PubMed Health⌕ Search

Biomedical subjects

D V Goeddel

Publications and source records attributed to D V Goeddel.

At least 55 records · Page 3Linked to original sources
PubMed1994

Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice.

Tumour necrosis factor (TNF) elicits multiple biological effects through two distinct cell surface receptors, TNF-R1 (p55) and TNF-R2 (p75). Most TNF-mediated biological responses, such as cell death, gene induction, antiviral activity and cytokine production, have been attributed to TNF-R1 (refs 1-5). Gene targeting of this receptor confirms its role in the lethality attributable to low doses of lipopolysaccharide after sensitization with D-galactosamine; surprisingly, the toxicity of high doses of lipopolysaccharide was unaffected. The function of TNF-R2 is less well understood, although there are data supporting a role in T-cell development and the proliferation of cytotoxic T lymphocytes. To clarify the physiological role of TNF-R2, we have generated mice deficient in this receptor by gene targeting. The TNF-R2-/- mice show normal T-cell development and activity, but we find that they have increased resistance to TNF-induced death. Additionally, such mice injected subcutaneously with TNF show a dramatic decrease in tissue necrosis, indicating that this receptor plays a role in the necrotic effects of TNF.

Animals↗
PubMed1994

Determination of the solution structure of the peptide hormone guanylin: observation of a novel form of topological stereoisomerism.

Guanylin is a 15 amino acid mammalian hormone containing two disulfide bonds. Guanylin shares sequence similarity with the bacterial heat-stable enterotoxin (STa) and is capable of binding to and stimulating the STa guanylyl cyclase receptor. Biologically active peptides have been prepared by two methods: (1) enzymatic treatment of a 99 residue proprotein (denoted proguanylin) expressed in Escherichia coli and (2) solid-phase chemical synthesis. Although both sources yield material that is pure by high-performance liquid chromatography and mass spectrometry, analysis by nuclear magnetic resonance (NMR) indicates that peptides from both sources contain two conformationally distinct species present in a 1:1 ratio. The chemical shift differences between the two species are large, allowing unambiguous sequential NMR assignments to be made for both sets of resonances. Exchange between the two forms was not observed even at 70 degrees C. Structural restraints have been generated from nuclear Overhauser effects and scalar coupling constants and used to calculate structures for both forms using distance geometry and restrained energy minimization. The resulting structures for the first isoform are well defined (root-mean-square deviation from the average structure for backbone atoms of 0.47 A) and adopt a right-handed spiral conformation, similar to that observed for heat stable enterotoxin. The second isoform is less well defined (root-mean-square deviation from the average structure for backbone atoms of 1.07 A) but clearly adopts a very different fold consisting of a left-hand spiral. The differences in structure suggest that the two forms may have very different affinities toward the STa receptor. The observation of such isomerism has important implications for the common practice of introducing multiple disulfide bonds into small peptides to limit conformational flexibility and enhance bioactivity.

Amino Acid Sequence↗
PubMed1994

Role of the 75-kDa tumor necrosis factor receptor: inhibition of early hematopoiesis.

Biological effects of tumor necrosis factor alpha (TNF-alpha) are mediated through two cell surface receptors, the 55-kDa TNF receptor and the 75-kDa TNF receptor. The present study investigated the relative roles of the two TNF receptors in normal hematopoiesis. Using agonists (antibodies) specific for the 55- and 75-kDa TNF receptors, we demonstrate differential roles of the two TNF receptors in hematopoiesis in that only the 55-kDa TNF receptor mediates antiproliferative effects of TNF-alpha on mature Lin- hematopoietic progenitor cells responding to granulocyte colony-stimulating factor or interleukin 3 alone. In contrast, the 75-kDa TNF receptor is essential in mediating inhibition of primitive Lin-Sca-1+ high-proliferative-potential colony-forming cells and inhibition of the total number of proliferative clones of individually cultured Lin-Sca-1+Rh123lo and Lin-Sca-1+Rh123hi cells.

Animals↗
PubMed1994

A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor.

Mutational analysis identified a C-terminal region of 78 amino acids within the cytoplasmic domain of the human 75 kDa tumor necrosis factor receptor (TNF-R2) that is required for signal transduction. This region was subsequently shown to mediate the interaction of cytoplasmic factors with TNF-R2. Two of these factors were isolated and molecularly cloned using biochemical purification and the yeast two-hybrid system. TNF receptor-associated factor 1 (TRAF1) and TRAF2 are the first two members of a novel protein family containing a novel C-terminal homology region, the TRAF domain. In addition, TRAF2 contains an N-terminal RING finger motif. TRAF1 and TRAF2 can form homo- and heterotypic dimers. Our analysis indicates that TRAF1 and TRAF2 are associated with the cytoplasmic domain of TNF-R2 in a heterodimeric complex in which TRAF2 contacts the receptor directly. TRAF1 interacts with TNF-R2 indirectly through heterodimer formation with TRAF2.

Amino Acid Sequence↗
PubMed1994

The interferon-gamma receptor extracellular domain. Non-identical requirements for ligand binding and signaling.

The human interferon-gamma receptor (hIFN-gamma R) extracellular domain interacts in a species-specific manner with both its ligand and an accessory factor encoded on human chromosome 21. Mutant interferon-gamma receptors were constructed by homolog-scanning mutagenesis, replacing segments of the human extracellular domain with the corresponding murine sequence. Replacement of hIFN-gamma R amino acids 1-100, 100-132, 134-183, or 183-245 abolished binding to human interferon-gamma (hIFN-gamma). However, replacement of hIFN-gamma R amino acids 134-209, 183-209, 134-153, 153-167, or 167-183 or deletion of residues 156-165 affected hIFN-gamma binding only partially or not at all. Receptors that bound hIFN-gamma were tested for their ability to signal a functional response, induction of major histocompatibility complex class I antigen expression. Replacement of residues 134-209 greatly reduced the ability of the receptor to signal. This signaling defect could not be attributed solely to a reduction in affinity for ligand and could not be localized to any subregion.

Animals↗
PubMed1994

Cloning and characterization of HTK, a novel transmembrane tyrosine kinase of the EPH subfamily.

Using a polymerase chain reaction based strategy, we identified a novel transmembrane tyrosine kinase in CD34+ human bone marrow cells and a human hepatocellular carcinoma cell line, Hep3B. This protein, hepatoma transmembrane kinase or Htk, shares amino acid similarity with the EPH subfamily of tyrosine kinases. The HTK gene is located on human chromosome 7. The predicted 987-amino acid sequence of Htk includes a transmembrane region and signal sequence. In the predicted extracellular domain, a cysteine-rich region and tandem fibronectin type III repeats are present while a single uninterrupted catalytic domain is present in the intracellular domain. These features are consistent with other members of the Eph subfamily. Antibodies raised against Htk extracellular domain immunoprecipitated a 120-kDa protein from either in vitro translated HTK or Hep3B cells which localized primarily to the Hep3B membrane subcellular fraction. Purified in vitro translated Htk was enzymatically active and autophosphorylated on tyrosine in kinase assays. Furthermore, antibodies against Htk ECD were agonistic, inducing Htk tyrosine phosphorylation in transfected NIH3T3 cells. Northern blot analysis demonstrated a single HTK transcript abundantly present in placenta and in a range of primary tissues and malignant cell lines. HTK appears to be expressed in fetal but not adult brain and in primitive and myeloid but not lymphoid hematopoietic cells. The novel transmembrane protein, Htk, may function as a receptor with an expression pattern suggesting a role in events mediating differentiation and development.

3T3 Cells↗
PubMed1994

Enhanced synthesis of tumor necrosis factor-inducible proteins, plasminogen activator inhibitor-2, manganese superoxide dismutase, and protein 28/5.6, is selectively triggered by the 55-kDa tumor necrosis factor receptor in human melanoma cells.

We have demonstrated that A375 melanoma cells express mRNA for both types of tumor necrosis factor (TNF) receptors and receptor proteins on their plasma membranes. Specific agonist and blocking antibodies to either 55-kDa (TNF-R1) or 75-kDa (TNF-R2) TNF receptors combined with two-dimensional gel analysis were employed to determine which receptor type is responsible for mediating the induction of individual melanoma proteins. Our results indicate that the enhanced synthesis of proteins 21/>7 (M(r)/pI), 28/5.6, and 41/5.7 is selectively induced through TNF-R1. TNF induces these proteins; antagonist antibody to TNF-R1 prevents their induction by TNF, and TNF-R1 agonist induces them in the absence of TNF. Identification of these proteins by immunoblot analysis proved that 21/>7 is manganese superoxide dismutase, protein 28/5.6 is unrelated to 27/28-kDa heat shock protein, and protein 41/5.7 is plasminogen activator inhibitor-2. Furthermore, TNF cytotoxicity for A375 cells is also mediated by TNF-R1. These studies indicate that TNF-R1 is a critical signaling receptor for TNF action on A375 cells and demonstrate the potential use of TNF-R1 antibodies to selectively block or enhance specific effects of TNF on melanoma cells.

Base Sequence↗
PubMed1994

Activation of intestinal CFTR Cl- channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.

Heat-stable enterotoxins (STa) produced by pathogenic bacteria induce profound salt and water secretion in the gut, leading to diarrhea. Recently, guanylin, an endogenous peptide with properties similar to STa, was identified. While STa and guanylin bind to the same receptor guanylyl cyclase and raise cell cGMP, the signaling mechanism distal to cGMP remains controversial. Here we show that STa, guanylin and cGMP each activate intestinal Cl- secretion, and that this is abolished by inhibitors of cAMP-dependent protein kinase (PKA), suggesting that PKA is a major mediator of this effect. These agents induce Cl- secretion only in cells expressing the wild-type CFTR, indicating that this molecule is the final common effector of the signaling pathway. The involvement of CFTR suggests a possible cystic fibrosis heterozygote advantage against STa-induced diarrhea.

3T3 Cells↗
PubMed1994

Fas antigen and p55 TNF receptor signal apoptosis through distinct pathways.

The Fas Ag and the p55 TNF receptor (TNF-R1) are related molecules that can signal apoptosis. Some tumor cell lines are selectively killed by Fas activation and others by TNF-R1 activation even though both receptors are often co-expressed. TNF-R1-mediated cytotoxicity can be selectively inhibited under conditions in which Fas-mediated cell death is not affected. Activation of both receptors results in synergistic signaling of apoptosis. These results indicate that different biochemical pathways are activated by Fas and TNF-R1. Combination treatment with agonists of Fas and TNF-R1 may have therapeutic potential.

Animals↗
PubMed1994

Identification and characterization of a novel tyrosine kinase from megakaryocytes.

Protein-tyrosine kinases play pivotal roles in cell signal transduction. We have isolated a cDNA clone encoding a novel human intracytoplasmic tyrosine kinase, termed matk (megakaryocyte-associated tyrosine kinase). Expression of matk mRNA was predominantly found in cells of megakaryocytic lineage. The matk cDNA clone encodes a polypeptide of 527 amino acids and has closest sequence similarity to the csk tyrosine kinase. Sequence comparisons also indicate that matk contains src homology region 2 and 3 domains but lacks the NH2-terminal myristylation signal, the negative regulatory tyrosine (Tyr-527), and the autophosphorylation site (Tyr-416) corresponding to those found in src. Antibodies raised against the NH2 terminus of matk immunoprecipitated a 60-kDa protein from the CMK human megakaryocyte cell line. Expression of matk mRNA was up-regulated in megakaryocytic cells induced to differentiate by the phorbol ester. Based on its restriction in expression and its modulation during in vitro differentiation, it is likely that matk participates in signal transduction during megakaryocytopoiesis.

Base Sequence↗
PubMed1993

Stimulation of human T-cell proliferation by specific activation of the 75-kDa tumor necrosis factor receptor.

TNF-alpha can enhance the proliferation of human thymocytes stimulated by the comitogen Con A. To determine which of the two different TNF receptors is responsible for signaling this cellular response, we investigated the proliferation of human thymocytes in response to agonistic antibodies specific for the two TNF receptor types. In contrast to previously examined TNF activities in human cells, thymocyte proliferation was stimulated in response to rabbit polyclonal antibodies directed against the 75-kDa TNF receptor (TNF-R2), but not those directed against the 55-kDa TNF receptor (TNF-R1). Lymphotoxin (TNF-beta) was also shown to stimulate human thymocyte proliferation, demonstrating that TNF-beta can initiate a biologic response that is mediated by TNF-R2. TNF-R2-mediated T-cell proliferation was not restricted to the immature T cells within the thymus, as the anti-TNF-R2 antibodies also stimulated the proliferation of peripheral T cells. As a first step toward identifying a specific agonist of TNF-R2 with therapeutic potential, 10 anti-TNF-R2 mAb were examined for potential agonist activity. Nine of these significantly stimulated human thymocyte proliferation with maximal responses ranging from twofold to significantly greater than that obtained with TNF-alpha by itself.

Antibodies, Monoclonal↗
PubMed1993

Processing and characterization of human proguanylin expressed in Escherichia coli.

Guanylin is a 15-amino acid peptide hormone that was originally isolated from the jejunum of the rat small intestine and shown to be an endogenous activator of the intestinal heat-stable enterotoxin receptor-guanylyl cyclase. Guanylin is synthesized as a 115-amino acid prohormone, proguanylin, which is processed at a site yet to be determined, into a C-terminal bioactive fragment(s). In order to examine the processing of proguanylin in vitro, we have generated large quantities of the properly folded prohormone by constructing an expression vector that directs its secretion into the periplasmic space of Escherichia coli. The bacterially expressed human proguanylin was then processed to smaller C-terminal fragments by protease digestion. Digestion with trypsin or lysine-C generated C-terminal peptides of different length, which have been purified and characterized. Guanylin-22 and guanylin-32 have binding affinities and biological activities similar to guanylin-15, while guanylin-63 and the entire proguanylin have only minimal bioactivity. Circular dichroism spectroscopy reveals that proguanylin is a stably folded protein containing mostly beta-sheet and beta-turn structure.

Amino Acid Sequence↗
PubMed1993

A novel domain within the 55 kd TNF receptor signals cell death.

Deletion mutagenesis of the intracellular region of the 55 kd TNF receptor (TNF-R1) identified an approximately 80 amino acid domain near the C-terminus responsible for signaling cytotoxicity. This domain shows weak homology with the intracellular domain of Fas antigen, a transmembrane polypeptide that can also initiate a signal for cytotoxicity. Alanine-scanning mutagenesis of TNF-R1 confirmed that many of the amino acids conserved with Fas antigen are critical for the cytotoxic signal. This region of TNF-R1-Fas homology is therefore likely to define a novel domain (death domain) that signals programmed cell death. Mutations within the death domain of TNF-R1 also disrupted its ability to signal anti-viral activity and nitric oxide (NO) synthase induction. In addition, large deletions in the membrane-proximal half of the intracellular domain did not block signaling of cytotoxicity or anti-viral activity but did block induction of NO synthase.

Amino Acid Sequence↗
PubMed1993

Ligand passing: the 75-kDa tumor necrosis factor (TNF) receptor recruits TNF for signaling by the 55-kDa TNF receptor.

To understand the role of the 75-kDa tumor necrosis factor (TNF) receptor in non-lymphoid cells, the cytotoxic signaling and ligand binding activities of the 55-kDa (TNF-R1) and 75-kDa (TNF-R2) TNF receptors were investigated using agonist and antagonist antibodies specific for the two receptor types. This study indicates that although TNF-R2 can significantly reduce the TNF concentration required for cell killing, the mechanism by which this is accomplished is not through the generation of an intracellular signal by TNF-R2. Instead, TNF-R2 regulates the rate of TNF association with TNF-R1, possibly by increasing the local concentration of TNF at the cell surface through rapid ligand association and dissociation. We propose that other cell-surface receptors, such as the low affinity p75 nerve growth factor receptor, may utilize an analogous "ligand passing" mechanism.

3T3 Cells↗
PubMed1993

Interferon-gamma induces receptor dimerization in solution and on cells.

The extracellular domain (ECD) of the human interferon-gamma (IFN gamma) receptor was stably expressed in Chinese hamster ovary cells and purified to homogeneity. Scatchard analysis of 125I-IFN gamma binding to ECD preparations revealed the formation of a ligand-receptor complex which displayed a Ka of 6.4 +/- 0.9 x 10(8) M-1. Two types of complexes were identified by sucrose density gradient ultracentrifugation. The stoichiometry of the major ECD-ligand complex was determined by high performance liquid chromatography gel filtration. When IFN gamma was incubated with a 2-fold molar excess of ECD, a 190-kDa complex was isolated that contained 2 mol of ECD per 1 mol of IFN gamma. IFN gamma also induced dimerization of IFN gamma receptors expressed at the cell surface as detected by chemically cross-linking receptor bound ligand and analyzing cell lysates by SDS-polyacrylamide gel electrophoresis and immunoblotting. Finally, labeled forms of ECD bound to cells preincubated at 4 degrees C with excess amounts of IFN gamma indicating that the ligand could associate with more than one receptor molecule in the absence of chemical cross-linking agents. These results demonstrate that IFN gamma effects dimerization of its receptor under physiologic conditions and suggest that IFN gamma receptor dimerization may be an important event in inducing IFN gamma-dependent biologic responses.

Animals↗
PubMed1993

Human macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta chemokines attract distinct populations of lymphocytes.

Lymphocyte trafficking is an essential process in immune and inflammatory functions which can be thought to contain at least two main components: adhesion and migration. Whereas adhesion molecules such as the selections are known to mediate the homing of leukocytes from the blood to the endothelium, the chemoattractant substances responsible for the migration of specific subsets of lymphocytes to sites of infection or inflammation are largely unknown. Here we show that two molecules in the chemokine (for chemoattractant cytokine) superfamily, human macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta, do not share identical attractant activities for lymphocyte subpopulations. When analyzed in vitro in microchemotaxis experiments, HuMIP-1 beta tends to attract CD4+ T lymphocytes, with some preference for T cells of the naive (CD45RA) phenotype. HuMIP-1 alpha, when tested in parallel with HuMIP-1 beta, is a more potent lymphocyte chemoattractant with a broader range of concentration-dependent chemoattractant specificities. HuMIP-1 alpha at a concentration of 100 pg/ml attracts B cells and cytotoxic T cells, whereas at higher concentrations (10 ng/ml), the migration of these cells appears diminished, and the migration of CD4+ T cells is enhanced. Thus, in this assay system, HuMIP-1 alpha and -1 beta have differential attractant activities for subsets of immune effector cells, with HuMIP-1 alpha having greater effects than HuMIP-1 beta, particularly on B cells.

Chemokine CCL3↗