Showing papers on "Receptor published in 1985"
TL;DR: 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes, suggesting that such altered expression and amplification is a particular feature of certain human tumours.
Abstract: Epidermal growth factor (EGF), through interaction with specific cell surface receptors, generates a pleiotropic response that, by a poorly defined mechanism, can induce proliferation of target cells. Subversion of the EGF mitogenic signal through expression of a truncated receptor may be involved in transformation by the avian erythroblastosis virus (AEV) oncogene v-erb-B, suggesting that similar EGF receptor defects may be found in human neoplasias. Overexpression of EGF receptors has been reported on the epidermoid carcinoma cell line A431, in various primary brain tumours and in squamous carcinomas. In A431 cells the receptor gene is amplified. Here we show that 4 of 10 primary brain tumours of glial origin which express levels of EGF receptors that are higher than normal also have amplified EGF receptor genes. Amplified receptor genes were not detected in the other brain tumours examined. Further analysis of EGF receptor defects may show that such altered expression and amplification is a particular feature of certain human tumours.
1,511 citations
Journal Article•
TL;DR: The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation.
Abstract: With the use of an enzyme-linked immunoabsorbent assay to measure soluble human interleukin 2 receptors (IL 2R), certain human T cell leukemia virus I (HTLV I)-positive T cell lines were found to spontaneously release large quantities of IL 2R into culture supernatants This was not found with HTLV I-negative and IL 2 independent T cell lines, and only one of seven B cell-derived lines examined produced small amounts of IL 2R In addition to this constitutive production of soluble IL 2R by certain cell lines, normal human peripheral blood mononuclear cells (PBMC) could be induced to release soluble IL 2R by plant lectins, the murine monoclonal antibody OKT3, tetanus toxoid, and allogeneic cells Such activated cells also expressed cellular IL 2R measurable in detergent solubilized cell extracts The generation of cellular and supernatant IL 2R was: dependent on cellular activation, rapid, radioresistant (3000 rad), and inhibited by cycloheximide treatment NaDodSO4-polyacrylamide gel electrophoresis analysis of soluble IL 2R released from either the HTLV I-positive T cell line HUT 102B2 or normal phytohemagglutinin-activated PBMC demonstrated molecules of apparent Mr = 35,000 to 40,000, and 45,000 to 50,000, respectively, somewhat smaller than the mature surface receptor on these cells The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation
1,092 citations
TL;DR: This is the first report that TNF-α and -β share a common receptor and that the receptors can be up-regulated by interferon, and may explain previous observations regarding similar biological activities observed for these two cytotoxic proteins and also their synergistic action with interferons.
Abstract: Tumour necrosis factors, TNF-α and TNF-β (previously called lymphotoxin), are the products of activated monocytes and lymphocytes, respectively, and both have recently been purified, sequenced and cloned by recombinant DNA methods1–5, revealing 35% identity and 50% homology in the amino-acid sequence. Both proteins have been found to be specifically toxic to many tumour cells. Furthermore, it has been reported that various interferons are synergistic with TNF for anti-tumour effects in vitro6–8, while activities attributed to the two proteins have also been shown to necrotize various tumours in vivo2,3,9. We have now prepared 125I-labelled highly purified recombinant human TNF-α to study in detail its binding to the human cervical carcinoma cell line ME-180. Our results indicate that there is a single class of specific high-affinity receptors for TNF on this cell line which has a Kd of about 0.2 nM and an average of 2,000 receptor sites per cell. The binding of labelled TNF-α to these cells can be inhibited by both TNF-α and TNF-β but not by γ-interferon (IFN-γ). However, preincubation of cells with IFN-γ increases the total number of TNF receptors two to threefold without any significant change in the affinity constant. This is the first report that TNF-α and -β share a common receptor and that the receptors can be up-regulated by interferon. Our results may explain previous observations regarding similar biological activities observed for these two cytotoxic proteins and also their synergistic action with interferons.
907 citations
TL;DR: The findings indicate that Sm-C/IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity at nanomolar concentrations compatible with its granULosa cell receptor binding affinity, and can be added to a growing list of growth factors known to modulate granul Rosa cell growth and function.
Abstract: A relatively large body of evidence now appears to support the existence of the essential ingredients for novel intraovarian IGF-driven control mechanisms. Indeed, evidence presented in this communication is in keeping with the possibility that the granulosa cell may be the site of IGF production, reception, and action. Although the relevance of IGFs to ovarian cell types other than the granulosa cell is largely unknown, one cannot at the present time exclude the possibility of nongranulosa cell contributions to intraovarian IGF production, reception, and action. Indeed, preliminary affinity cross-linking studies (Adashi, Resnick, Svoboda, Van Wyk and D'Ercole; unpublished data) suggest the existence of type-I and type-II receptors in nongranulosa cell compartments. The above notwithstanding, IGFs of granulosa (and possibly circulatory) origins may interact with granulosa cell autoreceptors either independently or in synergy with other granulosa cell agonists. According to this view, IGFs may act in the autocrine mode to stimulate granulosa cell replication on the one hand and promote granulosa cell differentiation on the other. Although proliferation and terminal differentiation may prove mutually exclusive under some circumstances, coexistence of the two processes is being increasingly recognized. In this context, some studies of porcine granulosa cells support a dual role for IGFs in granulosa cell ontogeny. As such, the IGFs can be added to a growing list of growth factors known to modulate granulosa cell growth and function, including EGF, PDGF, and FGF. Our findings indicate that Sm-C/IGF-I synergizes with FSH in the induction of rat granulosa cell aromatase activity at nanomolar concentrations compatible with its granulosa cell receptor binding affinity (thus far studied only in porcine cells. A role for Sm-C/IGF-I in the regulation of this key granulosa cell function would be in keeping with the possibility that Sm-C/IGF-I may partake in the assertion and maintenance of dominance by the selected follicle(s) or in promoting juvenile and early follicular development. Moreover, the ability of Sm-C/IGF-I to potentiate this and other FSH-driven ovarian functions may also account, at least in part, for the puberty-promoting effect of growth hormone. This permissive action of growth hormone has been initially suggested by observation in growth hormone-deficient rats, mice (dwarf mutants, and humans (sporadic, hereditary or acquired growth hormone deficiency.(ABSTRACT TRUNCATED AT 400 WORDS)
905 citations
TL;DR: The results provide strong support for the hypothesis that the microglia are derived from monocytes and show thatmicroglia possess receptors which would allow them to play a part in the immune defence of the nervous system.
870 citations
TL;DR: The purification of cachectin to apparent homogeneity and characterization of its receptor should facilitate further investigations into the role of Cachectin and other macrophage mediators in the metabolic derangements that occur during infection and cachexia.
Abstract: Previous studies have indicated that endotoxin and other bacterial and protozoal products can stimulate macrophages to produce a factor that can suppress the activity of the enzyme lipoprotein lipase (LPL), in vivo and in vitro. In the present report we describe the purification of this factor, cachectin, to apparent homogeneity from the conditioned medium of endotoxin-stimulated RAW 264.7 cells. The isolated protein has an isoelectric point of 4.7 and a subunit molecular weight of 17,000. Although cachectin's isoelectric point and molecular weight are similar to those described for interleukin 1, pure cachectin has no leukocyte-activating factor (LAF) activity. Cachectin at a concentration of 10(-11) M has the ability to suppress the LPL activity of the 3T3-L1 adipocyte cell line by 80%. Binding studies using radio-labeled cachectin and 3T3-L1 adipocytes and C2 myotubules revealed approximately 10(4) high-affinity receptors per cell on both cell types (Ka, 3 X 10(9]. Cachectin receptors were also present on liver membranes but were absent on erythrocytes and lymphocytes. The isolation of cachectin and characterization of its receptor should facilitate further investigations into the role of cachectin and other macrophage mediators in the metabolic derangements that occur during infection and cachexia.
854 citations
Journal Article•
TL;DR: Electrophoretic analysis of tissues recovered from injected animals suggests that the hormone is very rapidly degraded after binding, andCachectin/TNF bioactivity was monitored in the same serum samples by measuring lipoprotein lipase (LPL) suppression in 3T3-L1 cells.
Abstract: A highly specific radioreceptor assay for cachectin/tumor necrosis factor (TNF) was utilized to measure the time course of lipopolysaccharide (LPS)-induced hormone production in rabbits. Cachectin/TNF bioactivity was monitored in the same serum samples by measuring lipoprotein lipase (LPL) suppression in 3T3-L1 cells. Cachectin/TNF is produced in large quantities by LPS-treated rabbits without priming by bacillus Calmette Guerin, C. parvum, or other agents. Nanomolar concentrations of the hormone are achieved, with peak levels occurring at 2 hr postinjection; the hormone is rapidly cleared thereafter. In separate studies, mice were used to assess the distribution and metabolic fate of cachectin/TNF. Radioiodinated hormone is cleared from the plasma with a half-life of 6 to 7 min. Studies of the tissue distribution of label after injection demonstrate that liver, kidneys, skin, and gastrointestinal tract take up most of the hormone. Electrophoretic analysis of tissues recovered from injected animals suggests that the hormone is very rapidly degraded after binding.
768 citations
TL;DR: By virtue of its dual role as a neurotransmitter and a hormone, DA provides perhaps the best example of neuroendocrine interactions and it also possesses a more universal property.
Abstract: A. Introduction DOPAMINE (DA) is an interesting and versatile compound. In the central nervous system (CNS) it is involved with the control of fine movements and mental processes. Its association with disorders such as Parkinsonism and schizophrenia is well recognized. In the hypothalamo-hypophysial axis, DA is the primary physiological inhibitor of PRL secretion. Currently, this catecholamine represents the only nonpeptidergic hypothalamic agent with a well defined hypophysiotropic function. By virtue of its dual role as a neurotransmitter and a hormone, DA provides perhaps the best example of neuroendocrine interactions. It also possesses a more universal property. In all hormonal systems studied thus far, whether at the hypothalamic, posterior pituitary, or anterior pituitary level, DA functions as an inhibitor. Perhaps the common denominator to these diverse cells is the presence of D2 DA receptors, which are negatively linked to the adenylate cyclase system.
730 citations
TL;DR: The combination of calcium ionophores and TPA bypasses the requirement for antigen- or lectin-induced signal at the onset of lymphocyte activation, and ligand-induced cellular response follows a rise in cytosolic calcium concentration and protein kinase C activation.
Abstract: Although it has been proposed that the activation of T lymphocytes is mediated by an early rise in cytosolic calcium concentration, it has not been possible to mimic antigen- or mitogen-induced mouse lymphocyte activation by calcium ionophores that bypass receptor-mediated processes. There is now evidence from other systems that the rise in cytosolic calcium which follows receptor triggering is preceded by the breakdown of phosphatidylinositol bisphosphate into 1,2-diacylglycerol and inositol trisphosphate. The latter is known to cause release of calcium from intracellular stores. The cellular target for the former is now widely accepted to be protein kinase C. Therefore, ligand-induced cellular response follows a rise in cytosolic calcium concentration and protein kinase C activation. Here we confirm that the calcium ionophores A23187 and ionomycin do not activate mouse T lymphocytes. However, either one in combination with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is structurally related to 1,2-diacylglycerol, induces in lymphoid cell populations the expression of receptors for interleukin-2 (IL-2), the secretion of IL-2 and cell proliferation as measured by 3H-thymidine uptake. The growth-promoting effect of IL-2 on an exogenous IL-2-dependent clone could not be substituted for by ionomycin either alone or with TPA. Thus, the combination of calcium ionophores and TPA bypasses the requirement for antigen- or lectin-induced signal at the onset of lymphocyte activation.
721 citations
TL;DR: It is demonstrated that a significant portion of the mitogenic activity for 3T3 cells secreted by cultured human alveolar and peritoneal macrophages is due to a molecule (or molecules) similar to platelet-derived growth factor (PDGF).
707 citations
TL;DR: New insights into the molecular mechanisms underlying desensitization have emerged from the study of the receptors coupled to the ubiquitous second messenger-generating system adenylate cyclase.
Abstract: Desensitization, the tendency of biological responses to wane over time despite the continuous presence of a stimulus of constant intensity, is observed in organisms as diverse as bacteria and mammals. Recently, new insights into the molecular mechanisms underlying these phenomena have emerged from the study of the receptors coupled to the ubiquitous second messenger-generating system adenylate cyclase. These mechanisms involve sequestration or down-regulation of the receptors from the cell surface as well as functionally significant covalent modifications of the receptors and / or guanine nucleotide regulatory proteins.
TL;DR: Insulin desensitizes rat hepatoma cells to the actions of insulin and IGFs, mediated by both insulin and insulin receptors, by mechanisms distal to hormone binding and possibly common to IGF and insulin effector pathways.
Abstract: Two subtypes of IGF receptors have been identified. Type I IGF receptors have a Mr greater than 300,000 and are composed of disulfide-linked 130,000-dalton (alpha) and approximately 90,000-dalton (beta) subunits. Type I receptors preferentially bind IGF-I but also bind IGF-II and, more weakly, insulin. Type II IGF receptors consist of a 250,000-dalton protein that contains internal disulfide bonds but is not linked to other membrane components. Type II receptors bind IGF-II with higher affinity than IGF-I. They do not interact with even very high concentrations of insulin. Type I IGF receptors and insulin receptors are homologous structures. Type II IGF receptors do not appear to be homologous to type I receptors. Type II receptors do not appear to be downregulated. Insulin acutely upregulates type II IGF receptors in intact rat adipose cells by effecting a redistribution of receptors cycling between a large intracellular pool and the plasma membrane. Insulin and the IGFs elicit the same biological responses, either by cross-reacting with one of the receptors for the heterologous ligand or by concurrent activation of convergent effector pathways by binding to the homologous receptor. Which mechanism is utilized appears to depend more on the tissue than on the biological response. Insulinmore » desensitizes rat hepatoma cells to the actions of insulin and IGFs, mediated by both insulin and IGF receptors, by mechanisms distal to hormone binding and possibly common to IGF and insulin effector pathways.« less
TL;DR: The results presented strongly suggest that mouse sperm bind to eggs via O-linked oligosaccharides present on ZP3, showing a role for carbohydrate, rather than polypeptide chain in receptor function.
TL;DR: It is suggested that the presence of a high intensity of staining for the EGF receptor in human bladder tumours is associated with poor differentiation and with invasion.
TL;DR: The results suggest a role for cyclic nucleotides in olfactory transduction13,20–22, and point to a molecular analogy between olfaction and visual15,16, hormone17,18 and neurotransmitter19 reception, and reveal new ways to identify and isolate Olfactory receptor proteins.
Abstract: The mechanism of the sense of smell has long been a subject for theory and speculation. More recently, the notion of odorant recognition by stereospecific protein receptors has gained wide acceptance, but the receptor molecules remained elusive. The recognition molecules are believed to be quite diverse, which would partly explain the unusual difficulties encountered in their isolation by conventional ligand-binding techniques. An alternative approach would be to probe the receptors through transductory components that may be common to all receptor types. Here we report the identification of one such transductory molecular component. This is an odorant-sensitive adenylate cyclase, present in very large concentrations in isolated dendritic membranes of olfactory sensory neurones. Odorant activation of the enzyme is ligand and tissue specific, and occurs only in the presence of GTP, suggesting the involvement of receptor(s) coupled to a guanine nucleotide binding protein (G-protein). The olfactory G-protein is independently identified by labelling with bacterial toxins, and found to be similar to stimulatory G-proteins in other systems. Our results suggest a role for cyclic nucleotides in olfactory transduction, and point to a molecular analogy between olfaction and visual, hormone and neurotransmitter reception. Most importantly, the present findings reveal new ways to identify and isolate olfactory receptor proteins.
TL;DR: The results support the notion that agonist is required to induce GppNHp binding and/or activation of the G proteins, and suggest that the muscarinic receptor and the IAch channel are separate molecular structures.
Abstract: Guanine nucleotide binding proteins, interchangeably called N or G proteins, seem to be the primary signal-transducing components of various agonist-induced cell membrane functions. In the heart, G proteins have been implicated in beta-adrenergic modulation of the slow inward Ca2+ current. We have investigated the role of G proteins in muscarinic activation of an inwardly rectifying, acetylcholine (ACh)-induced K+ current (IACh), and beta-adrenergic activation of an (isoprenaline)-induced Ca2+ current (Isi). Here we report that intracellular application of the non-hydrolysable GTP analogue 5'-guanylylimidodiphosphate (GppNHp) brought about an agonist-induced, antagonist-resistant, persistent activation of IACh and Isi. This functional uncoupling of channel from receptor suggests that the muscarinic receptor and the IACh channel are separate molecular structures. Membrane conductance responses to sequential activation of muscarinic and beta-adrenergic receptors demonstrate that in contrast to the muscarinic inhibition of Isi, muscarinic stimulation of IACh is mediated by a G protein via a pathway that does not involve adenylate cyclase. Taken together, the results support the notion that agonist is required to induce GppNHp binding and/or activation of the G proteins. Once triggered by agonist, the control system remains maximally activated, thereby transforming the cell so that it no longer responds to subsequent homologous receptor-mediated signals.
TL;DR: It is demonstrated that this ubiquitous BF4‐positive 90‐kd protein is in fact the chicken 90 kd heat‐shock protein (hsp 90): it increased in heat‐shocked chick embryo fibroblasts, and displayed identical migration in two‐dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90.
Abstract: Non-transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250-300 kd which includes a non-hormone binding 90-kd protein. A monoclonal antibody BF4 raised against the purified, molybdate-stabilized, 8S progesterone receptor (8S-PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90-kd protein present in great abundance in all chicken tissues, including that present in 8S-forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4-positive 90-kd protein is in fact the chicken 90 kd heat-shock protein (hsp 90): it increased in heat-shocked chick embryo fibroblasts, and displayed identical migration in two-dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone-binding subunits may play a role in keeping the receptor in an inactive form.
TL;DR: Detergent extracts of human osteosarcoma cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronECTin cell attachment sequence Arg- Gly- asp.
Abstract: Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities
TL;DR: The results suggest that 3H-raclopride labels dopamine D-2 receptors with high specificity in the rat brain both in vitro and in vivo, and thus, that it should be a useful tool in studies of central dopamine D -2 receptors.
TL;DR: The results suggest that the presence of EGF receptors is associated with metastatic potential and that the growth of a proportion of poor-prognosis oestrogen-receptor-negative tumours may be regulated by peptide growth factors interacting with the EGF receptor.
TL;DR: It is reported that whereas messenger RNAs encoding the β-and γ-subunits are relatively abundant in immature thymocytes, α mRNA levels are very low in mature T cells, suggesting a role for the γ gene in T-cell differentiation.
Abstract: In contrast to B cells or their antibody products, T lymphocytes have a dual specificity, for both the eliciting foreign antigen and for polymorphic determinants on cell surface glycoproteins encoded in the major histocompatibility complex (MHC restriction)1–4. The recent identification of T-cell receptor glycoproteins5–7 as well as the genes encoding T-cell receptor subunits will help to elucidate whether MHC proteins and foreign antigens are recognized by two T-cell receptors or by a single receptor. An important feature of MHC restriction is that it appears to be largely acquired by a differentiating T-cell population under the influence of MHC antigens expressed in the thymus8–10, suggesting that precursor T cells are selected on the basis of their reactivity with MHC determinants expressed in the host thymus9–11. To understand this process of ‘thymus education’, knowledge of the developmental regulation of T-cell receptor gene expression is necessary. Here we report that whereas messenger RNAs encoding the β-and γ-subunits are relatively abundant in immature thymocytes, α mRNA levels are very low. Interestingly, whereas α mRNA levels increase during further development and β mRNA levels stay roughly constant, γ mRNA falls to very low levels in mature T cells, suggesting a role for the γ gene in T-cell differentiation.
TL;DR: It is shown that the 90-kDa receptor-associated phosphoprotein is an abundant cytosolic protein that reacts with a monoclonal antibody that recognizes the 90 -kDa phosphop protein that binds steroid receptors in the chicken oviduct.
TL;DR: It is demonstrated that 1,25-(OH)2D3 can inhibit DNA synthesis and cell proliferation of the G-8 myoblast cells in a dose-dependent manner and the possibility that muscle is a target tissue for 1, 25-dihydroxyvitamin D3 and the hormone may act to initiate terminal differentiation of myOBlast cells is supported.
TL;DR: Data showed that vascular cells have insulin and IGF receptors, but have a differential response to these hormones, which could provide clues to understanding the pathogenesis of diabetic micro- and macroangiopathy.
Abstract: It has been suggested that elevated levels of insulin or insulin-like growth factors (IGFs) play a role in the development of diabetic vascular complications. Previously, we have shown a differential response to insulin between vascular cells from retinal capillaries and large arteries with the former being much more insulin responsive. In the present study, we have characterized the receptors and the growth-promoting effect of insulinlike growth factor I (IGF-I) and multiplication-stimulating activity (MSA, an IGF-II) on endothelial cells and pericytes from calf retinal capillaries and on endothelial and smooth muscle cells from calf aorta. We found single and separate populations of high affinity receptors for IGF-I and MSA with respective affinity constants of 1 X 10(-9) M-1 and 10(-8) M-1 in all four cell types studied. Specific binding of IGF-I was between 7.2 and 7.9% per milligram of protein in endothelial cells and 9.1 and 10.4% in the vascular supporting cells. For 125I-MSA, retinal endothelial cells bound only 1.7-2.5%, whereas the aortic endothelial cells and the vascular supporting cells bound between 5.6 and 8.5% per milligram of protein. The specificity of the receptors for IGF-I and MSA differed, as insulin and MSA was able to compete with 125I-IGF-I for binding to the IGF-I receptors with 0.01-0.1, the potency of unlabeled IGF-I, whereas even 1 X 10(-6) M, insulin did not significantly compete with 125I-MSA for binding to the receptors for MSA. For growth-promoting effects, as measured by the incorporation of [3H]thymidine into DNA, confluent retinal endothelial cells responded to IGF-I and MSA by up to threefold increase in the rate of DNA synthesis, whereas confluent aortic endothelial cells did not respond at all. A similar differential of response to insulin between micro- and macrovascular endothelial cells was reported by us previously. In the retinal endothelium, insulin was more potent than IGF-I and IGF-I was more potent that MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In the retinal and aortic supporting cells, no differential response to insulin or the IGFs was observed. In the retinal pericytes, IGF-I, which stimulated significant DNA synthesis beginning at 1 X 10(-9) M, and had a maximal effect at 5 X 10(-8) M, was 10-fold more potent than MSA and equally potent to insulin. In the aortic smooth muscle cells, IGF-I was 10-100 times more potent than insulin or MSA. In addition, insulin and IGF-I at 1 X 10(-6) and 1 X 10(-8) M, respectively, stimulated these cells to grow by doubling the number of cells as well. In all responsive tissues, the combination of insulin and IGFs were added together, no further increase in effect was seen. These data showed that vascular cells have insulin and IGF receptors, but have a differential response to these hormones. These differences in biological response between cells from retinal capillaries and large arteries could provide clues to understanding the pathogenesis of diabetic micro- and macroangiopathy.
TL;DR: Results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF, suggesting that degradation occurs intracellularly.
Abstract: Highly purified, Escherichia coli-derived recombinant human tumor necrosis factor (TNF) was labeled with 125I and employed to determine receptor binding, internalization, and intracellular degradation in murine L929 cells (highly sensitive to the cytotoxic action of TNF) and in diploid human FS-4 cells (resistant to TNF cytotoxicity). 125I-labeled TNF bound specifically to high-affinity receptors on both L929 and FS-4 cells. Scatchard analysis of the binding data indicated the presence of 2200 binding sites per L929 cell and 7500 binding sites per FS-4 cell. The calculated dissociation constants are 6.1 X 10(-10) M and 3.2 X 10(-10) M for L929 and FS-4 cells, respectively. In both L929 and FS-4 cells, incubation at 37 degrees C resulted in a rapid internalization of the bulk of the cell-bound TNF, followed by the appearance of trichloroacetic acid-soluble 125I radioactivity in the tissue culture medium, due to degradation of TNF. Degradation but not cellular uptake of TNF was inhibited in the presence of chloroquine (an inhibitor of lysosomal proteases) in both L929 and FS-4 cells, suggesting that degradation occurs intracellularly, probably within lysosomes. These results show that resistance of FS-4 cells to TNF cytotoxicity is not due to a lack of receptors or their inability to internalize and degrade TNF.
TL;DR: The data suggest that a region near the carboxyl-terminus of the alpha-chain of fibrinogen interacts with the fibrInogen receptor on activated platelets, and support the concept that the sequence Arg-Gly-Asp-Ser has been conserved for use in a variety of cellular adhesive processes.
TL;DR: A reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied is described and the results suggest a rationale for concomitant Ca 2+ mobilization and diacylglycerol formation in response to some hormones, neuro-transmitters and growth factors.
Abstract: The activation of protein kinase C by diacylglycerol and by tumour promoters has implicated this enzyme in transmembrane signalling and in the regulation of the cell cycle1,2. In vitro studies revealed that catalytic activity requires the presence of calcium and phos-pholipids with a preference f or phosphatidylserine3. Diacylglycerol and tumour promoters such as phorbol esters bind to the enzyme4–7, leading to its activation while sharply increasing its affinity for Ca2+ and phospholipid. Addition of diacylglycerol analogues or phorbol esters to intact cells results in the phosphorylation of specific polypeptides4,8–20. Several cellular processes, including hormone and neurotransmitter release and receptor down-regulation1–4, are modulated by the activation of protein kinase C, while phorbol ester-induced stimulation of the enzyme in whole cells has been associated with its translocation from the cytoplasm to the plasma membrane21,22. Moreover, the use of Ca2+ ionophores has revealed an apparent synergism between Ca2+ mobilization and protein kinase C activation2. This synergism has recently also been found to apply to receptor down-regulation (ref. 23 and accompanying paper24). Here we describe a reconstitution system in which intracellular translocation of protein kinase C and the synergism between Ca2+ and enzyme activators can be studied. The results suggest a rationale for concomitant Ca2+ mobilization and diacylglycerol formation in response to some hormones, neuro-transmitters and growth factors.
TL;DR: The molecular size of the plasma LDL receptor synthesized by cultured fibroblasts from a patient with the internalization-defective form of familial hypercholesterolemia (FH 274) was smaller by 10,000 daltons, and a 5-kilobase deletion appeared to be caused by a novel intrastrand recombination between two repetitive sequences of the Alu family that were oriented in opposite directions.
Abstract: The molecular size of the plasma LDL (low density lipoprotein) receptor synthesized by cultured fibroblasts from a patient with the internalization-defective form of familial hypercholesterolemia (FH 274) was smaller by 10,000 daltons than the size of the normal LDL receptor. The segment of the gene encoding the truncated portion of the FH 274 receptor was cloned into bacteriophage lambda. Comparison of the nucleotide sequences of the normal and FH 274 genes revealed a 5-kilobase deletion, which eliminated the exons encoding the membrane-spanning region and the carboxyl terminal cytoplasmic domain of the receptor. The deletion appeared to be caused by a novel intrastrand recombination between two repetitive sequences of the Alu family that were oriented in opposite directions. The truncated receptors lack membrane-spanning regions and cytoplasmic domains; they are largely secreted into the culture medium, but a small fraction remains adherent to the cell surface. The surface-adherent receptors bind LDL, but they are unable to cluster in coated pits, thus explaining the internalization-defective phenotype.
TL;DR: Both genetic and acquired abnormalities in the number of insulin receptors, the activity of the receptor kinase, and the various post-receptor steps in insulin action occur in disease states leading to tissue resistance to insulin action.
Abstract: Insulin initiates its action by binding to a glycoprotein receptor on the surface of the cell. This receptor consists of an alpha-subunit, which binds the hormone, and a beta-subunit, which is an insulin-stimulated, tyrosine-specific protein kinase. Activation of this kinase is believed to generate a signal that eventually results in insulin's action on glucose, lipid, and protein metabolism. The growth-promoting effects of insulin appear to occur through activation of receptors for the family of related insulin-like growth factors. Both genetic and acquired abnormalities in the number of insulin receptors, the activity of the receptor kinase, and the various post-receptor steps in insulin action occur in disease states leading to tissue resistance to insulin action.
TL;DR: IL-2-receptor interactions actually promote the loss of IL-2 responsiveness by diminishing the density of high-affinity binding sites at the time that Tac antigen levels are increased, resulting in a time- and temperature-dependent 8- to 10-fold enhancement of Tac epitope expression and a 20-30% diminishment of detectable high-Affinity IL- 2 binding sites.
Abstract: The cell surface density of high-affinity membrane receptors for the T-lymphocytotrophic hormone interleukin 2 (IL-2) determines the rate of T-cell-cycle progression. Since 10-fold greater numbers of IL-2 receptor molecules were found by using a radiolabeled monoclonal antibody reactive with IL-2 receptors (anti-Tac) compared with binding of IL-2, the functional relationship of the binding sites recognized by both of these ligands was assessed. In the presence of cycloheximide, IL-2 binding sites declined with a half-time (t1/2) of 2.6 hr, whereas the decay of anti-Tac binding sites was much slower (t 1/2 = 6.4 hr). Moreover, after limited membrane proteolysis, the half-time for the reappearance of IL-2 binding sites was remarkably similar to its decay (t 1/2 = 2.2 hr), while Tac antigen reappearance was markedly retarded, returning to only 20% of original levels within 5 hr after proteolysis. Addition of homogeneous immunoaffinity-purified IL-2 to cell populations that expressed equivalent IL-2 and anti-Tac binding sites resulted in a time- and temperature-dependent 8- to 10-fold enhancement of Tac epitope expression and, simultaneously, a 20-30% diminishment of detectable high-affinity IL-2 binding sites. As the magnitude of the IL-2-dependent proliferative response correlated with the density of high-affinity IL-2 binding sites, rather than Tac antigen levels, quantitation of Tac epitope density does not provide a reliable indication of IL-2-responsiveness among activated T-cell populations. Instead, IL-2-receptor interactions actually promote the loss of IL-2 responsiveness by diminishing the density of high-affinity binding sites at the time that Tac antigen levels are increased.