共查询到20条相似文献,搜索用时 9 毫秒
1.
Bradford O. Fanger Dace Viceps-Madore John A. Cidlowski 《Archives of biochemistry and biophysics》1984,235(1):141-149
It is reported that receptors for epidermal growth factor (EGF) in HeLa S3 cells exist in two forms, which differ in both affinity and capacity. Both the number of receptors and their distribution into low- and high-affinity forms are modulated by glucocorticoids. Scatchard analysis of saturation binding assays performed at 0 °C indicates that there is a low-affinity class of receptors (), which contains approximately 6 × 104 binding sites per cell, and a second, high-affinity class of receptors () containing approximately 5 × 103 binding sites per cell. Exposure of HeLa S3 cells to 10?7m dexamethasone for 24 h increased EGF binding to whole cells by increasing the numbers of low- and high-affinity receptors by 20 and 114%, respectively. The increase in EGF binding depends upon the dose of dexamethasone, being raised from 10?11 to 10?6m. EGF binding is half-maximal near 2–4 × 10?9m, a concentration equal to the Kd of dexamethasone for the glucocorticoid receptor in these cells. The increase in EGF binding is specific for glucocorticoids, occurring when the HeLa S3 cells are exposed to 10?7m cortisol or dexamethasone for 24 h, but not when the cells are similarly treated with testosterone, 5α-dihydroxytestosterone, 17β-estradiol, or progesterone. The effect on EGF binding appears to be biphasic; the initial rapid increase occurs between 8 and 12 h, is blocked by both 10?6m cyclohexamide and 0.1 μg/ml actinomycin D, and is followed by a more gradual increase thereafter. These data indicate that glucocorticoids are able to regulate both the number of EGF receptors and their distribution into high- and low-affinity components. Press, Inc. 相似文献
2.
Selective inhibition of high- but not low-affinity interleukin 2 binding by lectins and anti-interleukin 2 receptor alpha antibody 总被引:1,自引:0,他引:1
The present study demonstrated that various reagents can specifically reduce the affinity of high-affinity interleukin 2 receptor (IL-2R) but not that of low-affinity IL-2R. They included lectins such as wheat germ agglutinin (WGA), concanavalin A and phytohemagglutinin, and a chemical cross-linker, glutaraldehyde, in addition to anti-IL-2R monoclonal antibodies, HIEI and H-47. The affinity of the high-affinity IL-2R was reduced when the cells were treated with WGA or H-47 before, but not after, addition of 125I-labeled interleukin 2 (IL-2). Their inhibitory effects were also demonstrated by the chemical cross-linking method. On treatment with the reagents, the IL-2 binding to both IL-2R alpha and beta chains was inhibited and these inhibitory effects were seen only when the reagents were added before IL-2 addition, like their high-affinity reducing effects. These results support a supposition that the high affinity IL-2R is generated by assembly of the alpha and beta chains, and suggest that the IL-2 binding to IL-2R alpha and beta chains could induce stable constitution of the high-affinity state of IL-2R, but these affinity modulating reagents could perturb the optimum association between alpha and beta chains to generate the high-affinity IL-2R. 相似文献
3.
Peptide p1-30, which is composed of the 30 amino-terminal residues (alpha-helix A) of human interleukin-2 (IL-2), binds as a tetramer to the dimeric IL-2Rbeta2 receptor, whereas the entire IL-2 recognizes the tricomponent receptor IL-2Ralphabetagamma. p1-30 is an IL-2 mimetic that activates CD8 low lymphocytes and natural killer cells, because these cells produce IL-2Rbeta constitutively. It also induces a strong lymphokine-activated killer cell response. A series of truncated peptides were analyzed by circular dichroism and analytical centrifugation to elucidate the role of p1-30 residues. We propose a model where residues 10-30 of the p1-30 peptide form an alpha-helix with eight hydrophobic side chains on the same surface buried in a hydrophobic core when four anti-parallel helices combine to form a bundle. IL-2Rbeta dimerization was further studied, and three-dimensional models of the free IL-2Rbeta2 receptor and the p1-304.IL-2Rbeta2 complex were built by comparative modeling based on the crystal structure of the erythropoietin receptor complex, because this belongs to the same hematopoietin family as IL-2. These models suggest that binding of the p1-30 tetramer rotates the COOH-terminal domains and brings both transmembrane regions 50 A closer together, driving the association of the two intracytoplasmic domains that would transduce the signal into the cytoplasm. 相似文献
4.
S A Cannistra M Koenigsmann J DiCarlo P Groshek J D Griffin 《The Journal of biological chemistry》1990,265(21):12656-12663
Granulocyte-macrophage colony-stimulating factor (GM-CSF) activates a broad range of myeloid cells through binding to high affinity surface membrane receptors. The effects of this hematopoietin are dependent upon the differentiation status of the myeloid cell and range from proliferation of early myeloid progenitor cells to activation of neutrophil and monocyte function. In addition, many of the biological effects of GM-CSF are shared with interleukin-3 (IL-3), a distantly related lymphokine. In this study, we have characterized the GM-CSF receptor of myeloid cells at various stages of differentiation by comparing the binding characteristics and surface regulation of this receptor in early versus late myeloid cells. Scatchard analysis revealed a single class of high affinity receptors on normal neutrophils, monocytes, and myeloblasts from patients with acute myeloid leukemia. Neutrophils expressed significantly higher numbers of receptors, with an approximately 2-fold lower affinity, when compared with other myeloid cells. Two different patterns of GM-CSF receptor regulation and binding were observed. In the first pattern, the GM-CSF receptor of neutrophils was rapidly down-regulated by GM-CSF itself, by phorbol myristate acetate (PMA), and by the calcium ionophore A23187, and it was not competed for by IL-3 (class I receptor). In contrast to the neutrophil receptor, the GM-CSF receptor of the myeloblast demonstrated resistance to the down-regulatory effects of GM-CSF itself, PMA, and A23187, and it was completely competed for by IL-3 (class II receptor). In some cases of acute myeloid leukemia and monocytes, a mixed pattern of partial PMA responsiveness and partial competition by unlabeled IL-3 was observed, suggesting the coexpression of both class I and II receptors in these cells. In these cells, after down-regulation of the class I receptor by PMA, the remaining receptors were shown to be completely cross-competed for by IL-3, further supporting the hypothesis that these cells have a mixture of class I and II receptors. Chemical cross-linking of radiolabeled GM-CSF to myeloid cells revealed the labeling of three proteins (156, 126, and 82 kDa) which were identical in cells expressing either class I or II binding sites. These data show that there are differentiation-associated differences in the regulation of the GM-CSF receptor which may have important physiological consequences. 相似文献
5.
RIP-140 interacts with multiple nuclear receptors by means of two distinct sites. 总被引:6,自引:3,他引:3
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F L'Horset S Dauvois D M Heery V Cavaills M G Parker 《Molecular and cellular biology》1996,16(11):6029-6036
6.
The odorant-sensitive adenylate cyclase of olfactory receptor cells. Differential stimulation by distinct classes of odorants 总被引:27,自引:0,他引:27
We have characterized odorant-stimulated adenylate cyclase activity in isolated chemosensory cilia prepared from frog and rat olfactory epithelium. Cilia from both species exhibit high levels of adenylate cyclase activity. Basal activity is stimulated approximately 2-fold by GTP and approximately 5-fold by guanosine 5'-(3-O-thio)triphosphate and forskolin. Odorants augment enzyme activity 30-65% above the basal level in a tissue-specific and GTP-dependent manner. Calcium reduces GTP-stimulated activity with a 50% effective concentration at 10 microM. Odorants vary in their influence upon olfactory adenylate cyclase activity. Most fruity, floral, minty, and herbaceous odorants stimulate the enzyme. 3,7-Dimethyl-2,6-octadienenitrile (citralva), menthone, D-carvone, L-carvone, and 2-isobutyl-3-methoxypyrazine display similar potencies in activating the adenylate cyclase upto concentrations of 100 microM. Putrid odorants, such as isovaleric acid, triethylamine, pyridine, thiazole, and methoxypyrazine, and odorous chemical solvents, do not stimulate enzyme activity. In homologous series of pyrazine, thiazole, and pyridine odorants, compounds with the longest hydrocarbon side chains are best able to enhance enzyme activity. The failure of certain odorants to affect adenylate cyclase activity suggests that additional transduction mechanisms besides the formation of cAMP are involved in olfaction. 相似文献
7.
Human lymphocytes from elderly and young donors were cultured with phytohemagglutinin. Cultures from two groups of aged donors, recruited respectively from our ambulatory clinic and a nursing home, incorporated less tritiated thymidine (3H-TdR) and secreted less interleukin-2 than did young donors. Furthermore, as determined for the first time by a radioligand binding receptor assay, the aged lymphoblasts possessed significantly fewer high affinity IL-2 receptors per cell. Despite a decrease in the number of high affinity receptor cells the dissociation constant (Kd) was comparable for the three groups. It was also shown that the amounts of soluble IL-2 receptors that were released into the supernatants by mitogen stimulated cells did not differ for the aged and young donors. These data suggest that defects in IL-2 production and high affinity IL-2 receptor generation may both be responsible for immune deficiency in the elderly. 相似文献
8.
Structural characterisation and expression analysis of toll-like receptor 2 gene from catfish 总被引:2,自引:0,他引:2
Toll-like receptors (TLRs) are important components of innate immunity. They were found to recognise specific structures on pathogens termed pathogen-associated molecular patterns (PAMPs) and utilise conserved signaling pathways to activate pro-inflammatory cytokines and type-1 interferons. In spite of much understanding gained from the mammalian systems, many fish TLRs are unknown. Recent studies in Japanese flounder as well as in zebrafish suggested that the ligand binding and activation of inflammatory responses in fish may be different from and more complex than those found in mammals. In channel catfish, the major aquaculture species in the United States, only partial sequences of TLR3 and TLR5 were reported. As a part of efforts to characterise the innate immune components in channel catfish, here we cloned and sequenced both the cDNA and the gene for TLR2, a receptor believed mostly responsible for recognition of lipopeptides on the surface of most Gram-positive bacteria. However, expression analysis after infection with a Gram-negative bacterium, Edwardsiella ictaluri indicated that TLR2 was modestly down-regulated in the head kidney tissue of blue catfish, and with a similar pattern in the head kidney of channel catfish though the down-regulation in channel catfish was not statistically significant. In the spleen, an insignificant down-regulation was initially observed early after infection, with an increase of TLR expression later after infection. These results suggest the involvement of TLR2 in the responses after the bacterial infection. As LPS is believed to be the major PAMP for Gram-negative bacteria, additional research is warranted to determine the functions and mechanisms of TLR2 in infections of Gram-negative bacteria. 相似文献
9.
Suppression of interleukin-1 beta and LDL scavenger receptor expression in macrophages by a selective protein kinase C inhibitor. 总被引:2,自引:0,他引:2
A L Akeson K Schroeder C Woods C J Schmidt W D Jones 《Journal of lipid research》1991,32(10):1699-1707
A human monocytic cell line, THP-1, stimulated with 40 nM phorbol myristate acetate (PMA), differentiated to macrophage-like cells, and exhibited increased expression and release of interleukin-1 beta and expression of acetylated low density lipoprotein (ac-LDL) receptors. A selective inhibitor, MDL 29,152 (4-propyl-5-(4-quinolinyl)-2(3H)-oxazolone) was used to show that this induction required activation of protein kinase C. MDL 29,152 acts in the catalytic domain of protein kinase C and is at least 200-fold selective for protein kinase C over cAMP-dependent protein kinase in THP-1 cells. MDL 29,152 (50 microM) reduced levels of interleukin-1 beta mRNA in PMA-stimulated cells by 76% and eliminated detectable interleukin-1 beta in the media. Flow cytometric analysis showed that 48 h after THP-1 activation, approximately 50% of the cells expressed ac-LDL receptors, while in the presence of 100 microM MDL 29,152, less than 5% of the cells expressed receptors. The relationship between THP-1 differentiation and protein kinase C activation was determined by following the expression of the cell surface antigen MO-1. Expression of MO-1 antigen increases as monocytes differentiate to macrophages. After 48 h of phorbol activation, 90% of the THP-1 population was MO-1-positive; less than 16% of the population was MO-1-positive when 100 microM MDL 29,152 was present. By dual analysis, it was found that within the differentiated, MO-1-positive population, only approximately 50% of the cells also expressed ac-LDL receptors. Based on these findings, we conclude that protein kinase C promotes processes important in THP-1 activation and differentiation to macrophage-like cells including interleukin-1 beta expression and secretion, ac-LDL receptor and MO-1 expression. 相似文献
10.
B Parhami-Seren M Viswanathan R K Strong M N Margolies 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(9):5129-5135
Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain. However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding. 相似文献
11.
Surface plasmon resonance (SPR) biosensor technology has been a significant addition to the evolution and refinement of methods to study macromolecular interactions. Prior to the advent of SPR, we employed a variety of biochemical and biological techniques to study the interleukin-2/interleukin-2 receptor system (IL-2/IL-2R). By combining site-directed mutagenesis, equilibrium and kinetic radioligand binding, and competitive biological assays, we and others had begun to understand many aspects of the structure-activity relationships of the IL-2/IL-2R system. Due to the complexity of the IL-2R, cell-based assays proved limited in their ability to provide quantitative information on the binding characteristics of subclasses of the IL-2 receptor. SPR technology promised to be a new and powerful approach to the quantitative analysis of complex receptor systems. To demonstrate the feasibility of this technology, we employed Biacore analysis to investigate the ligand binding characteristics of novel, pre-assembled, IL-2R coiled-coil complexes. The results of these studies, although limited by instrumentation and data analysis, clearly established the utility of this method. Subsequently, by incorporating advancements in both of these areas, we have been able to carry out detailed kinetic analyses of the binding properties of individual IL-2R subunits as well as heteromeric complexes on the surface of a biosensor. Therefore, SPR biosensor analysis combined with other established analytical methods has proven to be a powerful tool for the analysis of complex hematopoietic receptor systems. Published in 1999 by John Wiley & Sons, Ltd. 相似文献
12.
The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca(2+)-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1 microM, and the low-affinity sites one of 36 microM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca(2+)-ATPase. These results indicate that 1 mol of Ca(2+)-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxy-ethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13 microM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca(2+)-ATPase. Vanadate binding to the solubilized Ca(2+)-ATPase was competitively inhibited by inorganic phosphate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
13.
Hidetoshi Nara Mizanur Rahman Farha M. Juliana Hironobu Asao 《Biochemical and biophysical research communications》2010,392(2):171-177
Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain (γc) receptor subunit. We identified novel IL-21R-binding molecule, WD-40 repeats containing SOCS-box-2, WSB-2. WSB-2 associated with the membrane-proximal intracytoplasmic region of IL-21R, including box1 and box2. Overexpression study of WSB-2 showed the reduction of IL-21R expression and IL-21-induced signal transduction. On the other hand, small interfering RNA for WSB-2 enhanced the expression level of IL-21R and IL-21-induced STAT3 activation, indicating that WSB-2 negatively controls the receptor expression. This report provides the first evidence that WSB-2 is a regulator of IL-21R expression and IL-21-induced signal transduction. 相似文献
14.
15.
S Dubois F Magrangeas P Lehours S Raher J Bernard O Boisteau S Leroy S Minvielle A Godard Y Jacques 《The Journal of biological chemistry》1999,274(38):26978-26984
16.
M C Miedel J D Hulmes D V Weber P Bailon Y C Pan 《Biochemical and biophysical research communications》1988,154(1):372-379
A purified soluble and functional form of recombinant human interleukin-2 receptor, engineered and expressed in Chinese hamster ovary cells, was structurally characterized. The primary sequence of this 224 amino acid recombinant protein which lacks most of the carboxy-terminal transmembrane and cytoplasmic portions of the intact protein was established by sequence analyses. The disulfide bonds were assigned by comparative peptide mapping of the reduced and non-reduced peptide digests. As in the case of natural interleukin-2 receptor they occur between cysteines 3-147, 46-104, 131-163, and 28/30-59/61. Based on assignment of the disulfide bonds, a structural model of the interleukin-2 receptor for interleukin-2 binding is proposed. 相似文献
17.
The Jurkat T cell line was stably transfected with an Epstein-Barr virus-based episomal replicon designed to express high levels of the HIV-1 Tat protein. After selection in hygromycin B, high-level Tat activity was detected in 3 of 18 transfected cell lines. After stimulation with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), Tat transfectants with high Tat expression showed diminished expression of interleukin-2 (IL-2) and the interleukin-2 receptor alpha chain (IL-2R) when compared to untransfected Jurkat cells or Jurkat cell lines transfected with the parent control plasmid. Sublines derived from the high-level Tat transfectants with reduced Tat activity showed normalization of PHA/PMA-induced IL-2 expression. Northern analysis showed diminished expression of IL-2 and IL-2R mRNA in the stimulated Tat transfectants. Inhibition of IL-2 and IL-2R expression by the HIV-1 Tat protein may contribute to the immune suppression that characterizes HIV-1 infection. 相似文献
18.
19.
Stephan D. Voss Jacquelyn A. Hank Catherine A. Nobis Paul Fisch Jeff A. Sosman Paul M. Sondel 《Cancer immunology, immunotherapy : CII》1989,29(4):261-269
Summary Expression of the low-affinity interleukin-2 (IL-2) receptor molecule (TAC) has been associated with lymphocyte activation, in vitro and in vivo [Greene WC (1987) Clin Res 35:439]. We have used an enzyme-linked immunosorbent assay (ELISA) to quantify the role of released and cell-bound IL-2 receptor following in vitro or in vivo activation of human lymphocytes with IL-2. In vitro experiments, culturing fresh peripheral blood lymphocytes in 30 U/ml IL-2 (corresponding to the steady-state IL-2 concentration achieved in patients receiving IL-2 in our clinical trials), showed that the levels of IL-2 receptor released into the culture media exceeded the levels of cell-associated receptor, with both rising in parallel to the cytotoxic activity of the peripheral blood lymphocytes (PBL) against cultured tumor cells. In 12 patients receiving high-dose IL-2 for the treatment of various malignant neoplasms, the levels of IL-2 receptor released into the serum rose dramatically during the IL-2 infusion, and then fell following cessation of the IL-2 infusion. This heightened release of IL-2 receptor into the serum occurred during the episodes of profound lymphopenia that developed within hours after patients began an IL-2 infusion. Following each 4-day infusion of IL-2, a rebound lymphocytosis was observed, as has been previously reported. Serum IL-2 receptor levels do not rebound in parallel; rather, they reach a plateau near the end of the 4-day infusion and then decrease upon cessation of IL-2. These changes in serum IL-2 receptor levels accompany changes in lytic activity of circulating PBL on Daudi target cells. These results suggest that lymphocyte populations exposed to IL-2 in vivo are activated to become cytotoxic, release TAC, and relocate in non-peripheral blood compartments. Following cessation of the IL-2 infusion these activated lymphocytes return to the peripheral circulation and do not secrete TAC as vigorously as while influenced directly by the IL-2 infusion.This research was supported by NIH contract CM-47669, NIH grants CA-32685, and RR-03186, American Cancer Society CH-237B, and a research fellowship of the Leukemia Society of America (P. Fisch), a Ewing Foundation Fellowship (S. Voss) and Lutheran Brotherhood/Life and Health Insurance Medical Research Fund M. D./Ph. D. Training Fellowship (S. Voss) 相似文献
20.
Addition of pentoxifylline to lymphocytes caused a dose-dependent decrease in PHA-induced interleukin-2 receptor (IL-2R) expression. Expression of IL-2R protein and mRNA were inhibited by 60% at a concentration of 1 mM. Pentoxifylline also inhibited release of IL-2R into the medium by 85%. Treatment with recombinant IL-2 (50 U/ml) did not abrogate the effect of pentoxifylline. In addition to inhibition of IL-2R expression, pentoxifylline also decreased the expression of transferrin receptors and class I MHC antigens. Pentoxifylline also inhibited cell proliferation. However, aphidicolin, an inhibitor of DNA polymerase alpha inhibited cell proliferation to the same extent as pentoxifylline, but had no effect on IL-2R expression, indicating that inhibition of cell proliferation does not necessarily lead to inhibition of IL-2R expression. The inhibitory effect on IL-2R expression was also noted with other methylxanthines, theophylline and isobutylmethylxanthine, and with dbcAMP and forskolin. The inhibitory activity of pentoxifylline was prevented by W-13, a calmodulin antagonist, but not by HA-1004, a cyclic AMP-dependent protein kinase inhibitor. This suggests that pentoxifylline might act in part through a Ca2+/calmodulin-dependent mechanism. Pentoxifylline and other methylxanthines may prove useful in delineating the biochemical pathways involved in induction and expression of cell surface receptors. 相似文献