©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Protein with Homology to hsp90 That Binds the Type 1 Tumor Necrosis Factor Receptor (*)

(Received for publication, August 15, 1994; and in revised form, November 28, 1994)

Ho Yeong Song James D. Dunbar Yuan Xin Zhang Danqun Guo David B. Donner (§)

From the Department of Physiology and Biophysics and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The yeast-based two hybrid has been used to identify a novel protein that binds to the intracellular domain of the type 1 receptor for tumor necrosis factor (TNFR-1IC). The TNF receptor-associated protein, TRAP-1, shows strong homology to members of the 90-kDa family of heat shock proteins. After in vitro transcription/translation and S labeling, TRAP-1 was precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, showing that the two proteins directly interact. The ability of deletion mutants of TNFR-1 to interact with TRAP-1 was tested using the two hybrid system. This showed that the amino acid sequences that mediate binding are diffusely distributed outside of the domain in the C terminus of TNFR-1IC that signals cytotoxicity. The 2.4-kilobase TRAP-1 mRNA was variably expressed in skeletal muscle, liver, heart, brain, kidney, pancreas, lung, and placenta. TRAP-1 mRNA was also detected in each of eight different transformed cell lines. Identification of TRAP-1 may be an important step toward defining how TNFR-1, which does not contain protein tyrosine kinase activity, transmits its message to signal transduction pathways.


INTRODUCTION

Tumor necrosis factor (TNF) (^1)is produced predominantly by macrophages activated by infections or malignancies(1, 2, 3) . This potent multifunctional cytokine was first characterized by its ability to induce the hemorrhagic necrosis and regression of cancers in animals and by the cytotoxic response that it can elicit from transformed cells in vitro. Subsequent studies have shown that through interactions with virtually every type of cell, TNF also promotes immunity, antiviral responses, inflammation, shock, and metabolic alterations, including cachexia, which accompany disease states(1, 2, 3) . Such diverse and profoundly important functions have made TNF the subject of intense investigation.

The first step in TNF action is binding to specific receptors that are expressed on essentially all cells(4, 5, 6, 7) . Two TNF receptors have been identified as proteins of 55-kDa (the type 1 receptor, TNFR-1) and 75-kDa (the type 2 receptor, TNFR-2)(8, 9, 10, 11) , and their cDNAs have been cloned(12, 13, 14) . The extracellular domains of the TNF receptors share homologies with one another and with a group of cell surface receptors that include the FAS antigen, the low affinity NGF receptor, the murine cDNA clone 4-1BB from induced helper and cytolytic T cells, the B cell surface antigen CD40, the OX40 antigen of activated CD4-positive rat lymphocytes, and the T2 antigen of the Shope fibroma virus(15) . The intracellular domains do not display sequence similarities and couple to different signal transduction pathways. For this reason, the receptors induce distinct responses: TNFR-1 promotes cytotoxicity, fibroblast proliferation, antiviral responses, and the host defense against microorganisms and pathogens(10, 16, 17, 18, 19) ; TNFR-2 plays a role in cytotoxicity that is still being defined(20, 21, 22) , inhibits early hematopoiesis(23) , is involved in the proliferation of monocytes(24) , and promotes the proliferation of T cells(25) .

Neither TNF receptor contains intrinsic protein tyrosine kinase activity or any recognizable motif(12, 13, 14) , which suggests a mechanism through which the signal inherent in the hormone-receptor complex can be transmitted to signaling mechanisms. This may suggest that associated proteins, rather than the intracellular domain of either TNF receptor, act as essential elements in signal transmission. Such accessory molecules may be cytoplasmic tyrosine kinases, exemplified by JAK2, which interacts with the erythropoietin and other receptors(26) , or non-tyrosine kinases, such as gp130, which associates with the interleukin-6 and other receptors(27) . Consistent with the hypothesis that TRAPS may be important to TNF action are recent reports describing co-precipitation of TNFR-1 with a protein kinase activity(28, 29) . The present studies were initiated to identify proteins that associate with the intracellular domain of TNFR-1 (TNFR-1IC) and might then initiate signal transduction.

To address this problem, we used the yeast-based two hybrid system, a method for studying protein-protein interactions(30, 31) . This method is based on the properties of the yeast GAL4 protein, which consists of separable domains that mediate DNA binding and transcriptional activation. Plasmids encoding two hybrid proteins, one consisting of the GAL4 binding domain (GAL4-BD) fused to protein X and the other consisting of the GAL4 activation domain (GAL4-AD) fused to protein Y, are cotransformed into yeast. Interaction between these proteins permits transcriptional activation of an integrated copy of the GAL4-lacZ reporter gene.

To identify proteins that interact with TNFR-1IC, a plasmid in which TNFR-1IC was fused with GAL4-BD (pGBT-TNFR1IC) was cotransformed into yeast together with a pool of plasmids encoding GAL4-AD/HeLa S3 cDNA library fusion proteins. This has led to identification of a gene encoding a novel protein that binds TNFR-1IC. The protein was precipitated using a fusion protein consisting of glutathione S-transferase and TNFR-1IC, demonstrating that it interacts directly with the TNF receptor. The protein shows significant homology to members of the 90-kDa family of heat shock proteins (hsp90) and is widely expressed in normal tissues and transformed cells. We also used the two hybrid system to assay the ability of deletion mutations of TNFR-1IC to interact with the newly discovered protein. These experiments show that the amino acid sequences necessary for interaction are diffusely distributed outside of the cytotoxicity domain in TNFR-1IC. Identification of this TNF receptor-associated protein, which we call TRAP-1, is an important step toward defining how TNFR-1 couples to signal transduction pathways and transduces TNF binding into responses.


MATERIALS AND METHODS

Bacterial and Yeast Strains

Yeast strains for two hybrid experiments were obtained from Clontech as components of the Matchmaker Two Hybrid System. Yeast strains SFY526 (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu 2-3, 112, can^r,gal4-542, gal80-538, URA3::GAL1-lacZ) and HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1HIS3, URA3::(GAL4 17-mers)(3)-CYC1-lacZ) were used to assay for protein-protein interactions and for library screening, respectively. SFY526 has the upstream-activating sequence and TATA sequences of the GAL1 promoter fused to the lacZ gene. In HF7c, HIS3 is fused to a GAL1 promoter sequence and LacZ is fused to 3 copies of a 17-mer GAL4 consensus sequence plus the TATA sequence of the CYC1 promoter. The Escherichia coli strain of XL1-blue (Stratagene) was employed in the cloning of plasmids unless otherwise noted.

DNA Manipulation

Yeast shuttle vectors pGBT9 (GAL4-BD), pGAD424 (GAL4-AD), pLAM5` (the lamin gene), pVA3 (the p53 gene), and pTD1 (the SV40 large T antigen gene) were from Clontech. PCR subcloning was used to amplify and insert TNFR-1IC into the unique EcoRI and BamHI of pGBT9 (pGBT-TNFR1IC) using a cDNA clone of TNFR-1 in pUC19 (a gift from Dr. H. Loetscher, Hofmann-LaRoche Inc., Geneva, Switzerland) as the template. The intracellular domains of TNFR-2 (TNFR-2IC) (a gift from Dr. H. Loetscher) and the human FAS antigen (a gift from Dr. S. Nagata, Osaka Bioscience Insitute, Osaka, Japan) (FAS-IC) were amplified and subcloned into pGBT9 as described for TNFR-1IC. PCR products were run on a low melting point gel, cut out, melted, cleaned using a DNA cleanup kit (Promega), digested with appropriate enzymes, and finally ligated to the appropriate vector. Plasmid isolation was accomplished using the Wizard Miniprep and Maxiprep kits from Promega.

Six primers were designed to amplify various regions within TNFR-1IC to make a series of deletion mutants. For oriented cloning, the 5` primers were linked to EcoRI, and the 3` primers were linked to BamHI. All constructs were sequenced at the fusion sites to confirm in frame fusion of TNFR-1IC and TNFR-1IC deletion mutants with GAL4-BD.

Two Hybrid Library Screening

The HeLa S3 Matchmaker cDNA library was purchased from Clontech. pGBT9-TNFR1IC was transformed into HF7c using the lithium acetate procedure. After determining that TNFR-1IC alone does not contain any latent transcriptional activity in HF7c, the transformant was grown overnight in Trp synthetic medium, to ensure that every cell contained pGBT9-TNFR1IC, and sequentially transformed with 500 µg of the HeLa S3 Matchmaker cDNA (prepared in the two hybrid activation vector pGADGH). Double transformed cells on 50 Leu, Trp, His plates were incubated for 5 days at 30 °C before positive colonies were picked, restreaked onto triple minus plates, and assayed for the lacZ phenotype. Library clones that activated the lacZ reporter gene only in the presence of pGBT9-TNFR1IC were chosen for sequencing, which was conducted using the Sequenase sequencing kit (U. S. Biochemical).

Color Development Assays

Yeast transformants were assayed for beta-galactosidase activity using filter and liquid assays. For filter assays, transformants were transferred to nitrocellulose filters, permeabilized in liquid nitrogen, and placed on Whatman No. 1 filter paper that had been soaked in Z buffer (60 mM Na(2)HPO4, 40 mM NaH(2)PO4, 10 mM MgCl(2), 50 mM beta-mercaptoethanol) containing 1.0 mg/ml 5 bromo-4-chloro-3-indolyl beta-D-galactoside at 30 °C. Color developed between 5 min and 10 h. For the liquid assay, cells were diluted 5-fold in rich media (YPD), grown to mid-log phase (A, 0.4-0.8), snap frozen in liquid nitrogen, thawed at 37 °C, and further disrupted by vortexing with glass beads. The procedure of Miller (32) was then used to quantitate beta-galactosidase activity; however, chlorophenyl-red-beta-D-glactopyranoside (Boehringer Mannheim) was used for color development, and cell pellets were resuspended in 900 µl of 100 mM HEPES, pH 7.0, 150 mM NaCl, 2 mM MgCl(2), 1% bovine serum albumin. 100 µl of 50 mM chlorophenyl-red-beta-D-glactopyranoside was added following cellular disruption, and the amount of liberated chlorophenyl-red-beta-D-glactopyranoside was determined by A. Numbers represent beta-galactosidase activity in Miller units and are expressed as the mean of triplicate determinations ± S.D.

5`-RACE PCR and cDNA Screen

The 2-kb cDNA insert of clone I-45 from the two hybrid library screen was sequenced, and the restriction sites were mapped. PCR was performed to amplify the sequence missing at the 5`-end of the TRAP-1 gene using 5`-RACE Ready cDNA from human liver (Clontech) as a template. Two TRAP-1 gene-specific primers (GSP1, GSP2) were designed based on the TRAP-1 partial sequence of I-45 that binds near the N terminus of the known sequence. The sequences of the gene-specific primers are 5`-CTTTGTCTCGGCCTGGAAC-3` (GSP1) and 5`-CGGGATCCCATGTTTGGAAGTGGAACCCT-3` (GSP2). Primary and secondary PCR was performed according to the protocol provided by Clontech.

Using the N terminal 0.6-kb PCR product of the TRAP-1 gene from I-45 as a probe, a HeLa S3 cDNA library in lambda ZAPII (Stratagene) was screened using standard techniques. The longest 2.2-kb insert was rescued as a phagemid (pBluescript-TRAP1) by coinfection with the helper phage.

Northern Blotting

Membranes pre-blotted with poly(A) RNA from a variety of human tissues and cancer cell lines were obtained from Clontech. A 0.6-kb N-terminal PCR product of clone I-45 and an 0.8-kb probe to hsp90-beta (Stressgen Biotech. Corp.), which were labeled using a Prime-A gene labeling kit from Promega, were used as probes. Hybridization, washing, and stripping of the blots were conducted according to instructions provided by Clontech.

Preparation of GST-TNFR-1IC Fusion Proteins

Three GST-TNFR1IC fusion constructs were prepared by amplifying and inserting desired portions of TNFR-1IC into the unique EcoRI and BamHI sites of pGEX-2T (Pharmacia Biotech. Inc.): 1) full-length TNFR-1IC (amino acids 205-413, GST-TNFR1IC); 2) part of the N-terminal half of TNFR-1IC (amino acids 243-315, GST-TNFR1NH(2)); and 3) the C-terminal half of TNFR-1IC (amino acids 316-413, GST-TNFR1COOH). After induction, cells were grown at 30 °C for 3 h, suspended in lysis buffer (20 mM Tris, pH 8.0, 200 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.1% 2-mercaptoethanol), and sonicated. The lysate was centrifuged (12,000 rpm, 30 min), and one-tenth volume of 50% GST-agarose slurry (Sigma) was incubated with the supernatant for 1 h at 4 °C. The beads were washed three times with lysis, buffer and the purity of the GST fusion proteins was confirmed by SDS-PAGE.

In Vitro Translation

The TnT-coupled rabbit reticulocyte lysate system (Promega) was used for one tube transcription/translation according to the instructions of the manufacturer. Rabbit reticulocyte was mixed with 1 µg of pBluscript-TRAP1 and T3 RNA polymerase, after which amino acid mixture without methionine plus [S]methionine was added to the incubate. After 2 h at 30 °C, expression of TRAP-1 was characterized by SDS-PAGE and molecular image analysis (Bio-Rad). In vitro translation yielded a dominant 65-kDa protein, which is the predicted size assuming that the first internal Met codon of the pBluescript-TRAP1 is the start site for most translation.

In Vitro Binding

Agarose-bound GST alone (10 µg), GST-TNFR1IC (10 µg), GST-TNFR1NH(2) (30 µg), or GST-TNFR1COOH (10 µg) was added to binding buffer (20 mM Tris, pH 7.7, 0.5% Nonidet P-40, 200 mM NaCl, 50 mM NaF, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.1% 2-mercaptoethanol) and 40 µl of reticulocyte lysate containing [S]methionine-labeled TRAP-1. After 1 h at 4 °C, the agarose beads were washed four times with binding buffer before addition of SDS-PAGE sample buffer, electrophoresis, and phosphoimage analysis.

Two Hybrid Mapping of the TRAP-1 Binding Site in TNFR-1IC

TNFR-1IC truncations spanning amino acids 205-280, 205-359, 278-359, 278-426, and 352-426 were fused in frame with the GAL4-BD by PCR subcloning. These constructs and I-45 were cotransformed into SFY526, which were assayed for the lacZ phenotype. Expression of the GAL4-BD/TNFR-1IC fusion proteins was determined by Western blotting using a polyclonal antibody directed against GAL4-BD (Upstate Biotechnology, Inc.). Briefly, yeast transformants were grown overnight in minimal media and then diluted 5-fold in YPD broth. At mid-log phase, cells were harvested by centrifugation, washed twice with ice-cold TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5), resuspended in TE buffer containing 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, and 2 µg/ml leupeptin, and lysed by agitation with glass beads. Supernatants were fractionated by SDS-PAGE and transferred to Immobilon-P, which was hybridized with the GAL4-BD antibody at 1:2000 dilution and detected by ECL (Amersham Corp.).


RESULTS

To identify proteins that interact with TNFR-1IC, plasmids encoding TNFR-1IC fused with the GAL4-BD (pGBT-TNFR1IC) and HeLa S3 Matchmaker cDNA were cotransformed into HF7c. Double transformant colonies were screened and selected for histidine prototrophy (Fig. 1a). Colonies isolated as His transformants were assayed for lacZ expression to eliminate false positives, an effective procedure as the His3 and lacZ reporter genes in HF7c are under control of dissimilar promoters. Activation domain library plasmids encoding potential TRAPs were isolated from the double positive colonies, and the GAL4-AD/TRAP gene fusion site was sequenced. We found two groups of cDNAs that encode distinct proteins: TNF receptor-associated protein 1 (TRAP-1) and a second receptor associated protein (TRAP-2), which will be described elsewhere. Two TRAP-1 clones, I-45 and I-57, were sequenced at the fusion site; both were in frame with GAL4-AD, and I-57 was shorter than I-45 by 9 bases (Fig. 1b). Sequence analysis of the cDNA insert of I-45 showed that the partial TRAP-1 gene spans 1996 bases with an open reading frame ending at base 1827. From the fusion site, this clone encodes a protein of 608 amino acids.


Figure 1: a, two hybrid library screen. pGBT9-TNFR1IC was used to screen a HeLa S3 cDNA library. Among the interacting clones, I-45 and I-57 encode partial sequences of TRAP-1 gene. b, schematic of two clones. Clone I-45 contains the partial TRAP-1 gene that spans 1996 bases up to the beginning of the poly(A) tail, which is in frame with GAL4-AD. Clone I-57 is shorter by 9 bases, initiating at base 10. c, specificity of interactions. Yeast strains transformed with combinations of plasmids fused to the GAL4-BD and with clone I-45 were assayed for activation of beta-galactosidase. The p53 gene (pVA3) and the SV40 large T antigen (pTD1) associate strongly and serve as a positive control. pGBT-FAS is a GAL4-BD/FAS-IC fusion; pGBT-TNFR2IC is a GAL4-BD/TNFR-2IC fusion.



Experiments were conducted to establish that interaction of TNFR-1IC with TRAP-1 is specific (Fig. 1c). I-45 by itself was incapable of activating beta-galactosidase activity, which shows that TRAP-1 does not contain a latent transcriptional activator. GAL4-BD alone, lamin, TNFR-2IC, or FAS-IC, which contains a cytotoxicity domain with homology to that found in TNFR-1IC, did not interact with TRAP-1. Only when the TNFR-1IC gene was cotransformed with I-45 was activation of the lacZ reporter gene detected. These observations rule out the possibility that TRAP-1 nonspecifically interacts with other proteins.

To identify the full-length size and distribution of expression of TRAP-1 RNA, we used a 0.6-kb N-terminal PCR product of I-45 as a probe to hybridize blots of multiple normal tissues and various transformed cell lines. As shown in Fig. 2, there was a single TRAP-1 transcript of about 2.4 kb. To obtain the full-length cDNA for TRAP-1, we used 5`-RACE PCR and also screened a lambda ZAPII cDNA library. The 5`-RACE PCR extended the known sequence of TRAP-1 by 180 bases. The combined sequence of I-45 with the 5`-RACE PCR product spans 2.2 kb (versus 2.4 kb, mRNA size) and is predicted to encode a protein of 661 amino acids (Fig. 3). A highly compressed GC-rich region of about 30 bases at the 5`-end of the PCR product may have prevented the reverse transcriptase from reading to the 5`-end of the message. Similarly, the longest phage clone isolated as a phagemid from the lambda ZAPII library search was 2.2 kb (pBluescript-TRAP1). However, it is most important that the sequence defined by the two hybrid screen and the 5`-RACE PCR encodes virtually the complete coding sequence of TRAP-1 and contains all of the elements necessary for binding to TNFR-1IC.


Figure 2: Expression of TRAP-1 and human hsp90-beta. Pre-blotted membranes containing mRNAs from human tissues (a, left) or cancer cell lines (b, right) were probed with a 0.6-kb random primed N-terminal PCR product of the TRAP-1 gene in I-45 (top) and a specific probe for human hsp90-beta (bottom). The cancer cell lines from right to left were HL-60 promyelocytic leukemia cells, HeLa S3 cells, k-562 chronic myelogenous leukemic cells, MOLT-4 lymphoblastic leukemia cells, Burkitt's lymphoma Raji, SW480 colorectal adenocarcinoma cells, A549 lung carcinoma cells, and G361 melanoma cells. Numbers to the left of the blots indicate relative size in kb. The line to the right of the toppanel indicates the 2.4-kb TRAP-1 mRNA. The line to the right of the bottompanel indicates the 2.7-kb hsp90-beta mRNA.




Figure 3: Sequence of TRAP-1 cDNA with predicted amino acid sequence. Clone I-45, which includes the poly(A) tail, and a cDNA segment obtained from 5`-RACE PCR were sequenced. The sequence spans 2156 bases without the poly(A) tail and encodes a protein of 661 amino acids.



To identify homologous proteins, the predicted amino acid sequence of TRAP-1 was used to search the National Center for Biotechnology Information (NCBI) data base using Blast command. TRAP-1 shares significant homology, 34% sequence identity and overall homology of about 60% when conserved substitutions are included, with members of the hsp90 family (33) (Fig. 4). Homologies between hsp90s and TRAP-1 are not restricted to a single sequence of amino acids but reside in at least six distinct domains. A striking difference between hsp90s and TRAP-1 is the absence of a highly charged domain in the latter, which is present in the former.


Figure 4: Amino acid sequence alignment of rat hsp90-beta with TRAP-1. Sequence alignment was obtained using Bestfit command in Genetics Computer Group software . The uppersequence is TRAP-1, and the lowersequence is rat hsp90. Straightlines between amino acids indicate identity, and dottedlines indicate conserved substitutions. The highly charged segment in rat hsp90 is underlined, while obviously conserved domains in TRAP-1 and hsp90 are boxed.



Northern blot analysis (Fig. 2, top) showed that TRAP-1 mRNA is variably present in eight different normal tissues and was in eight transformed cell lines. To rule out the possibility that the probe used to define the size and tissue distribution of TRAP-1 mRNA nonspecifically recognizes hsp90, the blots used for the experiments in Fig. 2were stripped and hybridized with a probe to hsp90 (Fig. 2, bottom). The hsp90 mRNA was distinguished from that of TRAP-1 by size (2.4 compared with 2.7 kb, respectively) and relative expression level in normal tissues and transformed cells.

An in vitro binding assay was developed to demonstrate direct interaction between TRAP-1 and TNFR-1IC and to establish that identification of TRAP-1 as a TNF receptor-associated protein was not an artifact of the two hybrid system. TRAP-1 expressed by in vitro translation and labeled with [S]methionine was incubated with agarose GST, and with GST fusion proteins containing full-length TNFR-1IC (GST-TNFR1IC), the N-terminal half of TNFR-1IC (GST-TNFR1NH(2)) and the C-terminal half of TNFR-1IC (GST-TNFR1COOH) (Fig. 5). GST-TNFR1IC and GST-TNFR1NH(2) precipitated TRAP-1, whereas GST or GST-TNFR1COOH did not. These observations show that TNFR-1IC binds TRAP-1 without the intermediacy of third proteins and does so in a mammalian cell-free system as well as in yeast. Thus, identification of TRAP-1 as a TNF receptor-associated protein cannot be an artifact associated with the use of the two hybrid system. These observations additionally show that the binding site for TRAP-1 resides in the N-terminal half of TNFR-1IC.


Figure 5: In vitro precipitation of TRAP-1. In vitro translated and [S]methionine-labeled TRAP-1 was precipitated using GST fusions of TNFR-1IC. Lane1, GST alone, control; lane2, GST-TNFR1IC; lane3, GST-TNFR1NH(2); lane4, GST-TNFR1COOH. The arrow indicates TRAP-1 precipitated by GST-TNFR1IC and GST-TNFR1ICOOH.



To further define the amino acid sequences important for interaction of TNFR-1IC with TRAP-1, we constructed C- and N-terminal truncations of TNFR-1IC. The deletion mutants were subcloned into the pGBT9 and cotransformed into SFY526 together with I-45 to assay their interactions using the two hybrid system. The relationship of these mutants to TNFR-1IC and their ability to interact with TRAP-1 and thereby activate beta-galactosidase is shown in Fig. 6a. The results obtained do not directly reflect the affinity of the interaction between TNFR-1IC domains with TRAP-1 since expression of the GAL4-BD/TNFR-1IC fusion proteins in yeast, assayed by Western blotting with an antibody directed against the GAL4-BD (Fig. 6b), was somewhat variable. However, the results can be used to determine whether any receptor domain contains recognition elements essential to the interaction between TNFR-1IC and TRAP-1, especially when interpreted together with observations made using GST-TNFR1IC fusion proteins (Fig. 5). Peptides encompassing amino acids 205-280 and 278-359 activated beta-galactosidase, showing that the elements that promote interaction of TNFR-1IC with TRAP-1 are diffusely distributed. The avidity with which amino acids 205-359 bound TRAP-1 (123 Miller units of beta-galactosidase activity) was more than predicted based on an additive response to amino acids 205-280 and 278-359 (45 Miller units), suggesting that the conformation of the peptide and perhaps the accessibility of crucial binding domains plays a role in regulating interaction. The inability of residues 352-426 to produce beta-galactosidase activity shows that the binding sites reside largely, if not entirely, outside of the cytotoxicity domain, a result consistent with the ability of GST-TNFR1NH(2) but not GST-TNFR1COOH to precipitate TRAP-1 (Fig. 5). A larger C-terminal peptide (amino acids 278-426) was also unable to activate beta-galactosidase, despite the capacity of the peptide encompassing residues 278-359 to do so. One explanation for this result is that the cytotoxicity domain negatively regulates interaction of TNFR-1IC with TRAP-1.


Figure 6: Mapping the TRAP-1 binding sites in TNFR-1IC. a, plasmids encoding GAL4-BD/TNFR-1IC deletion mutants were cotransformed with I-45, and beta-galactosidase activity was assayed. The topbar illustrates the structure of TNFR-1IC. The blackenedhorizontalbars represent the relative size and location of each deletion mutant in TNFR-1IC. b, expression of GAL4-BD/TNFR-1IC deletion mutants was assayed by Western blotting using antisera directed to GAL4-BD. Lanea, full-length TNFR-1IC (amino acids 205-426); laneb, TNFR-1IC amino acids 205-280; lanec, TNFR-1IC amino acids 205-359; laned, TNFR-1IC amino acids 278-359; lanee, TNFR-1IC amino acids 278-426; lanef, TNFR-1IC amino acids 352-426, which consistently appeared to overlap a nonspecific band. The arrow to the right of each lane in the Western blot indicates the position of the fusion protein.




DISCUSSION

Studies taking advantage of the strong species specificity of TNFR-2 for murine compared with human TNF (17) or employing agonist antibodies specific to either TNF receptor(10, 16, 17, 18, 19, 20) have shown that signaling through TNFR-1 promotes growth inhibition and cytotoxicity in transformed cells, antiviral activity, proliferation of fibroblasts, induction of NF-kB, accumulation of c-Fos, interleukin-6, and manganous superoxide dismutase mRNAs, prostaglandin E(2) synthesis, and HLA class I and II cell surface antigen expression(1, 2, 3, 10, 16, 17, 18, 19, 20) . Mice deficient in TNFR-1 are severely impaired in the ability to clear the bacterial pathogen Listeria monocytogenes and die rapidly from infections; however, these animals are resistant to lipopolysaccharide-mediated septic shock(19) . Thus, TNFR-1 plays a decisive role in the host's defense against microorganisms and their pathogenic factors. The significant role of this receptor in TNF action led us to initiate our search for TRAPs using TNFR-1IC as bait in a two hybrid screen of a cDNA library.

The two hybrid system detects proteins capable of interacting with a known protein by transcriptional activation of a reporter gene(30, 31) . This method has demonstrated interactions between Bcl-2 and R-ras p23(34) , Sos 1 and GRB2(35) , complex formation between Ras and Raf and other protein kinases(36) , binding of the human immunodeficiency virus type 1 GAP protein with cyclophilins A and B(37) , and also identified proteins that interact with the type 1 receptor for TGF-beta (38) and the type 2 receptor for TNF(39) . Previously, we used the two hybrid system to demonstrate self-association of TNFR-1IC(40) . Evidence for such aggregation was obtained from a screen of a HeLa S3 cDNA library using TNFR-1IC as bait; this yielded a clone encoding a protein encompassing the death domain at the C-terminal of TNFR-1IC(41) . Aggregation is independently suggested by the ability of non-functional TNFR-1 deletion mutants to suppress signaling by non-defective endogenous TNF receptors(42) . Thus, our previous studies (40) demonstrated that the two hybrid system can successfully identify proteins that interact with TNFR-1, one of which is TNFR-1IC itself.

In this and another study that describes a second TNF receptor-associated protein, TRAP-2, (^2)the two hybrid system was used to search for additional proteins that bind TNFR-1IC, leading to the discovery of TRAP-1 and TRAP-2. Both proteins interact specifically with TNFR-1 and not with unrelated proteins. mRNAs for both TRAPs are highly expressed in numerous transformed cell lines and are detected at different levels of expression in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas.

Fig. 7a summarizes the domain structure of TNFR-1IC, showing the relationships of the death/aggregation domain to the binding sites for TRAP-1 and TRAP-2. Each TRAP binds to sites in TNFR-1IC that reside outside of the death (41, 42) and/or aggregation (40) domain. Mutations in this domain disrupt the ability of TNFR-1 to signal not only cytotoxicity but antiviral activity and induction of nitric oxide synthase as well. These observations should not lead one to conclude that TRAP-1 and TRAP-2 are not involved in TNF action. While the cytotoxicity domain is undoubtedly important for induction of some responses mediated by TNFR-1, it is not the only domain involved in signaling. Recent observations show that the ability of TNF to activate an endosomal acidic sphingomyelinase is abrogated by C-terminal deletions encompassing all or part of the cytotoxicity domain in TNFR-1, whereas activation of a membrane-associated neutral sphingomyelinase is unaffected(43) . Ceramide produced by the neutral sphingomyelinase is important for activation of a proline-directed serine threonine kinase and phospholipase A(2). The acidic sphingomyelinase activates NF-kB. Thus, different domains in TNFR-1IC control important and distinguishable second messenger pathways and cellular responses.


Figure 7: a, map of TRAP binding sites in TNFR-1IC defined using the two hybrid system and by in vitro precipitation. b, relationship of hsp90, TRAP-1, and TRAP-2. Six domains that share homology in hsp90 and TRAP-1 are indicated by speckledboxes and Romannumerals. The blackboxes delineate a highly charged segment in hsp90 and TRAP-2. The length of each bar represents the relative size of each protein.



TRAP-1 shows strong homology to members of the hsp90 family. Fig. 4illustrates the presence of at least six domains of high amino acid identity and conservation that relate TRAP-1 and hsp90s. Interestingly, a well conserved, highly charged sequence of amino acids in hsp90 is absent from TRAP-1 but identified in TRAP-2. Fig. 7b illustrates the structural relationship of hsp90 to TRAP-1 and TRAP-2. While the significance of the relationship of the TRAPS to one another and hsp90 is not presently known, a basis exists for believing that such stress proteins are important to defining cellular responses to TNF. First, induction of the heat shock response renders transformed cells resistant to TNF and macrophage-mediated cytotoxicity(44) . Second, transfection of hsp70s into otherwise responsive cells induces a state of resistance(45) , and TNF-induced phosphorylation of hsp28 stress proteins is also associated with protection against cytotoxicity (46) . Third, the elements in TNFR-1IC necessary for binding to TRAP-1 and TRAP-2 reside outside of the cytotoxicity/aggregation domain. Large deletions outside of this domain(41) , where TRAP-1 and TRAP-2 bind, do not impair the ability of mutant receptors to induce cytotoxicity or antiviral activity.

Heat shock proteins are likely candidates for service as TRAPs as they act as cofactors or chaperones in cell growth-associated processes, protein folding and transport, and cell division and membrane function (47) . Hsp90s associate with steroid receptors, which are thereby stabilized in a non-DNA binding conformation, and also with protein kinases(47) . Hsp90s also modulate signaling through transduction cascades. Mutations in a member of the hsp90 family, hsp83, impairs signaling by the sevenless receptor tyrosine kinase, which is required for differentiation of the R7 photoreceptor neuron in Drosophila(48) . Also, lethal expression of vSrc in Saccharomyces cerevisiae is suppressed by a reduction in the level of Hsp82(49) . The diverse properties of hsps suggest that the discovery of TRAP-1, and also TRAP-2, is important and likely to provide insight into the mechanistic basis for TNF action.


FOOTNOTES

*
This work was supported by a grant from the Indiana Affiliate of the American Diabetes Association (to D. B. D.), by a postdoctoral fellowship from the Indiana Affiliate of the American Heart Association (to H. Y. S.), by a postdoctoral fellowship from the Walther Oncology Center (to Y. X. Z.), and by a predoctoral fellowship from the Indiana Affiliate of the American Heart Association (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U12595[GenBank], U12596[GenBank].

§
To whom correspondence should be addressed. Tel.: 317-278-2155; Fax: 317-274-3318.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; TNFR-1, 55-kDa type 1 tumor necrosis factor receptor; TNFR-2, 75-kDa type 2 tumor necrosis factor receptor; TNFR-1IC, intracellular domain of TNFR-1; TNFR-2IC, intracellular domain of TNFR-2; TRAP-1, TNF receptor-associated protein-1; TRAP-2, TNF receptor-associated protein-2; GAL4-BD, the GAL4 binding domain; GAL4-AD, the GAL4 transactivation domain; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; TnT, troponin T; PCR, polymerase chain reaction; kb, kilobase(s); RACE, rapid amplification of cDNA ends.

(^2)
Song, H. Y., Dunbar, J. D. Zhang, Y. X., Guo, D., and Donner, D. B., submitted for publication.


ACKNOWLEDGEMENTS

We gratefully acknowledge the assistance of Dr. Li Zhu (Clontech) in the use of the two hybrid system.


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