Two-hybrid cloning of a gene encoding TNF receptor-associated protein 2, a protein that interacts with the intracellular domain of the type 1 TNF receptor: identity with subunit 2 of the 26S protease.
A protein that binds the intracellular domain of the type 1 TNFR (TNFR-1IC) has been identified by two-hybrid cloning. The 97-kDa TNFR-associated protein, TRAP2, shows sequence identity with internal amino acid sequences from subunit 2 of the 26S protease. TRAP2 antiserum recognizes subunit 2 of the 26S protease, which is ... consistent with the identity of these proteins. TRAP2 antiserum interacted with the 97-kDa protein in HeLa cell lysates and cytosol, the latter observation showing that TRAP2 resides in the same cellular compartment as TNFR-1IC. A fusion of glutathione-S-transferase and TNFR-1IC (GST-TNFR-1IC) precipitated TRAP2 from a HeLa cell lysate; conversely, GST-TRAP2 precipitated TNFR-1 from such a lysate. These observations show that the proteins interact in the cellular milieu. After in vitro transcription/translation and 35S labeling, TRAP2 was precipitated from a cellfree system by GST-TNFR-1IC, showing that TNFR-1IC and TRAP2 interact directly. TRAP2 was also precipitated from the cellfree translation system by a GST fusion containing the N-terminal half of TNFR-1IC, but not by a GST fusion containing the C-terminal half of TNFR-1IC that contains a "death domain" that plays an obligatory role in signaling cytotoxicity. The ability of deletion mutants of TNFR-1IC to interact with TRAP2 was tested using the two-hybrid system. This also showed that the amino acid sequences that mediate binding reside outside of the death domain in TNFR-1IC. The demonstration that a subunit of the 26S protease binds TNFR-1 may identify a novel TNF-signaling pathway.
Mesh Terms:
Amino Acid Sequence, Base Sequence, Binding Sites, Carrier Proteins, Cloning, Molecular, Gene Expression, Hela Cells, Humans, Molecular Sequence Data, Peptide Hydrolases, Proteasome Endopeptidase Complex, Protein Binding, Proteins, RNA, Messenger, Receptors, Tumor Necrosis Factor, TNF Receptor-Associated Factor 2, Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
Amino Acid Sequence, Base Sequence, Binding Sites, Carrier Proteins, Cloning, Molecular, Gene Expression, Hela Cells, Humans, Molecular Sequence Data, Peptide Hydrolases, Proteasome Endopeptidase Complex, Protein Binding, Proteins, RNA, Messenger, Receptors, Tumor Necrosis Factor, TNF Receptor-Associated Factor 2, Tumor Necrosis Factor Receptor-Associated Peptides and Proteins
J. Immunol.
Date: May. 01, 1997
PubMed ID: 9126987
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