Biomedical subjects

W R Force

Publications and source records attributed to W R Force.

8 recordsLinked to original sources

PubMed2000

Discrete signaling regions in the lymphotoxin-beta receptor for tumor necrosis factor receptor-associated factor binding, subcellular localization, and activation of cell death and NF-kappaB pathways.

Lymphotoxin-beta receptor (LTbetaR), a member of the tumor necrosis factor receptor superfamily, is essential for the development and organization of secondary lymphoid tissue. Wild type and mutant LTbetaR containing successive truncations of the cytoplasmic domain were investigated by retrovirus-mediated gene transfer into HT29.14s and in 293T cells by transfection. Wild type receptors accumulated in perinuclear compartments and enhanced responsiveness to ligand-induced cell death and ligand-independent activation of NFkappaB p50 dimers. Coimmunoprecipitation and confocal microscopy mapped the TRAF3 binding site to amino acids PEEGDPG at position 389. However, LTbetaR truncated at position Pro(379) acted as a dominant positive mutant that down-modulated surface expression and recruited TRAF3 to endogenous LTbetaR. This mutant exhibited ligand-independent cell death and activated NF-kappaB p50 dimers. By contrast, truncation at Gly(359) created a dominant-negative mutant that inhibited ligand-induced cell death and activation of NF-kappaB p50/p65 heterodimers. This mutant also blocked accumulation of wild type receptor into perinuclear compartments, suggesting subcellular localization may be crucial for signal transduction. A cryptic TRAF-independent NF-kappaB activating region was identified. These mutants define discrete subregions of a novel proline-rich domain that is required for subcellular localization and signal transduction by the LTbetaR.

Amino Acid Sequence↗

PubMed1997

Dominant negative mutants of TRAF3 reveal an important role for the coiled coil domains in cell death signaling by the lymphotoxin-beta receptor.

Ligation of the lymphotoxin-beta receptor (LTbetaR) recruits tumor necrosis factor receptor-associated factor-3 (TRAF3) and initiates cell death in HT29 adenocarcinoma cells. The minimal receptor binding domain (TRAF-C) defined by two hybrid analyses is not sufficient for direct recruitment to the ligated receptor. A series of TRAF3 deletion mutants reveal that a subregion of the coiled coil motif is required for efficient recruitment to the LTbetaR. Furthermore, the ability of TRAF3 to self-associate maps to an adjacent subregion. A TRAF3 deletion mutant that lacks the N-terminal zinc RING and zinc finger motifs, but retains the coiled coil and TRAF-C motifs, competitively displaces endogenous TRAF3 from the LTbetaR. A second TRAF3 mutant that lacks the receptor binding domain, yet contains the TRAF3 self-association domain, prevents TRAF3 homodimers from being recruited to the LTbetaR. Both of these mutants have a dominant negative effect on cell death and demonstrate that the recruitment of TRAF3 oligomers is necessary to initiate signal transduction that activates the cell death pathway.

Apoptosis↗

PubMed1997

Lymphotoxin-beta receptor signaling complex: role of tumor necrosis factor receptor-associated factor 3 recruitment in cell death and activation of nuclear factor kappaB.

The binding of heterotrimeric lymphotoxin, LT alpha1 beta2, to the LTbeta receptor (LTbeta R), a member of the tumor necrosis factor receptor (TNFR) superfamily, induces nuclear factor kappaB (NF-kappaB) activation and cell death in HT29 adenocarcinoma cells. We now show that treatment with LT alpha1 beta2 or agonistic LTbeta R antibodies causes rapid recruitment of TNFR-associated factor 3 (TRAF3) to the LTbeta R cytoplasmic domain. Further, stable overexpression of a TRAF3 mutant that lacks the RING and zinc finger domains inhibits LTbeta R-mediated cell death. The inhibition is specific for LTbeta R cell death signaling, since NF-kappaB activation by LT alpha1 beta2 and Fas-mediated apoptosis are not inhibited in the same cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LTbeta R, suggesting that the mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LTbeta R death signaling complex and indicate that at least two independent signaling pathways are initiated by LTbeta R ligation.

Adenocarcinoma↗

PubMed1995

Mouse lymphotoxin-beta receptor. Molecular genetics, ligand binding, and expression.

Lymphotoxin (LT) -alpha beta heterotrimer is a membrane-anchored ligand expressed by activated T cells which binds specifically to the LT beta receptor (LT beta R), a member of the TNFR family. The LT beta R is implicated as a critical element in controlling lymph node development and cellular immune reactions. To address this hypothesis we have isolated a mouse cDNA encoding a single transmembrane protein of 415 amino acids with 76% identity to human LT beta R. The receptor function of this molecule was demonstrated by the ability of the extracellular domain, constructed as a chimera with the Fc region of IgG7, to bind to LT alpha beta complexes expressed on the surface of activated T cells or insect cells infected with baculoviruses containing LT alpha and LT beta cDNAs. The gene encoding mouse LT beta R, Ltbr, contains 10 exons spanning 6.9 kb and maps to mouse chromosome 6, which is closely linked to Tnfr1, consistent with the tight linkage of the human homologue of these genes on chromosome 12p13. Mouse LT beta R mRNA is expressed by cell lines of monocytic and epithelial origin but not by a CTL line, and in vivo it is constitutively expressed in visceral and lymphoid tissues. The delineation of the structure of the mouse LT beta R will aid investigations into the role of this cytokine-receptor system in immune function and development.

Amino Acid Sequence↗

PubMed1995

Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas.

A number of murine T-cell hybridomas undergo apoptosis within a few hours of activation by specific antigens, mitogens, antibodies against the T-cell antigen receptor, or a combination of phorbol ester and calcium ionophore. This phenomenon has been extensively studied as a model for clonal deletion in the immune system, in which potentially autoreactive T cells eliminate themselves by apoptosis after activation, either in the thymus or in the periphery. Here we show that the Fas/CD95 receptor, which can transduce a potent apoptotic signal when ligand, is rapidly expressed following activation of T-cell hybridomas, as is its functional, membrane-bound ligand. Interference with the ensuing Fas/Fas-ligand interaction inhibits activation-induced apoptosis. Because T-cell receptor ligation can induce apoptosis in a single T hybridoma cell, we suggest that the Fas/Fas-ligand interaction can induce cell death in a cell-autonomous manner.

Animals↗

PubMed1994

3,5,3'-L-triiodothyronine (thyroid hormone)-induced protein-DNA interactions in the thyroid hormone response elements and cell type-specific elements of the rat growth hormone gene revealed by in vivo dimethyl sulfate footprinting.

The cell type-specific element and 3,5,3'-L-triiodothyronine (thyroid hormone) (T3) response element of the rat growth hormone gene act synergistically to produce cell type-specific, T3-regulated expression. Pit-1 is a pituitary cell type-specific transcription factor that binds the cell type-specific element and is essential to its activity. T3 receptors bind as homodimers and heterodimers to the T3 response element and are essential for its activity. Here, we report the use of ligation-mediated polymerase chain reaction in vivo dimethyl sulfate footprinting to study the effects of T3 on protein-DNA interactions in the rat growth hormone gene promoter in vivo. T3-responsive guanine methylation was detected only in and near the T3 response element and the Pit-1 binding sites. The results indicate that within 2 h, T3 induces occupancy of the T3 response element and Pit-1 sites by their respective trans-acting factors in vivo.

Animals↗

PubMed1994

Homodimer and heterodimer DNA binding and transcriptional responsiveness to triiodothyronine (T3) and 9-cis-retinoic acid are determined by the number and order of high affinity half-sites in a T3 response element.

T3 (triiodothyronine) response elements (TREs) consist of pairs of strong and weak (S and W), 10-nucleotide T3 receptor (TR) monomer binding sites (half-sites). We report that the number and order of S and W half-sites in a direct repeat TRE determines whether it mediates ligand-dependent or independent transcriptional activation or inhibition in the presence of TR or TR and 9-cis-retinoic acid receptor (RXR); and whether a TRE is preferentially bound by TR homodimers, TR-RXR heterodimers, or CV1 cell TR accessory protein (TRAP)-TR heterodimers. TR homodimers bound equally to TREs composed of the 5'-S and 3'-W (SW) and the opposite (WS) arrangement of half-sites. TR-RXR gamma heterodimers bound SW better than WS. TR-TRAP heterodimers bound WS better than SW. Transcription of a reporter gene cis-linked to WS responded to unliganded TR and RXR, and either ligand stimulated expression 2-fold more. Reporter expression cis-linked to SW was not altered by unliganded receptors, and T3 stimulated transcription only in the presence of both TR and RXR. SS was strongly activated by liganded, but not by unliganded TR. SS was activated by unliganded TR and RXR gamma together, and T3 further stimulated transcription 2-fold. Under these conditions, transcription was inhibited 60% by 9-cis-retinoic acid.

Animals↗

PubMed1992

Positive and negative thyroid hormone response elements are composed of strong and weak half-sites 10 nucleotides in length.

The steroid-thyroid hormone receptors bind to imperfect repeats of two or more half-sites. It is generally accepted that a T3 response element (TRE) half-site consists of a six-nucleotide core motif (5'-AGGT(C/A)A-3'). It is less widely appreciated that the nucleotides flanking this core motif also have a major influence on the affinity of T3 receptor (TR) for its response element. We analyzed TR-DNA interactions under conditions in which the affinity of receptor monomers for individual TRE half-sites of the rat GH (rGH) gene was measured. These studies avoided the effects of half-site spacing and orientation on receptor binding. Variations in the nucleotides flanking the core sequence can modulate receptor binding by more than 15-fold. Systematic mutational analysis of TRE half-site structure demonstrated that at least two nucleotides flanking either side of the half-site core motif strongly influence TR binding affinity and activity, indicating that half-sites are approximately 10 nucleotides long. Thus, the half-sites of most TREs overlap, and mutations in one half-site may affect the activity of its partner. The TRE half-site sequence 5'-CTGAGGTAACG-3' was bound with highest affinity by TRs. The negatively T3-responsive promoter of the rGH gene was used to investigate the functional significance of the nucleotides flanking the core motif in vivo. A promoter consisting of only 22 rGH nucleotides, containing two functional TRE half-sites which overlap the rGH TATA box, directed T3-inhibited transcription. Mutation of nucleotides flanking the core sequence of the weaker half-site dramatically reduced the activity of the element, demonstrating that the flanking sequences of the half-sites can profoundly affect TRE activity.

Animals↗