TNF Recruits TRADD to the Plasma
Membrane But Not the trans-Golgi Network,
the Principal Subcellular Location of TNF-R1
This information is current as
of June 15, 2017.
Sally J. Jones, Elizabeth C. Ledgerwood, Johannes B. Prins,
Jenny Galbraith, David R. Johnson, Jordan S. Pober and
John R. Bradley
J Immunol 1999; 162:1042-1048; ;
http://www.jimmunol.org/content/162/2/1042
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References
TNF Recruits TRADD to the Plasma Membrane But Not the
trans-Golgi Network, the Principal Subcellular Location of
TNF-R11
Sally J. Jones,* Elizabeth C. Ledgerwood,*† Johannes B. Prins,*† Jenny Galbraith,*
David R. Johnson,‡ Jordan S. Pober,‡ and John R. Bradley2*
T
umor necrosis factor (TNF) mediates a broad range of
biological activities (1–3) through interaction with two
distinct membrane-bound receptors, TNF-R1 (;55 kDa)
and TNF-R2 (;75 kDa). TNF binding is followed by receptor
clustering and internalization of the TNFR complex by endocytosis (4). However, the role of ligand or receptor internalization in
the induction of cellular responses remains unclear. Inhibition of
receptor internalization reduces some aspects of TNF signaling (5,
6), but it is unlikely that the ligand needs to be internalized because
many effects of TNF can be mimicked by agonistic antireceptor
Abs (7–9).
Several proteins couple TNFRs to signaling cascades through
interaction with the intracellular domains of TNF-R1 and TNF-R2.
TNF-R1 interacts in a ligand-dependent process with a 34-kDa
TNFR-associated death domain protein (TRADD)3 leading to the
activation of NF-kB, Jun N-terminal, and p38 activated protein
kinases and programmed cell death (10, 11). In contrast, TNF-R2
has been shown to interact with heterocomplexes of TNFR-asso-
ciated factors 1 and 2 (TRAF1 and TRAF2) in a non-TNF-dependent manner (12).
TNF-R1 and TNF-R2 are traditionally regarded as cell surface
receptors, although we have reported that TNF-R1 is primarily a
Golgi-associated protein (13) with only a minor subpopulation of
receptors expressed in the plasma membrane. However, the precise
Golgi subcompartment (cis-, medial-, trans-Golgi cisternae) or the
trans-Golgi network (TGN) in which TNF-R1 resides had not been
characterized. With respect to TNF-signaling pathways, this may
be important since the TGN is responsible not only for sorting
proteins, endosomes and lysosomes, to the cell surface (14 –18),
but also for retrieving and reusing plasma membrane components
internalized by endocytosis (19 –21). In this study, we have defined
the precise Golgi subcompartment in which TNF-R1 is localized
and determined the potential of TRADD to associate with TNF-R1
in the Golgi compartment after TNF treatment.
Materials and Methods
†
Departments of *Medicine and Clinical Biochemistry, University of Cambridge,
Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom; and ‡Boyer Center
for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536
Received for publication June 23, 1998. Accepted for publication September
30, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the National Kidney Research Fund (U.K.), the Wellcome Trust, and the National Institutes of Health. J.R.B. is a National Kidney Research Fund (U.K.) Senior Fellow. J.B.P. is a Wellcome Trust International Fellow.
2
Address correspondence and reprint requests to Dr. John Bradley, Department of
Medicine, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, U.K. E-mail
address: [email protected]
3
Abbreviations used in this paper: TRADD, TNFR-associated death domain protein;
TRAF, TNFR-associated factors; TGN, trans-Golgi network; HRP, horseradish peroxidase; BAEC, bovine aortic endothelial cells; BFA, brefeldin A; PNS, post nuclear
supernatant; MTOC, microtubule organizing center; ER; endoplasmic reticulum.
Copyright © 1999 by The American Association of Immunologists
Materials
Medium 199, RPMI 1640 medium, FCS, L-glutamine, penicillin/streptomycin, trypsin-EDTA, brefeldin-A (BFA), sucrose, Trisma-Base, NaCl,
EDTA, Triton X-100, aprotinin, PMSF, leupeptin, and pepstatin A were all
purchased from Sigma-Aldrich (Poole, U.K.). Human rTNF was provided
by Autogen Bioclear (P30001A; Potterne, U.K.). The mouse monoclonal
anti-human TNF-R1 was obtained from Genzyme Diagnostic (1995-01;
Cambridge, MA), goat polyclonal anti-human TNF-R1 Abs were from
R&D Systems (AB-225-PB; Minneapolis, MN), mouse monoclonal antip58 was from Sigma (G2404; St. Louis, MO) and mouse monoclonal antiTRADD Ab was from Transduction Laboratories (T50320; Lexington,
KY). The rabbit polyclonal Ab to TGN46 was a kind gift from Dr. S.
Ponnambalam at the University of Dundee. Secondary FITC-conjugated
Abs were from Sigma and Texas Red-conjugated Abs from Dako (Carpinteria, CA). Horseradish peroxidase (HRP) -conjugated donkey anti-mouse
IgG was from Jackson ImmunoResearch Laboratories (West Grove, PA)
and HRP-conjugated porcine anti-rabbit from Dako. Protein G Sepharose
4 fast flow was from Pharmacia (Piscataway, NJ).
0022-1767/99/$02.00
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The subcellular localization of TNF-R1 to the Golgi apparatus, initially observed in endothelial cells, has been confirmed using
transfection of bovine aortic endothelial cells with a human TNF-R1 expression plasmid. The subcellular interactions of TNF-R1
and the TRADD (TNFR-associated death domain protein) adaptor protein have been analyzed in the human monocyte cell line
U937 and the human endothelial cell line ECV304 by confocal immunofluorescence microscopy and by Western blot analysis of
fractionated cell extracts. In untreated cells, in which TNF-R1 is found on the cell surface but principally localizes to the transGolgi network, TRADD is concentrated in the cis- or medial-Golgi region, but separates from the Golgi during cell fractionation.
Coimmunoprecipitation studies have shown that TRADD binds to TNF-R1 within 1 min of TNF treatment in a cell fractioncontaining plasma membrane. This association is followed by a gradual dissociation, which is prevented if receptor-mediated
endocytosis is inhibited by hypertonic medium. In contrast, no association is detected between TRADD and TNF-R1 in the Golgi
in response to exogenous TNF at any time examined. These results suggest that although TNF-R1 is predominantly a Golgiassociated protein and TRADD also localizes to the Golgi region, exogenous TNF causes TRADD to bind to TNF-R1 only at the
plasma membrane. The Journal of Immunology, 1999, 162: 1042–1048.
The Journal of Immunology
1043
Cell culture
ECV304 cells, a spontaneously immortalized cell line of human umbilical
vein origin (22) (a kind gift from I. Fritz, Babraham Institute, Cambridge,
U.K.) were cultured in medium 199 supplemented with 10% FCS, L-glutamine, and antibiotics. U937 cells, a human monocyte cell line, were
cultured in RPMI 1640 medium supplemented with 10% FCS, L-glutamine,
and antibiotics.
Immunofluorescence microscopy
ECV304 cells were seeded onto sterile glass coverslips in 24-well plates
(Costar, High Wycombe, U.K.) 16 –24 h before fixation. Cells were washed
in PBS, fixed in 2% paraformaldehyde for 2 min, and permeabilized with
0.1% Triton X-100 for 2 min. U937 cells were washed in PBS, fixed as
above, cytospun at 600 rpm for 15 s, and permeabilized as above. Cells
were incubated with primary Abs diluted in 1% BSA/PBS for 1 h at room
temperature. After they were washed, the cells were incubated with secondary conjugated Abs for 1 h at room temperature. Coverslips or slides
were mounted in Citifluor and examined using a Nikon Optiphot-II microscope (Nikon, Kingston upon Thames, U.K.) coupled to a Bio-Rad
(MRC1000; Hemel Hempstead, U.K.) confocal attachment and COMOS
software (BioRad).
Expression constructs encoding the TNFR (TNF-R1, pcDNA3, and TNFR2, pcDNA3) were generated by ligating NotI fragments containing the
coding sequences from an expression vector generously provided by Immunex (Seattle, WA) into NotI-digested pcDNA3.1 (Invitrogen, San Diego, CA). BAEC were cultured in DMEM supplemented with 10% FCS, 5
mM HEPES, L-glutamine (1:100) sodium phosphate, penicillin, and streptomycin (all from Life Technologies, Gaithersburg, MD). Subconfluent
BAEC in 35 mm tissue culture dishes (C-6) were transfected with the
TNFR expression constructs (3 mg/0.2 ml Optimen with 6 ml lipofectamine/0.2 ml according to the manufacturer’s directions (Life Technologies). After culture overnight, cells were transferred to 10-cm-plates
and transfectants were selected with 0.5 mg ml21 G418 (Life
Technologies).
FIGURE 1. Transfection of human TNF-R1 and TNF-R2 into BAECs.
BAECs transfected with human TNF-R1 exhibit a Golgi immunofluorescence pattern (A) when stained with a mouse monoclonal anti-human
TNF-R1 Ab, whereas BAEC transfected with human TNF-R2 demonstrate
diffuse cytoplasmic and surface membrane staining (B). Staining of nontransfected cells was not observed with either Ab.
Coimmunoprecipitation
Subcellular fractionation
ECV304 cells (detached using PBS containing 0.05% EDTA) and U937
cells were washed three times in PBS (600 3 g for 5 min). All subsequent
steps were performed at 4°C. The washed cell pellets were resuspended
(1 3 108 cells ml21) in homogenization buffer (10 mM Tris-HCl (pH 7.4),
containing 3% (w/v) sucrose and proteinase inhibitors 2 1 mM EDTA, 10
mg ml21 aprotinin, 1 mg ml21 leupeptin, 1 mg ml21 pepstatin A and 1 mM
PMSF) and allowed to swell in the hypotonic buffer for 20 min. The cells
were then homogenized in buffer containing 0.25 M sucrose using 20 –30
strokes in a Dounce homogenizer. The homogenate was centrifuged
(1300 3 g for 5 min) to obtain a nuclear-cell debris pellet and postnuclear
supernatant (PNS). The PNS was further centrifuged (10,000 3 g for 15
min) and the postmitochondrial/lysosomal supernatant (10K supernatant)
was further centrifuged (100,000 3 g for 1 h) to yield a cytosolic fraction
(100K supernatant) and a microsomal pellet (100K pellet). The 100K pellet
was resuspended in 1.5 M sucrose and overlaid with 1.15 M, 0.9 M, 0.6 M,
and 0.25 M sucrose. The discontinuous sucrose gradient was centrifuged at
100,000 3 g for 2 h. The individual interfaces were removed, diluted in
0.25 M sucrose, and centrifuged (100,000 3 g for 1 h). The pellets were
resuspended in 500 ml of 0.25 M sucrose and assayed for alkaline phosphodiesterase activity (plasma membrane) and a mannosidase II activity
(Golgi marker) as described by Storrie and Madden (23) and the protein
concentrations were determined using the Bradford assay (24). Samples
were used immediately or stored at 220°C.
SDS-PAGE and Western blot analysis
Equivalent amounts of fractionated protein (5–25 mg) were resolved by
10% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were
blocked in buffer containing Tris-HCl (pH 7.4), 5% dried milk powder, and
0.05% Tween 20. After blocking, the membrane was immunoblotted with
mouse monoclonal anti-TNF-R1 (1:1500), mouse monoclonal anti-p58 (1:
500), mouse monoclonal anti-TRADD (1:500), or rabbit anti-TGN46 (1:
5000) for 2 h at room temperature or overnight at 4°C. The bound Abs were
visualised by chemiluminescence (Kirkegaard & Perry Laboratory,
Gaithersburg, MD).
U937 cells (5 3 107 ml21) were washed three times in PBS (600 3 g for
5 min) and incubated in the presence or absence of TNF (100 ngml21) for
variable time periods. The cells were washed once in PBS (600 3 g for 5
min) and either lysed immediately in 500 ml of lysis buffer (20 mM Tris
(pH 7.5), 100 mM NaCl, 1% Triton X-100, and proteinase inhibitors) for
1 h on ice or chased for variable time periods in PBS then lysed or to study
the effect of inhibiting receptor mediated endocytosis, the cells were chased
in prewarmed PBS or 0.45 M sucrose for variable time periods before lysis.
In the meantime, prewashed Protein G Sepharose beads (40 ml) were incubated with 20 ml of goat anti-TNF-R1 Ab in 500 ml of lysis buffer using
an upright rotator (1 h, 4°C). The U937 lysate was centrifuged (10,000 3
g for 15 min at 4°C) and the supernatant incubated with the Protein G/Ab
complex for 3 h at 4°C. The Sepharose beads were then washed four times
with lysis buffer and twice with lysis buffer lacking Triton X-100. The
precipitates were resuspended in nonreducing Laemmli sample buffer,
boiled, resolved by SDS-PAGE, and transferred onto nitrocellulose. After
blocking, the membrane was immunoblotted with the mouse monoclonal
anti-TRADD Ab (1:500) for either 2 h at room temperature or overnight at
4°C and visualized with HRP-conjugated donkey anti-mouse IgG using the
chemiluminescence detection system.
Results
Subcellular localization of TNF-R1
In our previous report (13), we showed that in HUVEC, TNF-R1
is principally localized in the Golgi apparatus. Although several
different Abs revealed Golgi staining, not all Abs reacted with all
cells. Therefore, we transfected expression plasmids for TNF-R1
and TNF-R2 to confirm that there was specific recognition of
TNF-R1 in the Golgi. BAECs transfected with human TNF-R1
exhibited a compact juxtanuclear staining pattern (Fig. 1A) characteristic of Golgi-associated proteins (25) when stained with
monoclonal anti-human TNF-R1 Ab (R&D Systems). This Ab did
not stain untransfected BAEC. In contrast, BAEC transfected with
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Transfection of human TNF-R1 and TNF-R2 into bovine aortic
endothelial cells (BAEC)
1044
SUBCELLULAR LOCALIZATION OF TNF-R1 AND TRADD
FIGURE 2. Recognition of TNF-R1, p58, and TGN46 in the Golgi enriched 0.9/1.15 M sucrose interface. The microsomal pellet of ECV304
cells was centrifuged on a discontinuous sucrose gradient and 20 mg of
protein from each interface fractionated by SDS-PAGE under reducing
conditions, transferred to nitrocellulose, and blotted with either: mouse
monoclonal p58 Ab or mouse monoclonal TNF-R1 Ab (A) or rabbit polyclonal control Ab or rabbit polyclonal TGN46 Ab (B).The mouse monoclonal anti-TNF-R1 Ab recognizes a band of approximately 60 kDa which
is enriched in the 0.9/1.15 M sucrose interface. The a mannosidase II
activity at this interface is 7-fold higher than the PNS. The rabbit polyclonal anti-TGN46 recognizes a close set of bands of approximately 100 to
120 kDa predominantly in the 0.9/1.15 M interface. The mouse monoclonal
anti-p58 Ab recognizes a 58-kDa band in the PNS, 0.9/1.15 M interface,
and the 1.15/1.5 M interface, with enrichment in the 0.9/1.15 M interface.
One of six experiments with similar results. C, A similar enrichment of
TNF-R1, p58, and TGN-46 in the 0.9/1.15 M interface was observed in
U937 cells analyzed under the same conditions. One of three experiments
with similar results was performed.
human TNF-R2 demonstrated diffuse cytoplasmic and surface
membrane staining with anti-human TNF-R2 Abs (Fig. 1B). Because untransfected BAECs do not stain with the Ab to human
TNF-R1, the Golgi-associated staining can be unequivocally attributed to TNF-R1 localization to this subcellular compartment.
To further analyze the location and function of TNF-R1, we
extended our studies to two other cell types, the ECV304 human
endothelial and the U937 human monocyte cell lines that provide
convenient sources of material for subcellular fractionation and
coimmunoprecipitation studies (Fig. 2). Both ECV304 and U937
cells exhibit a staining pattern characteristic of Golgi-associated
proteins when labeled with a mouse mAb to TNF-R1 (Figs. 3 and
4). We extended this morphological approach by immunoblotting
TNF-R1 in fractionated cells. In both ECV304 (Fig. 2, A and B)
and U937 cells (Fig. 2C) TNF-R1 was enriched in the 0.9/1.15 M
interface together with p58, a medial-Golgi marker, and TGN46, a
trans-Golgi marker. In Fig. 2B, the TGN46 polyclonal Ab recognizes a set of closely spaced bands of ;100 –120 kDa attributable
to TGN46 being highly glycosylated (26). a mannosidase II specific activity was 7-fold higher in this interface than the PNS. The
enrichment of TNF-R1 in this Golgi-containing fraction provides
biochemical evidence for the Golgi association of TNF-R1, complementing the previous immunocytochemical approaches.
Sub-Golgi localization of TNF-R1
To investigate with which Golgi subcompartment (cis-, medial-,
trans-Golgi cisternae or TGN) the receptor is associated, cells
were cultured in the presence or absence of BFA and again
analyzed by confocal microscopy. This fungal metabolite
causes the different Golgi compartments to redistribute in a
characteristic manner. In many cell types, the TGN collapses
around the microtubule organizing center (MTOC) giving a
spotlike appearance (27), whereas other Golgi compartments
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FIGURE 3. Effect of BFA on the subcellular localization of TNF-R1.
ECV304 cells incubated in the presence or absence of BFA were fixed,
permeabilized and labeled with either the mouse monoclonal anti-TNF-R1
Ab, the rabbit polyclonal anti-TGN46 Ab or the mouse monoclonal antip58 Ab. In the absence of BFA all three Abs exhibited perinuclear immunofluorescence in a Golgi-like distribution. In the presence of BFA the
immunofluorescence pattern exhibited by the anti-TNF-R1 and antiTGN46 Abs was of a collapse to the MTOC, characteristic of trans-Golgiassociated proteins. However, anti-p58 exhibited a diffuse punctate cytoplasmic staining pattern. No immunofluorescence staining was observed
with the nonimmune mouse and rabbit IgG controls.
The Journal of Immunology
1045
FIGURE 4. Colocalization of TNF-R1 with TGN46, a trans-Golgi
marker. ECV304 cells were fixed, permeabilized, and double labeled by
simultaneous incubations of cells with the mouse monoclonal anti-TNF-R1
Ab (A) and the rabbit polyclonal anti-TGN46 (B). Bound Ab was visualized using secondary Ab coupled to FITC (A) or Texas Red (B). The
merged image (C) confirms colocalization.
redistribute into the endoplasmic reticulum (ER) giving a cytoplasmic staining pattern (28). After treating ECV304 cells for
4 h with BFA, the immunofluorescence pattern associated with
TNF-R1 and TGN46 was that of a compact center of staining at
the nuclear membrane, representative of a collapse to the
MTOC, whereas the immunofluorescence pattern associated
with p58 was now dispersed throughout the cytoplasm consistent with the redistribution of p58 into the ER (Fig. 3). In all
cases, the collapse to the MTOC and the redistribution to the
ER was rapid, occurring within 10 min. Merged images following costaining with the rabbit polyclonal anti-TGN46 Ab and
the mouse monoclonal anti-TNF-R1 Ab demonstrated colocalization (Fig. 4), although anti-TNF-R1 Abs additionally exhibited weak cytoplasmic staining. Identical staining patterns were
also observed in U937 cells (Fig. 5). We conclude that most
TNF-R1 resides in the TGN, but smaller receptor pools also
exist.
Subcellular localization of TRADD
The principal signal transduction pathways initiated by ligand
binding to TNF-R1 are mediated by the recruitment of TRADD.
To determine whether the TGN was involved in TNF signaling, we
investigated whether TRADD also demonstrated a Golgi distribution. By indirect immunofluorescence using U937 cells (Fig. 5)
and ECV304 cells (data not shown), the monoclonal anti-TRADD
Ab exhibited a compact perinuclear stain characteristic of Golgiassociated proteins and similar to that observed with anti-TNF-R1
Abs on cells cultured both in the presence and absence of TNF.
After BFA treatment, the immunofluorescence pattern associated
with anti-TRADD Abs dispersed throughout the cytoplasm (Fig. 5)
suggesting redistribution to the ER. This pattern suggests a medialor cis-Golgi association, unrelated to the distribution of TNF-R1.
In contrast to the staining pattern, Western blot analysis of subcellular fractions derived from cells cultured in the absence of TNF
suggested that TRADD was a cytosolic protein (Fig. 6A), whereas
under the same conditions TNF-R1 was associated with membrane
containing fractions (Fig. 6B). These data suggest that in untreated
cells, TRADD is loosely associated with the Golgi but not the
TGN in which TNF-R1 is localized.
TNF recruitment of TRADD to TNF-R1
In U937 cells, TRADD associates with TNF-R1 in a TNF-dependent process (11). Therefore, we used U937 cells to examine
whether TRADD could be recruited to TNF-R1 in the Golgi. First,
we examined the ability to coimmunoprecipitate TRADD with
TNF-R1. TRADD coimmunoprecipitated with TNF-R1 in TNFtreated, but not mock-treated, cells from the whole cell lysate and
10K pellet, but poorly from the 100K membrane pellet that contains TGN-derived membranes (Fig. 7). Because plasma membranes are found to a greater extent in the 10K pellet, these results
are consistent with the conclusion that TRADD coimmunoprecipitated in the 10K pellet is associated with plasma membrane TNFR1. The failure to find TRADD in immunoprecipitates from the
100K membrane pellet of TNF-treated cells, suggests that the
Golgi complex is not involved in signaling in the response to
exogenous TNF.
After ligand binding, TNF-R1 is internalized (29) probably via
endosomes (present in the 100K pellet) and then lysosomes
(present in 10K pellet). Thus our failure to find TRADD associated
with TNF-R1 in the 100K pellet not only indicates that TRADD
does not associate with TNF-R1 in the Golgi, but also suggests that
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FIGURE 5. Subcellular localization of TRADD. U937 cells incubated
in the presence or absence of BFA were fixed, permeabilized, and labeled
with either anti-TNF-R1 or anti-TRADD Ab. In the absence of BFA, both
Abs exhibited perinuclear immunofluorescence in a Golgi-like distribution.
In the presence of BFA, the immunofluorescence pattern exhibited by the
anti-TNF-R1 Ab was of a collapse to the MTOC, whereas anti-TRADD Ab
exhibited a diffuse punctate cytoplasmic staining pattern.
1046
SUBCELLULAR LOCALIZATION OF TNF-R1 AND TRADD
and investigated the ability to coimmunoprecipitate TRADD with
TNF-R1. In PBS, TRADD dissociates from the receptor in between 30 and 60 min, but if receptor-mediated endocytosis is in-
FIGURE 6. Recognition of TRADD and TNF-R1 in different subcellular fractions. ECV304 cells were homogenized and fractionated, and 15 mg
of protein from the PNS, 10K, and 100K pellet and supernatants were
separated by SDS-PAGE. A, Anti-TRADD Ab specifically recognized a
band of approximately 34 kDa in the cytosolic (10K and 100K supernatants), but not in the membrane fractions (10K and 100K pellets). B, In
contrast, anti-TNF-R1 Ab recognized a band of approximately 60 kDa in
membrane fractions, but not in cytosolic fractions.
TRADD molecules, recruited to TNF-R1 in the plasma membrane,
may dissociate as TNF-R1 moves to endosomes.
To examine this possibility the kinetics of TNF-dependent
TRADD coimmunoprecipitation at both 4°C (inhibits receptor mediated endocytosis) and room temperature was determined. The
recruitment of TRADD to TNF-R1 in U937 cells is rapid, occurring within 1 min even at 4°C (Fig. 8A). Therefore, we pulsed
U937 cells with TNF for 1 min at 4°C and chased in PBS for 1 or
60 min at 20°C, prepared the 10K and 100K pellets, and examined
the ability to coimmunoprecipitate TRADD from each of these
subcellular fractions. TRADD was coimmunoprecipitated from the
10K pellet after 1 min, but not after 60 min, whereas TRADD was
not coimmunoprecipitated from the 100K pellet at either time
point (Fig. 8B). These data are consistent with the conclusion that
TRADD blotted in the 10K pellet was attributable to the presence
of plasma membrane TNF-R1.
To determine more directly whether TRADD was dissociating
from TNF-R1 after internalization, the effect of an inhibitor of
endocytosis was studied. Incubation of cells in hypertonic medium
has been shown to greatly reduce receptor-mediated endocytosis
(5, 6). Therefore, we pulsed cells for 1 min with TNF at 4°C,
chased for various time periods in either PBS or 0.45 M sucrose,
FIGURE 8. A, The kinetics of TNF-R1 and TRADD association at 4°C
and 20°C. U937 cells were treated with TNF for up to 30 min as indicated.
The cells were lysed and TNF-R1 was immunoprecipitated from the cell
lysate with the goat polyclonal anti-TNF-R1 Ab. The immunoprecipitates
(IP) were electrophoresed and immunoblotted with mouse monoclonal antiTRADD Abs. B, Subcellular localization of TNF-R1 and TRADD association after a pulse and chase study. U937 cells were incubated with TNF
for 1 min and chased in PBS for 1 and 60 min. TNF-R1 was immunoprecipitated from either 10K or 100K pellets and supernatants. The immunoprecipitates (IP) were electrophoresed and immunoblotted with mouse
monoclonal anti-TRADD Abs. The 34 kDa TRADD band is only recognized in the 10K pellet after a 1 min chase time point.
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FIGURE 7. Subcellular localization of TNF-R1 and TRADD association after TNF treatment. U937 cells were incubated in the presence or
absence of TNF for 30 min. TNF-R1 was immunoprecipitated from either
the cell lysate, 10K or 100K pellets, and the immunoprecipitates (IP) were
electrophoresed and immunoblotted with mouse monoclonal anti-TRADD
Abs. TRADD is only specifically recognized as a 34 kDa-band in the cell
lysate and 10K pellet of TNF-treated cells.
The Journal of Immunology
1047
FIGURE 9. Effect of hypertonic media on the
rate of dissociation of TRADD from TNF-R1.
U937 cells were incubated with TNF for 1 min at
4°C and chased for the various time periods indicated at 20°C in PBS (permits ligand receptor
internalization) or 0.45 M sucrose (inhibits ligand
receptor internalization). TNF-R1 was immunoprecipitated from cell lysates and the IP were
electrophoresed and immunoblotted with mouse
monoclonal anti-TRADD Abs.
hibited by hypertonic medium, TRADD remains associated with
the receptor for at least 150 min (Fig. 9).
Discussion
Acknowledgements
We thank Paul Luzio and Barbara Reave for helpful discussions.
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This study advances the findings of our previous report describing
the association of TNF-R1 with the Golgi apparatus in HUVEC
(13) using confocal immunofluorescence microscopy. In the
present study, we have confirmed that transfected TNF-R1 localizes to the Golgi and have defined the localization more precisely
as the TGN. We also extended the observation to two human cell
lines, the ECV304 and U937 cells, so that cell fractionation could
be employed to support the morphological studies. We have analyzed the intracellular distribution of TRADD, the initial adaptor
molecule involved in TNF-R1 signaling, and performed coimmunoprecipitaion studies to determine the function of TGN associated
TNF-R1. These findings suggest that TNF-R1 in the TGN is not
responsive to exogenous TNF and that internalization of plasma
membrane TNF-R1 may terminate signal transduction.
We have studied the localization of TNF-R1 in different cell
types with diverse biologic responses to TNF. Cultured human
endothelial cells are resistant to TNF-induced cell death, but respond to this cytokine by activating a number of proinflammatory
genes (30). In other cell types, TNF is cytotoxic and U937 cells are
reported to be sensitive to TNF-induced apoptosis (31). As
TNF-R1 is predominantly localized in the Golgi of both endothelial cells and U937 cells, this cellular distribution is unlikely to be
important in determining these different responses to TNF.
Our findings indicate that in U937 cells exogenous, TNF causes
TRADD to rapidly associate with TNF-R1 at the plasma membrane. It is believed that trimeric TNF induces trimeric clusters of
TNF-R1 and the death domains of the clustered receptor create a
high affinity binding site for TRADD (11). Our studies demonstrate that subsequent internalization of the TNF-TNF-R1 complex
results in the dissociation of TRADD. This could be attributed to
ligand dissociation when the endocytosed receptor-ligand complex
comes into contact with the acidic environment of endosomes.
When TNF is released from its receptor, the receptor clusters dissociate causing TRADD to be released. As most of the signaling
events are completed within minutes of the exposure of cells to
TNF, the inability to coimmunoprecipitate TRADD from Golgienriched subcellular fractions (100K pellet) within 30 min suggests that exogenous TNF does not induce TRADD recruitment to
the Golgi. Our observations do not exclude the possibility that
endogenously synthesized TNF may interact with the Golgi-associated TNF-R1. Expression of a transfected-TNF gene in TNFsensitive tumor cell lines confers resistance to TNF-mediated cell
lysis (32), which is correlated with down-modulation of cell surface TNFR (33). Secreted TNF is processed through the Golgi and
released by a BFA-sensitive mechanism (34). Further studies will
determine whether endogenous TNF can modify cellular response
to TNF through interaction with a Golgi receptor. Interestingly, the
indirect immunofluorescence studies demonstrated that, in cells
which had not been treated with TNF, TRADD was associated
with a different Golgi subcompartment to TNF-R1. The mechanism by which the cytosolic protein TRADD associates with the
Golgi is unknown, but their different localizations within the Golgi
may prevent constitutively synthesised TRADD and TNF-R1
interacting.
The significance of the TGN association of TNF-R1 remains
unknown. The TGN packages newly synthesized proteins into
transport vesicles that are directed to pre-lysosomal/lysosomal
compartments, secretory granules, or the plasma membrane (14,
15, 16, 17) and it participates in retrieving and reusing plasma
membrane components internalized by endocytosis such as the
mannose-6-phosphate and transferrin receptors (35, 36). As
TNF-R1 after TNF binding is also rapidly endocytosed, the internalized receptor ligand complex may recycle to the TGN or other
intracellular compartments and act locally to generate TNF-driven
signals. Alternatively, the TGN-associated receptor may act as a
pool from which the receptor is delivered in a regulated manner to
other sites.
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