Cytokine Receptor Function Research Articles

Histone Deacetylases (HDACs) Roles in Inflammation-mediated Diseases; Current Knowledge.

The histone acetyl transferases (HATs) and histone deacetylases (HDACs), which are mostly recognized for their involvement in regulating chromatin remodeling via histone acetylation/deacetylation, have been shown to also change several non-histone proteins to regulate other cellular processes. Acetylation affects the activity or function of cytokine receptors, nuclear hormone receptors, intracellular signaling molecules, and transcription factors in connection to inflammation. Some small-molecule HDAC inhibitors are utilized as anticancer medications in clinical settings due to their capability to regulate cellular growth arrest, differentiation, and death. Here, we summarize our present knowledge of the innate and adaptive immunological pathways that classical HDAC enzymes control. The aim is to justify the targeted (or non-targeted) use of inhibitors against certain HDAC enzymes in inflammatory diseases such as arthritis, inflammatory bowel diseases (IBD), airways inflammation and neurological diseases.

Identifying C‐Mannosylated Proteins in RAW264.7 Cells

C‐mannosylation is a specific type of glycosylation in which α‐mannose is covalently bound to the C2 atom of a tryptophan via a carbon‐carbon bond. Presently, it is known to occur on proteins that contain the W‐x‐x‐W‐x‐x‐W/C motif found within the Thrombospondin Type‐1 Repeat (TSR), with ‘x’ denoting any other amino acid. C‐mannosylation has been implicated in a few cellular functions, including protein secretion and type I cytokine receptor function. Previous research has shown that exogenous C‐mannosylated peptides derived from TSR containing proteins upregulate the production of the pro‐inflammatory cytokine Tumor Necrosis Factor alpha (TNF‐α) in RAW264.7 macrophage cells. This upregulation is substantially increased in the presence of Hsc70. When these peptides are internalized by RAW264.7 cells, a binding interaction occurs between the two proteins, which also correlates with increased TNF‐α levels. With the knowledge that exogenous C‐mannosylated peptides and Hsc70 can influence cytokine production, this study has focused on identifying endogenous C‐mannosylated proteins in RAW264.7 cells that interact with Hsc70. The potential of a naturally occurring interaction between these two proteins could elucidate the importance of C‐mannosylation in the innate immune response. Through a Hsc70 co‐immunoprecipitation, a potentially C‐mannosylated protein has been identified in Junctophilin‐1 (JPH1). JPH1 forms a junctional membrane complex (JMC) between the endoplasmic reticulum and plasma membrane of excitable cells, facilitating communication between calcium ion release channels. When comparing the nano‐LC MS/MS results for JPH1 against a mouse database, 69 separate PSMs were shown where JPH1 carries a hexose. MS/MS data suggests that the hexose appears on a single tryptophan in a peptide bearing the sequence: E‐G‐E‐W‐A‐N‐N‐K. This indicates that JPH1 may be an example of non‐canonical C‐mannosylation. To further explore the status of JPH1 as a C‐mannosylated protein, the protein was overexpressed in HEK293T cells. This experiment showed that JPH1 was not C‐mannosylated in HEK293T cells. Noting the lack of modification, RTPCR was performed with both RAW264.7 and HEK293T RNA. These experiments showed that all four DPY19L homologs were expressed in HEK293T cells, while only DPY19L1 and L4 were expressed in RAW cells. Further work is being conducted to overexpress JPH1 in RAW264.7 cells to determine why, or if, JPH1 is C‐mannosylated in this particular cell line.Support or Funding InformationThis work was supported by NSF grant OIA‐1738707.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor

Background/Aims: The cytokine interleukin-11 (IL-11) has important pro- and anti-inflammatory functions. It activates its target cells through binding to the IL-11 receptor (IL-11R), and the IL-11/IL-11R complex recruits a homodimer of glycoprotein 130 (gp130). N-linked glycosylation, a post-translational modification where complex oligosaccharides are attached to the side chain of asparagine residues, is often important for stability, folding and biological function of cytokine receptors. Methods: We generated different IL-11R mutants via site-directed mutagenesis and analyzed them in different cell lines via Western blot, flow cytometry, confocal microscopy and proliferation assays. Results: In this study, we identified two functional N-glycosylation sites in the D2 domain of the IL-11R at N127 and N194. While mutation of N127Q only slightly affects cell surface expression of the IL-11R, mutation of N194Q broadly prevents IL-11R appearance at the plasma membrane. Accordingly, IL-11R mutants lacking N194 are retained within the ER, whereas the N127 mutant is transported through the Golgi complex to the cell surface, uncovering a differential role of the two N-glycan sequons for IL-11R maturation. Interestingly, IL-11R mutants devoid of one or both N-glycans are still biologically active. Furthermore, the IL-11RN127Q/N194Q mutant shows no inducible shedding by ADAM10, but is rather constitutively released into the supernatant. Conclusion: Our results show that the two N-glycosylation sites differentially influence stability and proteolytic processing of the IL-11R, but that N-linked glycosylation is not a prerequisite for IL-11 signaling.

Requirement of Lipid Raft Integrity for Signaling by JAK2-V617F

AbstractAbstract 4191JAK2 is a cytoplasmic tyrosine kinase that plays an important role in signaling following activation of various cytokine receptors. JAK2 activation promotes growth, survival, and differentiation of various cell types. Mutation of JAK2 is seen in numerous hematopoietic diseases, most notably in myeloproliferative neoplasms (MPNs). JAK2-V617F is a frequent mutation found in the classical MPNs: polycythemia vera, essential thrombocythemia, and primary myelofibrosis. The single amino acid change of valine to phenylalanine occurs in the pseudokinase domain of JAK2, relieving auto-inhibition of the kinase domain and allowing constitutive kinase activity. Numerous mouse models have demonstrated that JAK2-V617F can induce MPN-like disorders in mice. Thus, this point mutation, as well as other less common JAK2 mutations, is believed to play an important etiologic role in the development of MPNs in humans. The development and use of JAK2 inhibitors in clinical trials has shown promising results, again demonstrating the important role JAK2 plays in MPNs. While the JAK2-V617F mutation, as well as other JAK2 mutations, decreases auto-inhibition of JAK2 kinase activity, it is clear that mutated JAK2 still requires the expression of cytokine receptors to induce activation of transforming signals in hematopoietic cells. Normally, JAK2 binds to homodimeric and heterodimeric cytokine receptors through specific receptor motifs and is activated by various structural changes induced by cytokine stimulation. Following activation it utilizes receptor tyrosines as substrates for phosphorylation, leading to recruitment of downstream signaling molecules, such as STAT5, among others. JAK2 then activates STAT5 via phosphorylation and STAT5 then translocates to the nucleus to regulate transcription of target genes. JAK2-V617F does not require ligand for activation, but still requires the scaffolding function of cytokine receptors to facilitate its full activation and activation of downstream signaling via phosphorylation. Lipid rafts are microdomains of the plasma membrane that are enriched in cholesterol and sphingolipids. They have gained appreciation in signal transduction as sites of localization of signaling mediators, including membrane-bound receptors. Congregation of signaling proteins in lipid rafts within the plasma membrane promotes complex formation and signaling cascade activation. We have recently demonstrated that JAK2 is present in lipid rafts during erythropoietin signaling and that lipid raft integrity is required for erythropoietin-mediated signal transduction (Blood 2009, 114: 292). In our current study, we demonstrate that constitutive JAK-STAT signaling driven by JAK2-V617F is sensitive to lipid raft disruption. Human erythroleukemia (HEL) cells express constitutive activation of JAK-STAT signaling due to the presence of JAK2-V617F. Treatment of these cells with methyl-beta-cyclodextrin to disrupt lipid rafts abolished JAK2, STAT5, and STAT3 activation. Similar results are obtained in other cell lines harboring JAK2-V617F and that exhibit JAK-STAT activation that is dependent on this activated form of JAK2. We also demonstrate that JAK2-V617F co-localizes with lipid rafts, as shown by immunofluorescence, and that this co-localization is abolished by lipid raft disruption. This suggests the loss of JAK2-V617F-mediated JAK-STAT activation we observe following lipid raft disruption may be due to an inhibition of properly localized protein complex formation in the plasma membrane that is necessary for JAK2-V617F signaling. Lipid rafts may provide a site for an accumulation of JAK2-V617F-containing signaling complexes and may be necessary for the cellular signals initiated by JAK2-V617F. Our data show JAK2-V617F-driven JAK-STAT pathway activation is vulnerable to lipid raft disrupting agents and suggest lipid raft integrity as a potential therapeutic target for JAK2-V617F positive neoplasms. Targeting lipid rafts in combination with JAK2 kinase inhibitors may allow for more effective kinase inhibition at lower doses, potentially decreasing undesirable side effects associated with kinase inhibitor treatment. Disclosures:No relevant conflicts of interest to declare.

Molecular basis of cytokine receptor activation

Cytokines are secreted soluble peptides that precisely regulate multiple cellular functions. Amongst these the GM-CSF/IL-3/IL-5 family of cytokines controls whether hematopoietic cells will survive or apoptose, proliferate, differentiate, migrate, or perform effector functions such as phagocytosis or reactive oxygen species release. Their potent and pleiotropic activities are mediated through binding to high affinity membrane receptors at surprisingly low numbers per cell. Receptor binding triggers a cascade of intracellular signaling events, including reversible phosphorylation of receptor subunits and associated signaling molecules, leading to multiple biological responses, with the prevention of apoptosis or "cell survival" being a key cellular function that underpins all others. Many chronic inflammatory diseases and a number of haematological malignancies are driven by deregulated GM-CSF, IL-3, or IL-5 cytokine receptor signaling, highlighting their importance in disease. A major step in understanding how these cytokine receptors function is to elucidate their three dimensional structure and to relate this to the many signaling pathways emanating from their receptors. We have recently solved the structure of the human GM-CSF receptor complexed to GM-CSF which revealed distinct forms of receptor assembly: a hexamer that comprises two molecules each of GM-CSF, GM-CSF receptor alpha chain and GM-CSF receptor beta chain; and an unexpected dodecamer in which two hexameric complexes associate through a novel site 4. This latter form is necessary to bring JAK2 molecules sufficiently close together to enable full receptor activation. In this review we focus on the most recent insights in cytokine receptor signaling, and in receptor assembly. The stage is now set to link distinct forms of cytokine receptor assembled structures to specific forms of cytokine receptor signaling and function. Armed with this knowledge it may be possible to map distinct cytokine receptor signaling pathways from the cell surface to the cell nucleus which may themselves become new therapeutic targets.

Histone Deacetylase Inhibitors In Inflammatory Disease

Lysine acetylation is becoming increasingly appreciated as a key post-translational modification in the endogenous regulation of protein function. The so-called histone acetyl transferases (HATs) and histone deacetylases (HDACs), best known for their roles in controlling chromatin remodeling via histone acetylation/deacetylation, are now known to modify a large number of non-histone proteins to control diverse cell processes. In relation to inflammation, acetylation modulates the activity or function of cytokine receptors, nuclear hormone receptors, intracellular signaling molecules and transcription factors. Small molecule inhibitors of HDACs have been found to trigger both pro- and anti-inflammatory effects in a range of inflammation-relevant cell types. Although their inflammatory profiles have only just begun to be elucidated, some HDAC inhibitors are already showing therapeutic promise in animal models of inflammatory diseases such as arthritis, inflammatory bowel diseases, septic shock, ischemia-reperfusion injury, airways inflammation and asthma, diabetes, age-related macular degeneration, cardiovascular diseases, multiple sclerosis and other CNS and neurodegenerative diseases. This article describes those HDAC inhibitors which have been most examined to date for their potentially beneficial effects on inflammatory cells or in animal models of inflammatory disease.

Functional Determination of the Interaction Interface Between the Kinase and Pseudokinase Domains of JAK2 V617F

The Janus kinases (JAKs) are cytosolic protein tyrosine kinasess mediating cytokine receptor function essential for blood formation and the immune response. The V617F mutation in the pseudokinase domain of JAK2 is present in over 95% of Polycythemia Vera patients and in 50% of Essential Thrombocythemia and Primary Myelofibrosis patients. The V617F mutation leads to constitutive kinase activity, which activates downstream STAT5, STAT3, MAP-kinase and phosphatidylinositol-3-kinase pathways. In the absence of a complete three-dimensional structure of JAK2, the structural mechanism of activation by the V617F mutation has remained elusive. Position 617 of JAK2 is located within one of the two predicted interfaces between the pseudokinase and the kinase domains of JAK2. We have shown that large hydrophobic residues such as Trp, Leu, Ile and Met also activate JAK2 when substituted for Val at position 617. These V617 mutations might either remove an inhibitory interaction exerted by the pseudokinase domain or stabilize an active conformation of the kinase domain. To investigate the nature of the conformational change promoted by the V617 mutations, we employed cysteine-scanning mutagenesis of residues in the activation loop of JAK2, and their predicted interaction partners on the pseudokinase side, and characterized their constitutive and ligand-dependent activity in proliferation and dual luciferase assays. The solved structure of the JAK2 kinase domain in its active form places residues Q1003, D1004 and K1005 on the tip of the activation loop. These residues were individually mutated to cysteine and stably expressed in murine pro-B Ba/F3 cells expressing the erythropoietin receptor. We hypothesized that cysteine residues placed in the activation loop might promote interactions with the pseudokinase domain or form disulfide bonds with naturally occurring Cys616 and Cys618 of the pseudokinase domain, if, as predicted, close proximity would occur between activation loop residues and the pseudokinase domain loop beta4-beta5 that contains V617F. GFPsorted cells expressing the K1005C mutant were able to proliferate in a medium lacking cytokines, similar to cells expressing V617F, but to a lower degree. This proliferation advantage could be explained by the formation of a disulfide bond between C1005 and C618 of JAK2, or by a conformational change induced by placing the smaller Cys residue at position 1005, which would lock the activation loop in an active conformation. Substitution of V617 to Cys abolished the constitutive signaling by JAK2 K1005C, and decreased the weak activity of JAK2 V617C, suggesting that the active conformations of V617 mutants and K1005C are different. Cysteine mutants at positions 1003 or 1004 could not synergize with V617C or induce constitutive activation. We also investigated whether JAK2 V617F activation simply required dimerization, or rather a particular dimer orientation of EpoR cytosolic domains. We took advantage of fusion proteins where a 28 amino acid coiled coil was used to replace the extracellular domain of EpoR in order to impose discrete dimeric orientations to the transmembrane and cytosolic domains of the EpoR. Two out of the seven fusion proteins were constitutively active when co-expressed with WT JAK2 in STAT5-dependent luciferase assays. In contrast, all dimers were active in the presence of JAK2 V617F, suggesting that dimerization and not a particular dimeric orientation promotes JAK2 V617F activation.

Functional IL-12 and IFNg receptors, and isolated low IgM, in an eight-year-old boy with disseminated nontuberculous mycobacterial infection otherwise consistent with the phenotype of IL-12 and IFNg receptor deficiency

RATIONALE: Children with systemic nontuberculous mycobacterial infections are described with absent or defective IFNg or IL-12 receptors, and conversely there are no reports of patients with this phenotype who do express these receptors; as such we assayed for receptor presence and functionality in a patient with progressive disseminated nontuberculous mycobacterial infection and unidentified inflammatory skin lesions, and report novel results.METHODS: Laboratory analysis of patient skin and blood samples included leukocyte phenotyping, cytokine response, cellular signaling, phosphorylation, and PCR genotyping studies over multiple time periods.RESULTS: IL-12 and IFNg receptor are both present and responsive to LPS and IFNg, but not to PHA. Phosphorylation of Stat 4 occurred with IL-12, but no downstream IFNg production could be detected. Decreases observed in all lymphocyte types with an isolated defect in IgM only, with normal levels of IgG, IgA, and IgE.CONCLUSIONS: The IL-12 and IFNg receptor defect phenotype can occur without the absence of either receptor's expression or function, and this may represent an as yet undescribed disease process involving novel mechanisms of antibody expression as well as cytokine receptor function. RATIONALE: Children with systemic nontuberculous mycobacterial infections are described with absent or defective IFNg or IL-12 receptors, and conversely there are no reports of patients with this phenotype who do express these receptors; as such we assayed for receptor presence and functionality in a patient with progressive disseminated nontuberculous mycobacterial infection and unidentified inflammatory skin lesions, and report novel results. METHODS: Laboratory analysis of patient skin and blood samples included leukocyte phenotyping, cytokine response, cellular signaling, phosphorylation, and PCR genotyping studies over multiple time periods. RESULTS: IL-12 and IFNg receptor are both present and responsive to LPS and IFNg, but not to PHA. Phosphorylation of Stat 4 occurred with IL-12, but no downstream IFNg production could be detected. Decreases observed in all lymphocyte types with an isolated defect in IgM only, with normal levels of IgG, IgA, and IgE. CONCLUSIONS: The IL-12 and IFNg receptor defect phenotype can occur without the absence of either receptor's expression or function, and this may represent an as yet undescribed disease process involving novel mechanisms of antibody expression as well as cytokine receptor function.

Gp130 cytokine signaling in the pituitary gland: a paradigm for cytokine-neuro-endocrine pathways.

Unlike most other classes of receptors, where the cytoplasmic domain is necessary for signal transduction, receptors for IL-6 and other related cytokines signal solely by virtue of their ability to form a larger receptor complex with a common subunit, the transmembrane glycoprotein gp130 (1). For instance, binding of IL-6 to IL-6Rα, the α subunit of the functional receptor, triggers the association of IL-6Rα with gp130 (1, 2). Indeed, even a soluble form of IL-6Rα, entirely lacking the intracellular region of the protein, can bind to its ligand and to surface-expressed gp130, producing a normal IL-6 signal (1). The ligands of these various receptors are therefore referred to as the gp130 cytokine receptor family. As summarized in Figure ​Figure1,1, gp130 functions as a common cytokine signal transducer for IL-6, leukemia inhibitory factor (LIF), IL-11, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), and cardiotropin-1 (CT-1), all of which bind specific receptors (their α subunits) but use the gp130 protein as the initial cellular signal transducer (2, 3). Because of this broad physiological role, the term gp130 is commonly used, in addition to its official human genome organization (HUGO) designation as the IL-6 signal transducer, or IL6ST. Figure 1 Schematic model of the IL-6/gp130 receptor system. The specific cytokine-binding subunits and gp130 belong to a cytokine receptor superfamily characterized by four positionally conserved cysteine residues and a WSXWS motif. Functional receptor complexes ... The human gp130 consists of an extracellular domain of 597 amino acids, with a single transmembrane domain of 22 amino acids, and a cytoplasmic domain of 277 amino acids. As with other hemopoietic cytokine receptors, gp130 contains a cytokine receptor homology region that includes fibronectin type III domains as well as four positionally conserved cysteine residues and a WSXWS motif (1, 3) (Figure ​(Figure1).1). While the expression of the gp130 cDNA alone does not confer the binding of IL-6 or the other family cytokines, gp130 and IL-6Ra together form a high-affinity IL-6 binding site. In the mouse, gp130 is ubiquitously expressed in adult organs, as well as in embryonic stem cells and in embryos as early as day 6 of gestation (3). Expression of gp130 therefore does not parallel that of the α subunit of any of the receptors of the cytokine family, nor of any specific cytokine of the IL-6 family. These factors show some functional redundancy in the immune, hematopoietic, nervous, and neuro-endocrine systems. For example, macrophage differentiation, expression of acute-phase proteins by hepatocytes, and neuronal survival and differentiation can all be induced by multiple gp130 cytokines. Conversely, as is extensively reviewed in refs. 3–5, these cytokines also exhibit specific biological activities. Considering that gp130 is ubiquitously expressed, the time and place at which gp130 functions in vivo appears to be determined by spatially and temporally regulated expression of specific cytokine-binding receptor chains or of the cytokines themselves. In addition, soluble gp130, probably translated from an alternative spliced mRNA, can neutralize receptor signaling complexes, thereby acting as an antagonist (6). Below, I discuss the regulation of cytokine and cytokine receptor function in the pituitary gland and its importance for neuro-endocrine–stress responses.

Tissue Factor Signaling

Tissue Factor Signaling

Crystal structure of a cytokine-binding region of gp130.

The structure of the cytokine-binding homology region of the cell surface receptor gp130 has been determined by X-ray crystallography at 2.0 A resolution. The beta sandwich structure of the two domains conforms to the topology of the cytokine receptor superfamily. This first structure of an uncomplexed receptor exhibits a similar L-shaped quaternary structure to that of ligand-bound family members and suggests a limited flexibility in relative domain orientation of some 3 degrees. The putative ligand-binding loops are relatively rigid, with a phenylalanine side chain similarly positioned to exposed aromatic residues implicated in ligand binding for other such receptors. The positioning and structure of the N-terminal portion of the polypeptide chain have implications for the structure and function of cytokine receptors, such as gp130, which contain an additional N-terminal immunoglobulin-like domain.

Cytokines in Hematopoiesis: Specificity and Redundancy in Receptor Function

This chapter outlines the role of cytokine receptor signaling in the regulation and maturation of myeloid cells: erythrocytes, macrophages, granulocytes, megakaryocytes, and eosinophils. Some cytokine receptors are involved in unique and lineage-specific effects, whereas others appear to have multiple, overlapping, or redundant effects. The biochemical events following receptor activation are identified. Each receptor activates a number of signaling molecules, and in many cases the same molecules are activated by a number of different receptors. Experiments with mutant mice demonstrate that growth factors with overlapping cellular functions nevertheless do have specific unique roles under certain circumstances. The chapter also describes experimental models available for the study, the principal ways in which cytokine receptors are thought to contribute to hematopoietic cell differentiation and survival, and the pathological effects of aberrant cytokine receptor function. Alterations in cytokine receptor structure and function can induce severe pathologies, including immunodeficiency, severe congenital neutropenia and leukemia, dwarfism, or obesity.

Cytokine Receptors: Structures and Signal Transduction

The characteristic features of cytokines are functional pleiotropy and redundancy. Each cytokine is produced by a variety of cell types and acts on a wide range of target cells and tissues. Many cytokines have overlapping biological activities in the same cells. It was originally thought that each cytokine has a specific receptor and a unique signal transduction system. However, extensive studies on cytokines and their receptors revealed that many cytokines share receptor subunits and signal transduction system, and that biological functions of a single cytokine can vary depending on the status of the cells. Therefore, it is important to know the structure and function of cytokine receptors to understand the pleiotropy and redundancy as well as specificity of cytokines. Among signal transduction pathways, recently identified Jak/STAT pathway, which connects activation of the receptor complexes and transcription of various genes directly, would give us further insights in the mechanisms of cytokine action.

Crossregulation between Th1 and Th2 cells.

Th1 and Th2 subsets have been characterized on the basis of the cytokines they secrete and the immune functions they mediate. Th1 cells secrete IL-2, IFN-gamma, and lymphotoxin and are important in the cell-mediated response; Th2 cells secrete IL-4, IL-5, IL-10, and IL-13 and are important in the control of macrophage function and in the stimulation of particular immunoglobulin isotypes. Cytokines secreted by Th1 and Th2 cells regulate the growth and differentiation of Th1 and Th2 cells in both positive and negative ways; this has been termed crossregulation. Much work has concentrated on the factors important in the differentiation of these Th subsets, and it has been established that Th cells become committed to a Th1 or Th2 phenotype within 48 hrs of antigenic stimulation. During the differentiation process irreversible changes in the expression and function of cytokine receptors occur that provide an explanation for the observed crossregulatory features of Th1 and Th2 cells. In this review we summarize the crossregulation between Th1 and Th2 cells in terms of the changes in cytokine receptor expression and function that occur during differentiation.

Signaling by IL-2 and related cytokines: JAKs, STATs, and relationship to immunodeficiency.

Cytokines that bind to the interleukin-2 (IL-2) receptor common gamma chain (gamma c), including IL-2, IL-4, IL-7, IL-9, and IL-15, are important for the growth and differentiation of T and B lymphocytes, natural killer cells, macrophages, and monocytes. These cytokines have overlapping biological effects that in part result from the use of the shared receptor subunit gamma c. Recently it has become clear that these cytokines activate a number of important intracellular signaling molecules, including the Janus kinases JAK1 and JAK3 and members of the transcription factor family of signal transducers and activators of transcription (STATs). The discovery of these signaling pathways has led to important new insights into their role in lymphocyte maturation, as it has emerged that mutations in the genes encoding both gamma c and JAK3 result in similar forms of severe combined immunodeficiency (SCID). In this review we examine the structure and function of cytokine receptors and the signaling pathways involved in their regulation of gene expression. Furthermore, we discuss recent advances that have led to a better understanding of how cytokines elicit intracellular responses, as well as their role in normal lymphoid development.

Inhibition of cellular responsiveness to interferon-gamma (IFN gamma) induced by overexpression of inactive forms of the IFN gamma receptor

Herein, we report that overexpression of either human or murine interferon-gamma (IFN-gamma) receptors lacking their entire intracellular domains in cells bearing functionally active IFN gamma receptors ablates responsiveness to homologous ligand in a dominant negative manner. Unresponsiveness could also be induced in murine cells overexpressing murine IFN gamma receptor mutants that either lack 39 COOH-terminal amino acids or contain an alanine substitution for a functionally critical tyrosine. Overexpression of the full-length receptor did not alter cellular responsiveness to IFN gamma. The inhibitory activities of the receptor mutants were dose-dependent, generalizable to a variety of cellular responses, and specific. Cells expressing 100:1 ratios of mutant to wild-type receptor were unresponsive to IFN gamma even at doses 30,000 times greater than that required to induce a maximal response in wild-type cells. These results provide an example of a dominant negative mutation that effects the complete inactivation of a transmembrane receptor lacking a kinase domain and suggest a more general utility for dominant negative mutations in the study of cytokine receptor function.

Tumor necrosis factor receptor signaling. A dominant negative mutation suppresses the activation of the 55-kDa tumor necrosis factor receptor.

To investigate the signaling mechanism of the 55-kDa tumor necrosis factor (TNF) receptor a functional transfection based assay was developed. The human 55-kDa TNF receptor, stably expressed in mouse L929 cells, was demonstrated to be activated specifically by agonist antibodies and to initiate a signal for cellular cytotoxicity. A deletion mutant of the human TNF receptor lacking most of the cytoplasmic domain was found to be completely defective in generating the signal for cytotoxicity. Additionally, expression of the truncated receptor substantially suppressed signaling by endogenous mouse TNF receptors in response to TNF, but not in response to specific anti-murine TNF receptor antibodies. These results suggest that aggregation of 55-kDa TNF receptor intracellular domains, which are not associated in the absence of ligand, is an important component of the signal for cellular toxicity. This work also provides an example of a dominant negative mutation in a transmembrane receptor that lacks a tyrosine kinase domain, and suggests a more general utility of dominant negative mutations in the investigation of cytokine receptor function.