Aggregation of the intracellular domain of the type 1 tumor necrosis factor receptor defined by the two-hybrid system.
The yeast-based two hybrid system has been used to determine whether oligomerization of the intracellular domain of the 55-kDa type 1 tumor necrosis factor (TNF) receptor may occur during TNF action. This assay depends upon reconstitution of the function of the GAL4 transcriptional activator through interaction of a protein fused to the GAL4 DNA binding domain with a protein fused to the transcriptional activation domain of GAL4. Fusion of the type 1 TNF receptor intracellular domain with the DNA binding domain and the transactivation domain of GAL4 led to activation of the lacZ indicator gene, demonstrating interaction of the receptor intracellular domain with itself. A HeLa cell cDNA library was searched for proteins that interact with the intracellular domain of the type 1 TNF receptor. A protein corresponding to amino acids 329-426 in the type 1 TNF receptor intracellular domain was identified by this screen. The aggregation domain was further defined by testing the ability of deletion mutants of the type 1 TNF receptor intracellular region to interact with the complete intracellular domain. These experiments map the aggregation domain to a sequence of amino acids previously shown to be responsible for mediating TNF-induced cytotoxicity. These results suggest that aggregation of type 1 TNF receptor intracellular domains may be important in TNF signal transduction.
Song
H Y
HY
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis 46202.
Dunbar
J D
JD
Donner
D B
DB
D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D014157:Transcription Factors; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D001616:beta-Galactosidase