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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.

Pubmed ID: 9126987

Authors

  • Dunbar JD
  • Song HY
  • Guo D
  • Wu LW
  • Donner DB

Journal

Journal of immunology (Baltimore, Md. : 1950)

Publication Data

May 1, 1997

Associated Grants

  • Agency: NCI NIH HHS, Id: CA 67891

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