R cDNA encodes the entire functional receptor and that the cytoplasmic domain is required for signal transduction but not ligand binding.     

Interaction of fibronectin with cultured human endothelial cells: characterization of the specific receptor

In this study we provide a characterization of the fibronectin (FN) binding to endothelial cells (EC), and we identify the FN binding site on these cells. 125I-FN binding to EC in suspension was time dependent and reached a plateau at 4 h. Cold FN inhibited this interaction in a concentration-dependent way, but vitronectin, fibrinogen, and IgG were poorly effective. About 80% of the total FN associated to EC at the equilibrium was specifically bound; of this, 60% was reversibly bound, while 20% appeared to be internalized. The FN binding was saturable and an apparent dissociation constant of about 0.23 x 10(-6) mol/L and a maximal number of binding sites of about 9.8 x 10(5) was estimated from binding isotherms. Autoradiography data showed that EC-associated 125I- FN was all in high mol wt form that did not enter the gel. We then characterize the FN receptor (FNR) in EC. An antiserum to the FNR isolated from human placenta inhibited FN binding to EC by 89%, and using the immunoblotting technique, it recognized two bands in the EC detergent extract of mol wt 125/160 Kd. This antiserum also recognized the EC membrane protein complex eluted from the FN affinity column by an arg-gly-asp (RGD) peptide. When this complex was included into liposomes, it poorly bound to FN. However, the binding was strikingly increased by addition of Mn in the buffer and was specific for FN in respect to other substrata. These data define the FN binding site in EC and indicate that it is functionally and structurally related to that isolated from human placenta.     

Nuclear triiodothyronine receptor in differentiating preadipocytes cloned from obese and lean mice

The nuclear T3 receptor was characterized in two T3-responsive preadipocyte cell lines cloned from the epididymal fat pads of ob/ob adult mice (ob 17 cells) and their lean counterpart (HGFu cells). Isolated nuclei from confluent or differentiating cells bound [125I]T3 to one class of high affinity sites exhibiting kinetic properties, stereospecificity, and salt extractibility of the nuclear T3 receptor. The solubilized T3 binding sites behave like the hepatic nuclear T3 receptor considering physicochemical and DNA binding properties. At confluence, no significant difference could be detected in equilibrium apparent affinity constant (Ka) and maximum binding capacity (MBC) for T3 whether nuclei were prepared from cells originating from ob/ob mice [Ka: 1.7 +/- (SE) 0.5 X 10(10) M-1; MBC: 432 +/- 29 fmol/mg DNA] or from lean mice (Ka: 1.6 +/- 0.2 X 10(10) M-1; MBC: 487 +/- 39 fmol/mg DNA). MBC values were in the range found in several T3-responsive tissues. This suggests that the primary defect in ob/ob mice is probably not at the level of the nuclear T3 receptor. Furthermore, during differentiation into adipose cells in both cell series, and roughly paralleling the amplifying effect of T3 on several lipogenic enzymes in the course of their development, the nuclear T3 receptor concentration significantly increased, attaining about twice the initial values after completion of the differentiation without any significant change in the affinity for T3.