Signaling mechanisms through gp130: A model of the cytokine system

The interleukin-6 cytokine family plays roles in a wide variety of tissues and organs, including the immune hematopoietic and nervous systems. Gp130 is a signal-transducing subunit shared by the receptors for the IL-6 family of cytokines. The binding of a ligand to its receptor induces the dimerization of gp 130, leading to the activation of JAK tyrosine kinase and tyrosine phosphorylation of gpl30. These events lead to the activation of multiple signal-transduction pathways, such as the STAT, Ras-MAPK and PI-3 kinase pathways whose activation is controlled by distinct regions of gp130. We propose a model showing that the outcome of the signal transduction depends on the balance or interplay among the contradictory signal transduction pathways that are simultaneously generated through a cytokine receptor in a given target cell.     

Interleukin-4 proliferative signal transduction involves the activation of a tyrosine-specific phosphatase and the dephosphorylation of an 80-kDa protein

Interleukin-4 (IL-4) is a cytokine that expresses its biological effects by binding to specific membrane receptors. Although the diverse biological properties of this molecule have been characterized extensively the biochemical mechanisms by which extracellular binding events lead to biological responses remain unclear. IL-4 can stimulate the proliferation of several hemopoietic cell types, and we have taken advantage of its ability to induce the growth of leukemic cell lines to investigate the role that protein phosphorylation events might play in IL-4 mitogenic signal transduction. We show that the addition of IL-4 to several leukemic cell lines of different origin causes the rapid dephosphorylation of an 80-kDa phosphoprotein (p80) from tyrosine residues. This event occurs in a dose-responsive manner closely correlating to that of biological activity, and both are blocked by an anti-IL-4-specific antiserum. The ability of sodium orthovanadate to prevent IL-4-induced dephosphorylation of p80 suggests that this event is mediated by a protein-tyrosine-phosphatase (EC 3.1.3.48). The importance of the role that tyrosine-specific dephosphorylation plays in mediating IL-4 mitogenic signal transduction is substantiated by the ability of sodium orthovanadate in cell culture to block effectively IL-4-induced proliferation at doses that enhance the proliferation stimulated by either granulocyte-macrophage colony-stimulating factor or interleukin-3.     

Ras-mediated cell proliferation and cell death: some clues from the interleukin-2 receptor system

Oncoproteins of the Ras family have been extensively studied because of their implication in human cancer. Their roles have been primarily assigned to the commandment of cell proliferation and suppression of apoptosis, which has also been demonstrated by the involvement of Ras activation in the signal transduction pathways triggered by most cytokine receptors. Nevertheless, the functions of Ras proteins have been extended in the last years by the findings showing that they can also act as promoters or enhancers of apoptosis in various systems and conditions. These considerations have raised the issue as to how the signals delivered by Ras are regulated and translated in terms of cellular responses, suggesting that signal complementation may direct the final fate of cells. As an example, the interleukin-2 receptor system may represent a useful model in which the meaning of Ras signals may be evaluated in terms of interactions with other simultaneous signalling events, since knowledge of the biochemical events triggered by the interaction of interleukin-2 with its cell surface receptor in lymphocytes has allowed the proposal of a complete signalling model arranged in three independent channels, one of which is mediated by Ras.This work was supported by grants from CICYT and Pharmacia-Upjohn.     

The molecular action of tumor necrosis factor-alpha.

Tumor necrosis factor-alpha (TNF-alpha) is a polypeptide hormone newly synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators (C5a anaphylatoxin), or cytokines such as interleukin-1 and, in an autocrine manner, TNF itself. The net biological effect of TNF-alpha may vary depending on relative concentration, duration of cell exposure and presence of other mediators which may act in synergism with this cytokine. TNF-alpha may be relevant either in pathological events occurring in cachexia and endotoxic shock and inflammation or in beneficial processes such as host defense, immunity and tissue homeostasis. The biological effects of TNF-alpha are triggered by the binding to specific cell surface receptors. The formation of TNF-alpha-receptor complex activates a variety of biochemical pathways that include the transduction of the signal at least in part controlled by guanine-nucleotide-binding regulatory proteins (G proteins), its amplification through activation of adenyl cyclase, phospholipases and protein kinases with the generation of second messenger pathways. The transduction of selected genes in different cell types determines the characteristics of the cell response to TNF-alpha. The full understanding of the molecular mechanisms of TNF-alpha will provide the basis for a pharmacological approach intended to inhibit or potentiate selected biological actions of this cytokine.     

Targeted receptor trafficking affects the efficiency of retrovirus transduction

We describe the development of an experimental system to test the hypothesis that the efficiency of retrovirus transduction is dependent on the pathway of virus entry into the host cell and the intracellular trafficking itinerary of the cellular receptor with which it interacts. The experimental system consists of three model target cell lines, derived from HeLa cells, that stably express one of three interleukin-2 receptor alpha chain (CD25) chimeras, TAC, TAC-CD16, and TAC-DKQTLL, which have identical extracellular domains but different intracellular trafficking itineraries, and a targeted amphotropic murine leukemia retrovirus whose envelope proteins were modified to include a binding site for TAC at their N-termini. We found that the efficiency of retrovirus transduction was affected by the distribution and trafficking itinerary of the TAC receptors. Transduction of cells that expressed TAC-DKQTLL was nearly 4-fold lower than transduction of control cells that did not express any of the TAC receptors. In contrast, transduction of cells that expressed TAC was 1.6-fold higher than transduction of control cells, whereas transduction was not significantly affected by the expression of TAC-CD16. Our results suggest that in the course of designing a targeted retrovirus it may be prudent to target only those receptors that internalize retroviruses via pathways that most efficiently support post-binding steps of infection.     

A point mutation uncouples human interleukin-1 beta biological activity and receptor binding

Interleukin-1 proteins elicit a number of biological activities, but the molecular events following formation of a cell surface receptor-ligand complex have not been well defined. Conversion of Arg127 to Gly127 in the mature human interleukin-1 beta protein reduces bioactivity by 100-fold while the receptor binding affinity decreases by only 25%. The results suggest that the mutant IL-1 beta protein is defective in activating signal transduction events and indicate that binding of interleukin-1 beta protein to receptor is necessary but insufficient for biological activity. The finding that the features of the IL-1 beta protein responsible for receptor binding and biological activity are at least in part distinct may be clinically relevant to the design of interleukin-1 antagonists.     

Structure and mechanism of immunoreceptors: New horizons in T cell and B cell receptor biology and beyond

Immunoreceptors, also named non-catalytic tyrosine-phosphorylated receptors, are a large class of leukocyte cell-surface proteins critically involved in innate and adaptive immune responses. Their most characteristic defining feature is a shared signal transduction machinery where binding events of cell surface-anchored ligands to the small extracellular receptor domains are translated into phosphorylation of conserved tyrosine-containing cytosolic sequence motifs initiating downstream signal transduction cascades. Despite their central importance to immunology, the molecular mechanism of how ligand binding activates the receptors and results in robust intracellular signaling has remained enigmatic. Recent breakthroughs in our understanding of the architecture and triggering mechanism of immunoreceptors come from cryogenic electron microscopy studies of the B cell and T cell antigen receptors.     

Average density and size of microclusters of epidermal growth factor receptors on A431 cells.

For some hormone receptors, the early events of signal transduction depend on their molecular arrangement and interactions at the cell surface. An understanding of the mechanism of signal transduction in general needs a careful analysis of the receptor distribution. Here, we present the first quantitative measurement of epidermal growth factor receptor distribution on A431 cells obtained by scanning fluorescence correlation spectroscopy. Prior to epidermal growth factor binding, the A431 cell membrane presents an average surface density of 7.7-8.4 microclusters/microns 2, each containing an average of 130 receptors.     

ANALYSIS OF SPATIAL DISTRIBUTIONS OF CELLULAR MOLECULES DURING MECHANICAL STRESSING OF CELL SURFACE RECEPTORS USING CONFOCAL MICROSCOPY

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.     

The functional basis of granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 receptor activation, basic and clinical implications

The cytokines granulocyte-macrophage colony stimulating factor, interleukin-3 and interleukin-5 have overlapping activities on cells expressing their receptors. This is explained by their sharing a receptor signal transduction subunit, beta c. This communal signaling subunit is also required for high affinity binding of all three cytokines. Therapeutic approaches attempting to interfere or modulate haemopoietic cells using cytokines or their analogues can in some instances be limited due to functional redundancy amongst cytokines using shared receptor signaling subunits. Therefore, a better approach would be to develop therapeutics against the shared subunit. Studies examining the GM-CSF, IL-3 and IL-5 receptors have identified the key events leading to functional receptor activation. With this knowledge, it is now possible to identify new targets for the development of a new class of antagonist that blocks the biological activity of all the cytokines utilizing beta c. This approach may be extended to other receptor systems such as IL-4 and IL-13 where receptor activation is dependent on a common signaling and binding subunit.     

Signal transduction and endocytosis: close encounters of many kinds

Binding of hormones, growth factors and other cell modulators to cell-surface receptors triggers a complex array of signal-transduction events. The activation of many receptors also accelerates their endocytosis. Endocytic transport is important in regulating signal transduction and in mediating the formation of specialized signalling complexes. Conversely, signal-transduction events modulate specific components of the endocytic machinery. Recent studies of protein tyrosine kinases and G-protein-coupled receptors have shed new light on the mechanisms and functional consequences of this bidirectional interplay between signalling and membrane-transport networks.     

Kinases and G proteins join the Wnt receptor complex

Wnt proteins form a family of secreted signaling proteins that play a key role in various developmental events such as cell differentiation, cell migration, cell polarity and cell proliferation. It is currently thought that Wnt proteins activate at least three different signaling pathways by binding to seven transmembrane receptors of the Frizzled family and the co-receptor LRP6. Despite our growing knowledge of intracellular components that mediate a Wnt signal, the molecular events at the membrane have remained rather unclear. Now several publications(1-4) indicate that Frizzled receptors are G-protein coupled and kinases were identified that phosphorylate the co-receptor LRP6. These data deepen our understanding of Wnt-mediated signal transduction and provide more insight into how specificity may be achieved.     

Interleukin-2 binding to activated human T lymphocytes triggers generation of cyclic AMP but not of inositol phosphates

Human T lymphocytes stimulated with phytohaemagglutinin undergo a single round of cell division. Further proliferation is dependent on the lymphokine interleukin-2 (IL2) [(1987) Immunology 60, 7-12]. We show here that binding of IL2 to its receptors on the lymphocyte surface triggers the generation of cyclic AMP. In contrast, generation of inositol phosphates from the breakdown of inositol lipids was not detected. We suggest that cyclic AMP may play a role in the transduction of the IL2 proliferative signal in T lymphocytes.     

The human serotonin<Subscript>1A</Subscript> receptor exhibits G-protein-dependent cell surface dynamics

Seven transmembrane domain G-protein-coupled receptors constitute the largest family of proteins in mammals. Signal transduction events mediated by such receptors are the primary means by which cells communicate with and respond to their external environment. The major paradigm in this signal transduction process is that stimulation of the receptor leads to the recruitment and activation of heterotrimeric GTP-binding proteins. These initial events, which are fundamental to all types of G-protein-coupled receptor signaling, occur at the plasma membrane via protein–protein interactions. As a result, the dynamics of the activated receptor on cell surfaces represents an important determinant in its encounter with G-proteins, and has significant impact on the overall efficiency of the signal transduction process. We have monitored the cell surface dynamics of the serotonin_1A receptor, an important member of the G-protein-coupled receptor superfamily, in relation to its interaction with G-proteins. Fluorescence recovery after photobleaching experiments carried out with the receptor tagged to the enhanced yellow fluorescent protein indicate that G-protein activation alters the diffusion properties of the receptor in a manner suggesting the activation process leads to dissociation of G-proteins from the receptor. This result demonstrates that the cell surface dynamics of the serotonin_1A receptor is modulated in a G-protein-dependent manner. Importantly, this result could provide the basis for a sensitive and powerful approach to assess receptor/G-protein interaction in an intact cellular environment.     

Kinetics and amplification in neutrophil activation and adaptation

Neutrophil activation and adaptation are beginning to be understood in quantitative and mechanistic detail. Neutrophil responses to chemoattractants involve cell surface receptors, guanine nucleotide binding (G) proteins and intracellular second messengers which are generated within a few seconds of the presentation of the ligand. Novel real-time fluorescent methods have made it possible to examine the dynamics of assembly and disassembly of receptors and G proteins during cell activation and to model, by computer, the complex sequence of ligand-receptor events. Rapid amplification cascades of signals are observed in both stimulatory and inhibitory receptor pathways. The adaptation of neutrophils involves uncoupling between receptors and G proteins, both by sequestering receptors from the transduction sequence and by reducing the activatibility of the G proteins.     

Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A

Macrophage scavenger class A type I and type II receptors (SR-A) are trimeric, integral membrane glycoproteins that bind an unusually broad array of macromolecular ligands. These ligands include modified proteins and lipoproteins, nucleic acids, and a variety of plant and microbial cell wall constituents, such as fucoidan and lipoteichoic acid. Early studies of SR-A functions indicated that the receptors bound, internalized, and degraded their ligands without provoking any macrophage activating signaling events. More recent studies have provided evidence that several SR-A ligands can activate macrophage gene expression via utilization of a receptor-linked, PI3-kinase pathway. To investigate the role of SR-A in engaging signal transduction events, we employed macrophages taken from mice lacking these receptors. Using either fucoidan or lipoteichoic acid, we confirm that both ligands stimulate tyrosine phosphorylation of PI3-kinase and production of modest levels of the cytokine, TNFalpha. However, macrophages taken from SR-A null mice did not differ from wild type macrophages in these responses, indicating that these signaling events arise independently of SR-A activity. Employing mice lacking CD14, a GPI anchored receptor that binds bacterial lipopolysaccharide and signals via activation of Toll-like receptors, we show that the fucoidan and lipoteichoic acid responses are largely abrogated when CD14 is absent. These data do not provide support for direct SR-A involvement in signal transduction events and suggest that the early characterization of these receptors as initiators of a non-phlogistic, pathogen clearance pathway was correct.     

Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.     

Abscisic acid receptors: multiple signal-perception sites

BACKGROUND AND AIMS: The phytohormone abscisic acid (ABA) plays a vital role in various aspects of plant growth and development and in adaptation of plants to various environmental stresses. Cell response to ABA is initiated by ABA perception with a cell receptor. Recently, three distinct ABA receptors have been identified, opening a door to uncover the initial events of ABA signal transduction. The aim of this Botanical Briefing is to present a perspective of the ABA receptors identified. SCOPE: This Briefing offers an introduction to the three ABA receptors identified and an analysis of the complexity and multiplicity of ABA receptors, and provides some viewpoints on future research.