A Novel p75TNF Receptor Isoform Mediating NFkappa B Activation*

Carola Seitz, Peter Müller, René C. Krieg, Daniela N. MännelDagger, and Thomas Hehlgans

From the Department of Pathology/Tumor Immunology, University of Regensburg, D-93042 Regensburg, Germany

Received for publication, February 12, 2001, and in revised form, March 1, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report the identification of a novel p75TNF receptor isoform termed icp75TNFR, which is generated by the use of an alternative transcriptional start site within the p75TNFR gene and characterized by regulated intracellular expression. The icp75TNFR protein has an apparent molecular mass of ~50 kDa and is recognized by antibodies generated against the transmembrane form of p75TNFR. The icp75TNFR binds the tumor necrosis factor(TNF) and mediates intracellular signaling. Overexpression of the icp75TNFR cDNA results in TNF-induced activation of NFkappa B in a TNF receptor-associated factor 2 (TRAF2)-dependent manner. Thus, our results provide an example for intracellular cytokine receptor activation.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TNF1 is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation and cytotoxicity in a variety of different cell types. Two distinct receptor molecules with an apparent molecular mass of 55 kDa (p55TNFR, TNFR type 1) and 75 kDa (p75TNFR, TNFR type 2) have been identified, and their corresponding cDNAs have been cloned (1-4). The p55TNFR is expressed rather constitutively on a broad spectrum of different cell types and has been shown to mediate most of the commonly known biological effects of TNF (5, 6). In contrast, expression of the p75TNFR seems to be modulated by various stimuli, and there are only a few cellular responses that can be attributed exclusively to signaling via the p75TNFR, e.g. proliferation of NK cells (7), B cells (8), thymocytes (9), and mature T cells (10), and GM-CSF secretion of T lymphocytes (11). Moreover, the p75TNFR has been shown to be preferentially activated by membrane-bound TNF (12). Although the intracellular receptor domains show only little similarity, they share activities like NFkappa B activation. Although p55TNFR is capable of mediating these effects when expressed at physiologically relevant levels, induction of NFkappa B via the p75TNFR alone is observed only in cells overexpressing this receptor subtype (13, 14).

In our report we describe the identification of a novel p75TNFR isoform, termed icp75TNFR, generated by the use of an additional transcriptional start site. The elucidated open reading frame of icp75TNFR revealed that the leader sequence in exon 1 of p75TNFR is replaced by an alternative exon. Immunohistochemical staining indicated intracellular expression of the icp75TNFR protein. We further present evidence that expression of icp75TNFR induces NFkappa B activation in TNF-stimulated cells, thus providing an example for intracellular cytokine receptor activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents, Cell Lines-- The rat anti-human p75TNFR antibody had been purchased from Genzyme (Genzyme, Cambridge, MA). The polyclonal antiserum M80 as well as the monoclonal antibodies (mAbs) 80M2, 80A5, and MR2-1 had been kindly provided by M. Grell (Stuttgart, Germany). Mouse L929 cells (fibrosarcoma cells) and the human cell lines HEK 293, HepG2, KYM-1, THP1, U937, and HeLa (ATCC, American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Sigma) supplemented with 10% heat-inactivated fetal calf serum (Life Technologies Inc.) and 0.05 mg/ml gentamicin (PAA Laboratories Linz, Austria). HUVECs and HMEC were kindly provided by G. Eisner (Regensburg, Germany).

Primer Extension Analysis-- Total RNA was isolated from THP-1 cells, and primer extension analysis was performed as described previously (15) using the following primers: Primer 1, 5'-CGGCCTGTTTAGACTCTAGAGCCAGACCACCTGGGTCTGG-3'; Primer 2, 5'-GGCGACGGGCGCCATGGGTGCGGGCGGGGTCCGG-3').

PCR amplification was performed using sense and antisense primers for icp75TNFR (5'-GGGAAGCTTGACAACATGGCGAAACCCCAT-3' and 5'-GGGTCTAGAGGTTAACTGGGCTTCATCCCAGCATCAGGC-3').

p75TNFR expression plasmids (p75TNFR, icp75TNFR, icp75TNFR Delta TRAF, and icp75TNFR Delta TNF) were constructed by inserting the corresponding cDNAs into pcDNA 3.1Hygro (Invitrogen, Groningen, The Netherlands). All expression constructs have been verified by sequencing.

Protein Expression and Western Blot Analysis-- Twenty-four h after transfection, cells were lysed in 1 ml of lysis buffer (1% Nonidet P-40, 0.1% Triton X-100, 0.1% SDS, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) for 30 min on ice. Supernatants were incubated overnight at 4 °C with 50 µl of NTA-agarose (Qiagen), washed twice with 1 ml of denaturing wash buffer (8 M urea, 0.1 M Na2HPO4, 0.01 M Tris base, pH 5.9). Eluted proteins were subjected to SDS polyacrylamide electrophoresis and blotted. Immunoblot analysis was performed with a horseradish peroxidase coupled-anti-V5 epitope antibody (Invitrogen) using the Enhanced Chemiluminescence (ECL) Western Blot Detection System (Energene, Regensburg, Germany).

Immunofluorescence Microscopy-- HeLa cells seeded on glass slides were transfected with expression plasmids using LipofectAMINE (Life Technologies Inc.) according to the manufacturer's instructions. Nuclei of cells were stained with Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) at 37 °C for 10 min. To visualize mitochondria cells were incubated with MitoTracker Red CMXRos (Molecular Probes) for 30 min at 37 °C. Cells were fixed with 2% paraformaldehyde for 10 min, permeabilized with acetone (-20 °C) for 15 min, and incubated in blocking solution (3% bovine serum albumin in PBS) for 2 h at 4 °C. Primary anti-Myc antibody (2 µg/ml in PBS containing 2% bovine serum albumin, Invitrogen) was added, and cells were incubated at 4 °C overnight. A secondary FITC-conjugated rabbit anti-mouse IgG (Dako, Hamburg, Germany) was added for 1 h at room temperature.

Colocalization of the Myc-tagged icp75TNFR protein with mitochondria was studied with the exhaustive photon reassignment (EPR) method.

To examine TNF binding of cells overexpressing p75TNFR or icp75TNFR, HeLa cells were seeded on glass slides and transfected with the corresponding expression plasmids as described above. Twenty-four h later, cells were washed with PBS and incubated with biotinylated TNF (100 ng/ml in PBS; R&D Systems, Wiesbaden, Germany) for 2 h on ice and then washed again with PBS and incubated at 37 °C under standard culture conditions. After 2 h, cells were fixed, permeabilized, and blocked as described before. Primary anti-Myc antibody (2 µg/ml (Invitrogen) in PBS containing 2% bovine serum albumin) was added, and cells were incubated at 4 °C overnight. The following day, cells were incubated with FITC-conjugated rabbit anti-mouse IgG (Dako) and Cy3-conjugated streptavidin (Dako) for 1 h at room temperature.

Transient Transfection and Reporter Assays-- Murine L929 cells stably expressing p75TNFR (L929 p75TNFR), icp75TNFR (L929 icp75TNFR), icp75TNFR Delta TNF (L929 icp75TNFR Delta TNF) or mock-transfected cells (L929 pcDNA3.1) were seeded at a density of 1 × 105. Cells were transiently transfected with an NFkappa B-dependent luciferase reporter plasmid or cotransfected with the NFkappa B-dependent luciferase reporter plasmid and the expression plasmids encoding icp75TNFR Delta TRAF or icp75TNFR Delta TNF deletion mutants using DOTAP (Roche Diagnostics) according to the manufacturer's instructions. Sixteen h after transfection cells were incubated with medium with or without 20 ng/ml mouse TNF for 6 h, and luciferase activity was assayed using the Luciferase Assay System (Promega) according to the manufacturer's directions. Each transfection was done in triplicate and repeated at least three times.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

While studying the transcriptional regulation of the human p75TNFR it was realized that this promoter sequence lacks a consensus TATA element within the first few hundred base pairs proximal to the translational start site ATG, whereas several TATA elements are located further upstream of the translational start site. To determine whether these TATA elements are active transcriptional initiation sites we performed primer extension analysis using RNA derived from THP-1 cells. Two oligonucleotides complementary to the nucleotides +16 to -19 in the cDNA and 5'-UTR (primer 2) and complementary to the nucleotides -795 to -835 in the 5'-flanking region (primer 1) were used as primers (Fig. 1, bottom). The primer extension analysis identified two transcriptional start sites within the 5'-flanking region of the p75TNFR gene. One of them is located at position -47 (TSI) relative to the translational start site, whereas a second transcriptional start site can be localized at position -870 (TSII) relative to the translational start site (Fig. 1, top). We previously reported about two functional major start sites of transcription in the promoter of the mouse p75TNFR gene (15).


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Fig. 1.   Analysis of the p75TNFR transcriptional start sites. RNA derived from THP1 cells was hybridized with either primer 1 or 2, and reverse transcription products were analyzed on a 10% sequencing gel along with a sequencing reaction serving as a size marker. The size of the reaction products are indicated on the left, and the positions of the corresponding transcriptional start sites are indicated on the right. Lane 1, product of the primer extension reaction performed with primer 1; lane 2, product of the primer extension reaction performed with primer 2. The annealing positions of primers 1 and 2 within the RNA template are given below. bp, base pair.

To identify the corresponding cDNA complementary to the mRNA originating from TSII we performed RT-PCR using a 5'-primer located at position -858 to -826 and a 3'-primer located at the 3'-end of the p75TNFR cDNA (Fig. 2A). Three independent cDNA clones were isolated and revealed sequence identity. Primary sequence analysis indicated that transcriptional initiation at TSII within the 5'-flanking region of the p75TNFR gene results in a novel p75TNFR cDNA. Comparison of the two p75TNFR cDNAs indicated sequence identity in the extracellular (ED), transmembrane (TM) and intracellular domain (ID). In contrast, the new p75TNFR cDNA isoform lacks the 5'-UTR and the first exon of p75TNFR (Fig. 2A). Alignment with the genomic sequence further shows that the mRNA transcript initiated at TSII generates a splice donor site at position -600, which fuses the newly transcribed exon 1a to the splice acceptor site at the 5'-end of exon 2 of the p75TNFR transcript. The fusion of this new exon 1a generates an open reading frame (ORF 1a) by positioning the ATG at -681 in frame with exon 2 of the p75TNFR transcript as a potential translational initiation codon (Fig. 2B). Thus, the ORF 1a of the new p75TNFR transcript generates a cDNA that encodes for a mature p75TNFR preceded by a stretch of 27 amino acids encoded by exon 1a (Fig. 2C). To address the question whether exon 1a encodes a putative signal sequence, we compared the predicted amino acid sequences of the p75TNFR cDNA (amino acids 1-26) and the new p75TNFR cDNA (amino acids 1-27) using two different signal peptide prediction methods (SignalP V1.1 and PSORT II, data not shown). Although the first 26 amino acids of p75TNFR were identified as a signal peptide capable of directing a nascent protein to the cell surface, no similarity to any known targeting sequence can be found within the first 27 amino acids of the new p75TNFR.


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Fig. 2.   Structure and sequence of the p75TNFR gene and the two p75TNFR mRNA splice variants. A), alignment of the two p75TNFR mRNA splice variants, mRNA I and mRNA II, originating at the transcriptional start sites TSI and TSII, respectively. Exons are indicated by boxes. SP, signal peptide; ED, extracellular domain; TM, transmembrane domain; ID, intracellular domain; 5'-UTR, 5'-untranslated region; 3'-UTR, 3'-untranslated region. Annealing positions of the sense and antisense primers used to amplify the icp75TNFR cDNA by PCR are indicated by arrows. B, nucleotide sequence of the p75TNFR gene containing the proximal promoter region, exon 1 and 1a, respectively, and the N terminus of exon 2. Translated nucleotide sequences are shown in capital letters. The A at the translational start site of exon 1 is designated as +1. The identified transcriptional start sites are denoted by arrows. 5'-splice sites are indicated by asterisks, the 3'-splice site is indicated by a triangle. The Kozak sequence at the N terminus of exon 1a is shown in italics. C, 5'-regions of p75TNFR cDNAs, alignment of the nucleotide sequences and the corresponding amino acid sequences of the two p75TNFR cDNAs; ORF 1, open reading frame starting at exon 1; ORF 1a, open reading frame starting at exon 1a. D, expression pattern of icp75TNFR mRNA. RT-PCR products were hybridized with a probe specific for the 5'-end of the icp75TNFR cDNA. Amplification of GAPDH cDNA served as an internal control. E, up-regulation of icp75TNFR mRNA expression in HUVEC and U937 cells after stimulation with lipopolysaccharide. RT-PCR was performed as described under "Experimental Procedures." bp, base pair; kb, kilobase.

To determine the expression pattern of icp75TNFR mRNA, we performed RT-PCR analysis using a primer specific for the 5'-end of icp75TNFR cDNA and a 3'-primer annealing in the second exon of the icp75TNFR cDNA resulting in a 360-base pair PCR product. From various cell lines cDNA was prepared and the PCR products were verified by hybridization with a probe specific for the 5'-untranslated region of the icp75TNFR cDNA. Whereas no icp75TNFR mRNA could be detected in HEK 293 and HepG2 cells, HeLa, Kym-1, THP-1, as well as HMEC cells expressed the icp75TNFR mRNA (Fig. 2D). Furthermore, stimulation of HUVEC and U937 cells with lipopolysaccharide resulted in an up-regulation of icp75TNFR mRNA suggesting that the icp75TNFR expression is regulated by proinflammatory stimuli (Fig. 2E). The same cell lines that were positive for the icp75TNFR were also found positive for p75TNFR mRNA expression in parallel experiments (data not shown). Compared with the p75TNFR mRNA expression level, the relative abundance of the icp75TNFR mRNA seemed much lower in all cell lines and primary cells we have tested so far.

Transient expression of the icp75TNFR cDNA with a C-terminal V5 His-tag in HeLa cells followed by purification and Western blot analysis using an antibody to V5 resulted in detection of a single protein band with an apparent molecular mass of ~50 kDa (Fig. 3A, lane 2), whereas no protein expression was detected in wild-type Hela cells (Fig. 3A, lane 1). Thus, the identified ORF 1a generated by the use of TSII and subsequent alternative splicing of the p75TNFR mRNA resulted in the icp75TNFR protein with an expected molecular mass. The elucidated molecular mass of icp75TNFR is very close to the calculated value of 48.5 kDa. In contrast, it has been shown in previous studies that the expression of the p75TNFR cDNA results in a protein signal ~75 kDa using Western blot analysis.


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Fig. 3.   Transient expression and characterization of the icp75TNFR protein. A, expression of icp75TNFR as a V5 His fusion protein. HeLa cells were transiently transfected with an expression plasmid containing the V5 His-tagged icp75TNFR cDNA. Twenty-four h after transfection cells were lysed and the icp75TNFR V5 His fusion protein was purified as described under "Experimental Procedures." An aliquot of the purified protein was separated by SDS-polyacrylamide gel electrophoresis and detected by Western blotting using an anti-V5 antibody. B, TNF binding capacity of icp75TNFR. HeLa cells were seeded on glass slides and transiently transfected with the expression plasmid encoding Myc-tagged icp75TNFR cDNA. Twenty-four h later, cells were incubated with biotinylated human TNF as described under "Experimental Procedures" and stained with an anti-Myc antibody and a secondary FITC-conjugated antibody (top) as well as with Cy3-conjugated streptavidin (bottom). Magnification factor of each panel is 1000×. C, recognition of icp75TNFR protein by different anti-p75TNFR antibodies. L929 cells were seeded on glass slides and transiently transfected with expression plasmids encoding Myc-tagged p75TNFR or icp75TNFR cDNA. Twenty-four h later, cells were stained with the indicated anti-p75TNFR antibodies as described under "Experimental Procedures."

HeLa cells were transiently transfected with Myc-tagged icp75TNFR cDNA and incubated with biotinylated human TNF followed by simultaneous staining with FITC-coupled anti-Myc antibody and Cy3-coupled streptavidin (Fig. 3B). In contrast to the staining of TNF bound to endogenous TNFRs in wild-type HeLa cells, which is visible as clustering in close proximity to the nucleus, the staining pattern of HeLa cells overexpressing icp75TNFR protein was characterized by a dispersed intracellular receptor staining that was colocalized with biotinylated TNF. This result indicates that the icp75TNFR protein is capable of binding TNF.

To further characterize the icp75TNFR protein, immunohistochemical staining of transiently transfected mouse L929 cells was performed using different mAbs as well as a polyclonal antiserum raised against the human p75TNFR protein. We observed positive staining in L929 cells transfected with either the p75TNFR or icp75TNFR cDNA indicating that the icp75TNFR is indistinguishable from p75TNFR by the use of a polyclonal anti-human p75TNFR serum and by all mAbs we have tested so far (Fig. 3C). No difference was observed in fluorescence-activated cell sorter staining experiments when U937 cells were either nontreated for staining of membrane p75TNFR or when the cell membrane was perforated for intracellular staining (data not shown). In both cases the staining of the p75TNFR protein was clearly enhanced after lipopolysaccharide stimulation.

To investigate the subcellular localization of the icp75TNFR protein we performed immunohistochemical staining of transiently transfected HeLa cells expressing either of the two p75TNFR cDNA isoforms fused to a C-terminal Myc-tag. Staining with an anti-Myc antibody revealed that the expression of p75TNFR can easily be recognized on the cell surface of the transiently transfected HeLa cells (Fig. 4A). In contrast, the expression of icp75TNFR was localized predominantly in the intracellular compartment (Fig. 4B), whereas no specific staining was detected in cells transfected with expression plasmid alone (Fig. 4C).


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Fig. 4.   Different subcellular localization of p75TNFR and icp75TNFR. HeLa cells were transiently transfected with expression plasmids encoding Myc-tagged p75TNFR (A), icp75TNFR (B), or the empty expression plasmid (C). Twenty-four h after transfection, cells were stained with anti-Myc antibody and a FITC-conjugated secondary antibody. Magnification factor of the left and middle panels is 1000-fold. HeLa cells transfected with p75TNFR demonstrate a cell membrane staining, whereas cells transfected with icp75TNFR show a diffuse intracellular staining. D, EPR analysis of the subcellular distribution of icp75TNFR. HeLa cells were transiently transfected with the Myc-tagged icp75TNFR expression plasmid. Cells were incubated with the mitochondria marker MitoTracker Red CMXRos for 30 min. Myc-tagged icp75TNFR was detected with a FITC-conjugated antibody as described above. A diffuse intracellular staining of icp75TNFR was confirmed using the EPR method as described under "Experimental Procedures." Colocalization of icp75TNFR (green) with the mitochondrial marker (red) can be seen only to a small extent using combined immunofluorescence (yellow).

The result of an EPR analysis, which provides a computer-based digital-confocal imaging, confirmed intracellular expression of the icp75TNFR protein characterized by a diffuse intracellular staining (Fig. 4D). Colocalization of icp75TNFR with a mitochondrial marker was recognized only to a small extent.

Because there have been several reports in the past correlating the expression level of p75TNFR with NFkappa B activation (16) we asked whether the expression of icp75TNFR would influence the TNF-induced activation of NFkappa B in L929 cells. L929 cells stably transfected with the cDNA of p75TNFR or icp75TNFR were tested for their ability to activate a NFkappa B-dependent reporter gene. Although expression of the p75TNFR protein in L929 cells resulted in only marginal activation of NFkappa B after treatment with TNF compared with a control transfection, a significant increase in NFkappa B activation was detected in L929 cells transfected with the cDNA for the icp75TNFR (Fig. 5A). In contrast L929 cells expressing icp75TNFR protein lacking the TNF binding domain are no longer capable of inducing NFkappa B after TNF treatment (Fig. 5B). This result indicates that ligand binding by the icp75TNFR protein is necessary for signal transduction and NFkappa B activation.


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Fig. 5.   TNF-mediated NFkappa B activation in L929 cells expressing different p75TNFR variants. A, enhanced NFkappa B activation in L929 expressing icp75TNFR. L929 cells were stably transfected with expression plasmids coding for p75TNFR or icp75TNFR, respectively, or were mock-transfected. Stable transfectants were assayed for NFkappa B activation by transient transfection of a NFkappa B luciferase reporter plasmid. Sixteen h after transfection, cells were stimulated with 20 ng/ml mouse TNF (hatched bars) or cultured in TNF-free medium for another 6 h (white bars). Luciferase activity was determined as described under "Experimental Procedures." B, impaired NFkappa B activation in L929 cells expressing an icp75TNFR deletion mutant lacking the TNF binding domain. Stable transfectants were assayed as described above in A. C, different effects on NFkappa B activation by overexpression of icp75TNFR deletion mutants lacking the TRAF2 binding domain or the TNF binding domain. L929 cells stably expressing icp75TNFR were cotransfected with a NFkappa B reporter plasmid and the expression plasmids for the icp75TNFR proteins without the TRAF2 binding domain (icp75 Delta TRAF) or without the TNF binding domain (icp75 Delta TNF). Sixteen h later, cells were stimulated with 20 ng/ml mouse TNF or cultured in TNF-free medium for another 6 h. Luciferase activity was determined as described under "Experimental Procedures."

It has been shown by mutational analysis within the cytoplasmic domain of p75TNFR that a C-terminal region responsible for the binding of TNF receptor-associated factor 2 (TRAF2) is indispensable for signal transduction and NFkappa B activation. Coexpression of a p75TNFR mutant, which lacks the TRAF2 domain, resulted in a dominant-negative effect implicating that this domain is essential for the response (17). Accordingly, we tested whether the TRAF2 domain of icp75TNFR is also involved in signal transduction of the icp75TNFR. In fact, coexpression of icp75TNFR lacking the TRAF2 binding domain in the corresponding stably transfected L929 cells resulted in inhibition of TNF-induced NFkappa B activation (Fig. 5C). This observation suggests that, also for the intracellularly expressed form of icp75TNFR, the TRAF2 domain is critical for signal transduction. In contrast, coexpression of icp75TNFR lacking the TNF binding domain did not reduce TNF-induced NFkappa B activation (Fig. 5C). This supports the result of our previous experiment (Fig. 5B) indicating that intracellular signaling by icp75TNFR seems to be ligand-dependent because coexpression of icp75TNFR Delta TNF, which is incapable of ligand-dependent oligomerization of its TRAF2 binding domains, did not interfere with TNF-induced NFkappa B activation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TNF exerts pleiotropic activities affecting proliferation, differentiation, or function in a wide variety of cell types (18). In the past two distinct receptors for TNF, referred here to as p55TNFR and p75TNFR, have been characterized (19). Our present data identify a novel human p75TNFR mRNA isoform termed icp75TNFR, which originates from an additional transcriptional start site in the 5'-flanking region of the p75TNFR gene. The deduced ORF is characterized by sequence identity concerning the extracellular, transmembrane, and intracellular receptor domains, but differs in the N-terminal sequence as well as in the 5'-untranslated region. Replacement of the leader sequence encoded by exon 1 by a novel sequence that does not contain characteristics for directing the protein into a specific cellular compartment suggests that the icp75 protein may not be transported to the cell membrane by the regular endosomal secretion pathway. The presence of the transmembrane domain in the icp75TNFR suggests that rather the protein remains inserted into a intracellular perinuclear membrane and not expressed in the cytoplasma. Our results indicate that the icp75TNFR protein is indistinguishable from p75TNFR using different mAbs as well as a polyclonal antiserum raised against human p75TNFR. This fact represents a major problem in the attempt to specifically detect endogenous icp75TNFR protein in cells that produce the membrane form of the p75TNFR at the same time. Immunohistochemical analysis revealed that the localization of the novel icp75TNFR is characterized by a diffuse intracellular staining pattern. Further evidence for intracellular expression of icp75TNFR was given by the observation that the expressed protein is detected with an apparent molecular mass of ~50 kDa, which is close to the calculated value of the primary amino acid sequence (48.5 kDa). This suggests that no posttranslational modifications, e.g. glycosylation, occur by which the transmembrane form of the p75TNFR is characterized. Interestingly, Ledgerwood et al. (20) described a 60-kDa protein localized in the inner mitochondrial membrane of umbilical vein endothelial cells, which was recognized by a specific mAb against human p75TNFR and was also capable of binding TNF. Our data show that the icp75TNFR protein was recognized by all mAbs raised against human p75TNFR that we have tested so far including the one used by Ledgerwood et al. EPR analysis showed that in addition to diffuse intracellular staining a small amount of icp75TNFR protein possibly was colocalized with a mitochondrial marker. The difference in the staining pattern may be due to overexpression of icp75TNFR in our experiments. Specific staining of endogenous icp75TNFR protein by cytochemistry or fluorescence-activated cell sorter analysis was not possible so far because no reagents are available that can distinguish between the membrane form of the p75TNFR and the icp75TNFR. More detailed localization studies have to be done to dissect the expression pattern of two p75TNFR isoforms.

Overexpression of icp75TNFR induced significant NFkappa B activation in L929 cells after TNF stimulation suggesting that this receptor subtype is capable of triggering signaling events in a TNF-dependent manner. L929 cells stably transfected with the p75TNFR cDNA encoding the transmembrane receptor subtype as confirmed by immunocytochemical staining and RT-PCR analysis show only a marginal TNF-induced increase in NFkappa B-dependent reporter gene activation compared with the control cells. It has been shown previously that p75TNFR is capable of inducing NFkappa B activation and that for this signal transduction the TRAF2 binding domain is indispensable (17). This also seems to be the case for NFkappa B activation by the icp75TNFR protein arguing for a direct involvement in TNF-mediated signaling. It remains to be analyzed whether TNF has to be internalized by the transmembrane receptors as described earlier (19), by a suggested TNFR-independent mechanism (20), and/or whether endogenously produced TNF is responsible for activation of icp75TNFR followed by intracellular signaling. In addition, the functional consequences of such intracellular TNF signaling via the icp75TNFR is currently under investigation.

    ACKNOWLEDGEMENTS

We thank Dr. Matthias Grell for the anti-p75TNFR antibodies and helpful discussions.

    FOOTNOTES

* This work was supported by Grant HE 3116 1-1 from the Deutsche Forschungsgemeinschaft (to T. H. and D. N. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. Pathology/Tumor Immunology, Univ. of Regensburg, F.-J.-Strauss-Allee, D-93042 Regensburg, Germany. Tel.: 49-941-944-6622; Fax: 49-941-944-6602; E-mail: daniela.maennel@klinik.uni-regensburg.de.

Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M101336200

    ABBREVIATIONS

The abbreviations used are: TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; mAb(s), monoclonal antibody(ies); PCR, polymerase chain reaction; PBS, phosphate-buffered solution; FITC, fluorescein isothiocyanate; EPR, exhaustive photon reassignment; ORF, open reading frame; RT, reverse transcription; TRAF2, TNF receptor-associated factor 2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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