Biomedical subjects

D V Goeddel

Publications and source records attributed to D V Goeddel.

At least 19 recordsLinked to original sources

PubMed1992

Antiviral activity of tumor necrosis factor is signaled through the 55-kDa type I TNF receptor [corrected].

Agonist antibodies (Ab) to the two TNF receptors, TNF-R1 (55 kDa) and TNF-R2 (75 kDa), have been shown to signal many of the distinct functions induced by TNF-alpha. We have found that anti-TNF-R1, but not anti-TNF-R2, Ab trigger antiviral activity in human hepatoma Hep-G2 cells and enhance the antiviral activity of IFN-gamma in human lung fibroblast A549 cells. Likewise, anti-human-TNF-R1 Ab had antiviral enhancing activity on murine L929 cells engineered to express human TNF-R1. However, L929 cells that express human TNF-R1 lacking most of the intracellular domain fail to respond to anti-human-TNF-R1 Ab. This demonstrates that the intracellular domain of TNF-R1 is necessary to generate antiviral activity. TNF-R1 but not TNF-R2 also signals killing of virus-infected cells by TNF-alpha. Thus, all the known antiviral activities of TNF-alpha are mediated through TNF-R1.

Animals↗

PubMed1992

Biochemical properties of the 75-kDa tumor necrosis factor receptor. Characterization of ligand binding, internalization, and receptor phosphorylation.

An expression plasmid encoding the human 75-kDa tumor necrosis factor (TNF) type 2 receptor (TNF-R2) was constructed and used to generate a stable human cell line (293/TNF-R2) overexpressing TNF-R2. Ligand binding analysis revealed high affinity binding (Kd = 0.2 nM) with approximately 94,000 +/- 7,500 sites/cell for 125I-TNF-alpha and approximately 5-fold lower affinity for TNF-beta (Kd = 1.1 nM) with 264,000 +/- 2,000 sites/cell. Cross-linking of 125I-TNF-alpha and 125I-TNF-beta to 293/TNF-R2 cells yielded predominant complexes with apparent molecular weights of 211,000 for TNF-alpha and 205,000 and 244,000 for TNF-beta, suggesting these complexes contain two or three TNF-R2 molecules. Immunoprecipitation of TNF-R2 from 32P-labeled 293/TNF-R2 cells demonstrated that the receptor is phosphorylated. The majority (97%) of 32Pi incorporation was found in serine residues with a very low level of incorporation (3%) in threonine residues. TNF-alpha treatment of 293/TNF-R2 cells did not significantly affect the degree or pattern of phosphorylation. Cell surface-bound 125I-TNF-alpha was slowly internalized by the 293/TNF-R2 cell line with a t1/2 = 25 min. Shedding of the extracellular domain of TNF-R2 was induced by 4 beta-phorbol 12-myristate 13-acetate but not by TNF-alpha or TNF-beta.

Chromatography, Affinity↗

PubMed1992

Precursor structure, expression, and tissue distribution of human guanylin.

Heat-stable enterotoxins (STa) are small, cysteine-rich peptides secreted by Escherichia coli that are able to induce diarrhea through the stimulation of an intestine-specific receptor-guanylyl cyclase known as STaR. A 15-amino acid peptide, guanylin, was recently purified from rat jejunum and proposed to be a potential endogenous activator of this receptor. We describe here the cloning and characterization of human and mouse cDNAs encoding precursor proteins of 115 and 116 amino acids, respectively, having guanylin present at their C termini. Expression of the human cDNA in mammalian cells leads to the secretion of proguanylin, an inactive 94-amino acid protein. Guanylin generated by either trypsin or acid treatment of proguanylin was purified and found to bind to, and activate, STaR. Northern blot and in situ hybridization show high-level expression of guanylin mRNA restricted to the intestine, with localization to Paneth cells at the base of the small intestinal crypts. These results demonstrate that guanylin is an endogenous activator of STaR.

Amino Acid Sequence↗

PubMed1992

Rational design of potent antagonists to the human growth hormone receptor.

A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially--a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.

Antibodies, Monoclonal↗

PubMed1992

Random PCR mutagenesis screening of secreted proteins by direct expression in mammalian cells.

We have developed a general method for screening randomly mutagenized expression libraries in mammalian cells by using fluorescence-activated cell sorting (FACS). The cDNA sequence of a secreted protein is randomly mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA is inserted into an expression vector encoding the membrane glycophospholipid anchor sequence of decay-accelerating factor (DAF) fused to the C terminus of the secreted protein. This results in expression of the protein on the cell surface in transiently transfected mammalian cells, which can then be screened by FACS. This method was used to isolate mutants in the kringle 1 (K1) domain of tissue plasminogen activator (t-PA) that would no longer be recognized by a specific monoclonal antibody (mAb387) that inhibits binding of t-PA to its clearance receptor. DNA sequence analysis of the mutants and localization of the mutated residues on a three-dimensional model of the K1 domain identified three key discontinuous amino acid residues that are essential for mAb387 binding. Mutants with changes in any of these three residues were found to have reduced binding to the t-PA receptor on human hepatoma HepG2 cells but to retain full clot lysis activity.

Amino Acid Sequence↗

PubMed1992

Tumor necrosis factor-alpha pretreatment is protective in a rat model of myocardial ischemia-reperfusion injury.

We have demonstrated that tumor necrosis factor-alpha (TNF-alpha) pretreatment protected the rat heart from ischemia-reperfusion injury. This effect was monitored by assaying for lactate dehydrogenase (LDH), an enzyme whose release correlates with loss of cell membrane integrity. Intact hearts removed from rats pretreated with TNF-released significantly lower amounts of LDH compared to control hearts after 20 min. of total global ischemia followed by reperfusion. Hearts from TNF-alpha-pretreated animals contained higher levels of manganous superoxide dismutase (MnSOD) mRNA than hearts from untreated rats. Because oxygen free radicals have been implicated as a major cause of reperfusion damage and the function of MnSOD is to detoxify superoxide anions in the mitochondria, a possible protective mechanism for TNF-alpha may be to induce expression of MnSOD in the heart and thus confer resistance to oxygen free radicals generated during reperfusion.

Actins↗

PubMed1992

Characterization of the recombinant human receptor for Escherichia coli heat-stable enterotoxin.

We report here the molecular characterization of a recombinant cell line (293-STaR) expressing the heat-stable enterotoxin receptor (STaR) from human intestine. We have compared the 293-STaR cell line with the human colonic cell line T84 that endogenously expresses STa binding sites. Scatchard analysis of displacement binding studies revealed a single STa binding site with an affinity (Ki) of 97 pM in 293-STaR compared with 55 pM in T84 cells. Saturation isotherms of STa binding gave a Kd of 94 pM for the cloned receptor expressed in 293 cells and 166 pM for the receptor present in T84 cells. Kinetic measurements of STa binding to 293-STaR gave an association rate constant, K1, of 2.4 x 10(8) M-1 min-1 and a dissociation rate constant, K2, of 0.016 min-1. The half-time of dissociation was 43 min, and the Kd calculated from the ratio of the kinetic constants was 67 pM. The pH profile of STa binding showed that the number of STa binding sites is increased 3-fold at pH 4.0 compared with pH 7.0, with no effect on binding affinity. A polyclonal antibody directed against the extracellular domain of STaR immunoprecipitated two proteins of approximately 140 and 160 kDa from both 293-STaR and T84 cells. Cross-linking of 125I-STa to 293-STaR cells resulted in the labeling of proteins with a molecular mass of approximately 153, 133, 81, 68, 56, and 49 kDa, the two smallest being the more abundant. Similar results have been reported for the STaR present on rat brush border membranes. These data suggest that the STaR-guanylyl cyclase identified by molecular cloning is the only receptor for STa present in T84 cells.

Bacterial Toxins↗

PubMed1992

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.

Animals↗

PubMed1992

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

Uncoupling of cytokine mRNA expression and protein secretion during the induction phase of T cell anergy.

The CD4+ T cell clone HA1.7 may be made specifically nonresponsive, or anergic, to its cognate Ag, an influenza hemagglutinin peptide (HA), by pretreatment with the superantigen Staphylococcus aureus enterotoxin B or with high concentrations of HA itself. We compare the patterns of mRNA expression and protein production of selected T cell cytokines during the first 24 h after treatments that induce anergy in HA1.7 and during the same period after treatments that simulate normal cellular activation. The cytokines examined include TNF-alpha, IL-8/neutrophil activating protein-1 and the RANTES/SIS cytokines, a family of small secreted proteins with inflammatory and potential antiproliferative and leukocyte regulating activities. Messenger RNA for TNF-alpha, human MIP-1 alpha, human MIP-1 beta, and IL-8 are all induced during the development of clonal anergy in HA1.7, and these levels are significantly higher than those seen during activation of the clone using an anti-CD3 antibody and IL-2. These high levels of mRNA also persist longer than those seen after anti-CD3 and IL-2 activation. However, the increased levels of mRNA are not typically accompanied by increased protein secretion. In all cases but one, the amount of cytokine secreted by HA1.7 cells was greater after anti-CD3 and IL-2 treatments than after anergy-inducing treatments. Thus, the induction of T cell anergy in HA1.7 does not appear to require a general inhibition of T cell cytokine mRNA expression, and, in fact, anergy treatments appear to superinduce certain cytokine transcripts, but anergy-specific posttranscriptional mechanisms may exist by which cytokine release is regulated.

Animals↗

PubMed1992

Two TNF receptors.

In the past few years, considerable progress has been made on the identification and characterization of tumor necrosis factor (TNF) receptors. The relative roles played by the two receptor types in signaling the diverse functions of TNF are less clear. Here, Louis Tartaglia and David Goeddel summarize progress to date and propose a model of TNF receptor triggering that reconciles the seemingly conflicting data.

Animals↗

PubMed1992

Molecular cloning of a retina-specific membrane guanylyl cyclase.

We have isolated and characterized cDNA clones encoding the human retinal guanylyl cyclase (retGC), a novel member of the membrane guanylyl cyclase gene family. Like other membrane guanylyl cyclases, the 1101 aa retGC is predicted to have a hydrophobic amino-terminal signal sequence followed by a large extracellular domain, a single membrane spanning domain, a kinase homology domain, and a guanylyl cyclase catalytic domain. In contrast to other membrane guanylyl cyclases, such as natriuretic peptide receptors, retGC has a relatively high basal level of activity when expressed in human 293 cells. cGMP production by retGC is unaffected by any of the known natriuretic peptides. In situ hybridization analysis of a variety of rhesus monkey tissues showed retGC transcripts to be localized exclusively along the retinal outer nuclear layer, corresponding to the nuclei of the rod and cone photoreceptor cells. Our results suggest that retGC may synthesize cGMP required for recovery of the dark state after phototransduction.

Amino Acid Sequence↗

PubMed1992

Conservation of the kinaselike regulatory domain is essential for activation of the natriuretic peptide receptor guanylyl cyclases.

The natriuretic peptide receptors, NPR-A and NPR-B, are two members of the newly described class of receptor guanylyl cyclases. The kinaselike domain of these proteins is an important regulator of the guanylyl cyclase activity. To begin to understand the molecular nature of this type of regulation, we made complete and partial deletions of the kinase domain in NPR-A and NPR-B. We also made chimeric proteins in which the kinase domains of NPR-A and NPR-B were exchanged or replaced with kinase domains from structurally similar proteins. Complete deletion of the kinase homology domain in NPR-A and NPR-B resulted in constitutive activation of the guanylyl cyclase. Various partial deletions of this region produced proteins that had no ability to activate the enzyme with or without hormone stimulation. The kinase homology domain can be exchanged between the two subtypes with no effect on regulation. However, structurally similar kinaselike domains, such as from the epidermal growth factor receptor or from the heat-stable enterotoxin receptor, another member of the receptor guanylyl cyclase family, were not able to regulate the guanylyl cyclase activity correctly. These findings suggest that the kinaselike domain of NPR-A and NPR-B requires strict sequence conservation to maintain proper regulation of their guanylyl cyclase activity.

Amino Acid Sequence↗

PubMed1992

Molecular biology of the natriuretic peptides and their receptors.

After the description in the past 5 years of BNP and CNP, interest in the natriuretic peptide family has dramatically increased. Molecular characterization of the receptors for this hormone family has identified a heterogeneity in the receptor subtypes not previously alluded to by pharmacological or biochemical studies. Much has been published on the physiology of ANP, but the major roles for BNP and CNP remain to be elucidated. Some experiments indicate that ANP and BNP may act synergistically, especially during cardiac stress; however, the high level of structural diversity of BNP among species and the ability of porcine BNP, but not human BNP, to activate human NPR-B suggest that an as yet unidentified receptor may exist that specifically recognizes BNP. Localization studies have implied that CNP is the most prominent neuropeptide in the natriuretic peptide family, and the restriction of its receptor, NPR-B, to the nervous system suggests that CNP and NPR-B may act in the brain to coordinate the central aspects of body fluid homeostasis. Of the three known NPRs, two, NPR-A and NPR-B, are capable of synthesizing their own second messenger, cGMP. The domain within these receptors that has high homology to protein kinases has been demonstrated to be essential for regulating this activity. No kinase activity has been measured in these proteins, but it is possible that this region is important for ATP regulation of guanylyl cyclase activity. This possibility raises interesting parallels with receptor-mediated cAMP signaling within cells. Seven transmembrane receptors, once activated by ligand, associate with G proteins to affect the activity of adenylyl cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Protection against endotoxic shock by a tumor necrosis factor receptor immunoadhesin.

Tumor necrosis factors (TNF) alpha and beta are structurally related cytokines that mediate a wide range of immunological, inflammatory, and cytotoxic effects. During bacterial infection of the bloodstream (sepsis), TNF-alpha induction by bacterial endotoxin is thought to be a major factor contributing to the cardiovascular collapse and critical organ failure that can develop. Despite antibiotic therapy, these consequences of sepsis continue to have a high mortality rate in humans. Here we describe a potent TNF antagonist, a TNF receptor (TNFR) immunoadhesin, constructed by gene fusion of the extracellular portion of human type 1 TNFR with the constant domains of human IgG heavy chain (TNFR-IgG). When expressed in transfected human cells, TNFR-IgG is secreted as a disulfide-bonded homodimer. Purified TNFR-IgG binds to both TNF-alpha and TNF-beta and exhibits 6- to 8-fold higher affinity for TNF-alpha than cell surface or soluble TNF receptors. In vitro, TNFR-IgG blocks completely the cytolytic effect of TNF-alpha or TNF-beta on actinomycin D-treated cells and is markedly more efficient than soluble TNFR (24-fold) or monoclonal anti-TNF-alpha antibodies (4-fold) in inhibiting TNF-alpha. In vitro, TNFR-IgG prevents endotoxin-induced lethality in mice when given 0.5 hr prior to endotoxin and provides significant protection when given up to 1 hr after endotoxin challenge. These results confirm the importance of TNF-alpha in the pathogenesis of septic shock and suggest a clinical potential for TNFR-IgG as a preventive and therapeutic treatment in sepsis.

Animals↗

PubMed1991

The two different receptors for tumor necrosis factor mediate distinct cellular responses.

The individual roles of the murine type 1 and type 2 tumor necrosis factor (TNF) receptors (TNF-R1 and TNF-R2) were investigated utilizing (i) the strong species specificity of TNF-R2 for murine TNF compared to human TNF and (ii) agonistic rabbit polyclonal antibodies directed against the individual TNF receptors. Proliferation of mouse thymocytes and the murine cytotoxic T-cell line CT-6 is stimulated by murine TNF but not by human TNF. Consistent with this observation, polyclonal antibodies directed against TNF-R2 induced proliferation in both of these cell types, whereas polyclonal antibodies directed against TNF-R1 had no effect. In contrast, cytotoxicity in murine LM cells (which are sensitive to murine and human TNF) was induced by antibodies against TNF-R1 but not by antibodies against TNF-R2. Also, the steady-state level of manganous superoxide dismutase mRNA in the murine NIH 3T3 cell line was induced by murine TNF, human TNF, and anti-TNF-R1 but not by anti-TNF-R2. These results suggest that TNF-R2 initiates signals for the proliferation of thymocytes and cytotoxic T cells, whereas TNF-R1 initiates signals for cytotoxicity and the induction of the protective activity, manganous superoxide dismutase. The nonredundant signaling observed for the two TNF receptors cannot be explained simply by the differential expression of the two TNF receptors in the various cell types, because LM cells express on their surface higher levels of TNF-R2 than TNF-R1, and LM cells, NIH 3T3 cells, and thymus cells all express mRNA corresponding to both receptor types. It is therefore likely that the two receptors initiate distinct signaling pathways that result in the induction of different cellular responses.

3T3 Cells↗

PubMed1991

Primary structure and functional expression of the human receptor for Escherichia coli heat-stable enterotoxin.

Heat-stable enterotoxin (STa) produced by Escherichia coli induces intestinal secretion in mammals by binding to the brush border membrane of the small intestine and activating guanylyl cyclase. We report here the cloning and expression of a cDNA encoding the human receptor for STa. The receptor contains both an extracellular ligand binding site and a cytoplasmic guanylyl cyclase catalytic domain, making it a member of the same receptor family as the natriuretic peptide receptors. Stable mammalian cell lines over-expressing the STa receptor specifically bind 125I-STa (Kd approximately 1.0 nM) and respond to STa by dramatically increasing (approximately 50-fold) cellular cGMP levels. Sequence comparisons between the human and the rat STa receptors show less conservation in the extracellular domain than similar comparisons of natriuretic peptide receptors. This divergence may indicate important species differences in ligand-receptor interaction.

Amino Acid Sequence↗

PubMed1991

Tumor necrosis factor alpha selectively sensitizes human immunodeficiency virus-infected cells to heat and radiation.

We report here that infection of the human T-cell line HUT-78 with human immunodeficiency virus (HIV) increases its sensitivity to heat and radiation toxicity. A possible explanation for this result may be the reduced expression of manganous superoxide dismutase (MnSOD) in HIV-infected cells compared to uninfected cells. Tumor necrosis factor alpha (TNF-alpha) further sensitizes HIV-infected cells but not uninfected cells to heat and radiation. This is consistent with the ability of TNF-alpha to induce the expression of MnSOD in uninfected but not in HIV-infected cells. HIV-infected HUT-78 cell lines engineered to overexpress MnSOD are more resistant to heat and radiation than HIV-infected cells that do not overexpress MnSOD. However, treatment with TNF-alpha still sensitizes these cells to heat and radiation.

Cell Line↗