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* Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206; and
Division of Pulmonary Sciences and Critical Care Medicine, Departments of Immunology, Medicine, and Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor, CD120a, has recently been shown to be
localized to both plasma membrane lipid rafts and to the
trans Golgi complex. Through a combination of both
confocal microscopy and sucrose density gradient ultracentrifugation,
we show that amino acid sequences located within the death domain (DD)
of CD120a are both necessary and sufficient to promote the appropriate
localization of the receptor to lipid rafts. Deletion of the DD
(CD120a.
321-425) prevented the receptor from being targeted to lipid
rafts and resulted in a uniform plasma membrane localization. A similar
loss of raft localization was also observed following pairwise deletion
of the six -helices that comprise the DD. In all situations, the
loss of the ability of CD120a to become localized to lipid rafts
following mutagenesis was paralleled by a failure of the receptor to
initiate apoptosis. Furthermore, introduction of the lpr
mutation into CD120a (CD120a.L351N) also resulted in both a loss in the
ability of the receptor to signal apoptosis and to be appropriately
localized to rafts. In contrast to CD120a, CD120b, which lacks a DD, is
mainly expressed in the bulk plasma membrane and to a lesser extent in
lipid rafts, but is absent from the Golgi complex. However, a chimeric
receptor in which the DD of CD120a was fused to the cytoplasmic domain
of CD120b was predominantly localized to lipid rafts. Collectively,
these findings suggest that in addition to its role in CD120a
signaling, an appropriately folded and functionally active DD is
required for the localization of the receptor to lipid
rafts. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interacts with two receptors, CD120a (p55) and
CD120b (p75) to stimulate responses critical to host defense,
inflammation, proliferation, differentiation, and programmed cell death
(1). Both receptors, while differing in the mechanisms
through which the initial signaling events are activated, share some
similarities in the scope of responses that are induced following their
ligation. The cytoplasmic domain of CD120a is organized as a Ser-,
Thr-, and Pro-rich membrane proximal region and an 
80 aa death
domain (DD)3 located
in the C-terminal region that is functionally conserved among death
receptors (Fas, DR3, DR4, DR5, DR6, and the more distantly related p75
neurotrophin receptor). The DD has been shown to be critical for the
induction of apoptosis as well as for the activation of NF-
B
(2, 3). Although less well studied, the membrane proximal
region of CD120a also participates in signaling through the binding of
several proteins, including factor associated with neutral
sphingomyelinase activation, growth factor receptor bound
protein-2, and 55.11/p97/TNFR-associated protein-2
(3).
Signaling by CD120a is initiated by receptor multimerization following
ligand binding and leads to the dissociation of silencer of DD
from, and TNFR-associated DD protein (TRADD) association with,
the DD of CD120a (4, 5). The CD120a-TRADD complex then
serves as a platform for the recruitment of other signaling
molecules such as Fas-associated DD protein (FADD),
receptor-interacting protein, and TNFR-associated factor
2, which in turn stimulate pathways leading to the activation of
I-B kinases, mitogen-activated protein kinases (MAPK), and to the
initiation of apoptotic death in certain responsive cell types
(2, 3). The mechanisms underlying the specificity in
CD120a signaling have long remained an enigma. Recent studies have
revealed novel regulatory mechanisms including FLICE inhibitory
protein-mediated inhibition of caspase-8 activation and its
consequences on apoptosis (6), and caspase-8-mediated
inhibition of NF-B activation through the cleavage of RIP
(7). Additionally, other studies have begun to
suggest that the spatial distribution of CD120a and other members of
the TNFR superfamily among subcellular compartments may contribute to
the regulation of death receptor signaling (8, 9, 10).
The plasma membranes of most cell types contain cholesterol and glycosphingolipid-containing microdomains or "lipid rafts" that are enriched in a variety of receptors and signaling molecules (11, 12). They are characterized by their resistance to solubilization at low temperature in nonionic detergents and by a punctate or focal staining pattern as detected by confocal microscopy. Recent studies have shown that members of the TNFR superfamily, including CD40, the p75 neurotrophin receptor, and CD120a are also associated with lipid rafts (13, 14, 15, 16), and that their localization to these structures is necessary for the initiation of signaling events. However, the mechanisms that mediate the localization of TNFR superfamily members to lipid rafts remain largely unknown.
In this study, we have investigated the mechanism of localization of CD120a to lipid rafts. In view of the importance of the DD in signaling programmed cell death, we hypothesized that this region of CD120a would also be important in localizing the receptor to rafts. Using a mutagenic approach combined with confocal microscopy and raft isolation strategies, we show that the DD of CD120a is necessary and sufficient for both the localization of the receptor to lipid rafts as well as for signaling apoptosis. In contrast, the DD was not found to be important in the localization of CD120a to the Golgi complex, implying that receptor localization to lipid rafts is an important event in the initiation of programmed cell death.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The hamster monoclonal anti-CD120a (p55) antagonist Ab (no.
80-4005-01) was purchased from R&D Systems (Minneapolis, MN). The
anti-
-adaptin-1 mouse mAb was from Sigma-Aldrich (St. Louis,
MO). The goat polyclonal anti-CD120a and anti-CD120b Abs were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Fluorescent
secondary Abs were purchased from Jackson Immunoresearch Laboratories
(West Grove, PA). FITC-conjugated wheat germ agglutinin was from
Molecular Probes (Eugene, OR).
Expression vectors
The expression vector encoding CD120a has been described (14). All deletion and point mutants were constructed using overlapping PCR (17) and ligated into EcoRI/SalI-digested pFLAG-CMV-1. The CD120b-(1-462)-CD120a-(308-425) chimeric protein was created by fusing the full-length CD120b to the DD of mouse CD120a (aa 308–425). A 1762-bp BamHI/EcoRI cDNA fragment encoding the chimera was amplified by PCR, digested with BamHI and EcoRI, and ligated into BamHI/EcoRI-digested pcDNA3 (Invitrogen, Carlsbad, CA). The fidelity of all the constructs was verified by restriction enzyme analysis and nucleotide sequencing. The expression vector for CD120b was a generous gift from Dr. H.-B. Shu (National Jewish Medical and Research Center, Denver, CO).
Transfections and confocal immunofluorescence microscopy
HeLa cells were maintained in DMEM containing 10% (v/v) FBS, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine. Approxiamately 6 x 104 cells/well were seeded in 12-well plates containing 18-mm glass coverslips and grown in 5% (v/v) CO2. Cells were transfected with 250 ng of DNA the following day using the Lipofectamine reagent (Life Technologies, Rockville, MD). Fourteen hours after transfection, the cells were washed with PBS, fixed for 15 min at room temperature in a solution containing 3% (w/v) paraformaldehyde and 3% (w/v) sucrose in PBS (pH 7.5). Cells were incubated for 15 min with wheat germ agglutinin (1/2000) in PBS, washed again extensively, and permeabilized with 0.2% (v/v) Triton X-100 for 10 min. The cells were then washed, blocked for 30 min in HBSS (without Mg2+, Ca2+, and phenol red, pH 7.2) containing 5% normal goat serum, and then incubated with the primary Ab (1/200) in blocking solution for 2 h. After washing with PBS, the cells were incubated for 1 h with Cy3- and/or fluorescein-conjugated F(ab')2 goat secondary anti-hamster IgG (1/200). The coverslips were incubated overnight in PBS supplemented with 0.02% sodium azide, and mounted in a solution containing 90% glycerol, 10% Tris-HCl (0.1 M, pH 8.5), and 20 mg/ml o-phenylenediamine as an antifading agent. To visualize the nuclei, cells were incubated with 10 µg/ml Hoechst 33342 (Hoechst-Sigma Chemical, St. Louis, MO) together with the secondary Abs. Cells were observed with a Leica DMR/XA confocal immunofluorescence microscope (Leica Microsystems, Bannockbum, IL) using a x100 Plan objective. Digital images were captured using a SensiCam camera, deconvolved using the software Slidebook 2.6 (Intelligent Imaging Innovations, Denver, CO) to remove out of focus fluorescence, and processed using Adobe Photoshop (Adobe Systems, Mountain View, CA).
For quantitative analysis, cells were transfected with the appropriate constructs, and stained in the absence of cell permeabilization as above. Analysis was performed using the software Slidebook 2.6. Briefly, stacks of 25 images were aquired in 0.4-µm steps throughout the cells. Stacks were deconvolved using the "nearest neighbors" algorithm, and the volume of the entire cell was defined manually. The amount of fluorescence was defined by the integrated intensity of fluorescence after correction for background fluorescence. Ten cells were analyzed per condition.
Flow cytometry analysis
HeLa cells (106) were seeded in 100-mm dishes and transfected with 5 µg of DNA the following day using the Lipofectamine PLUS reagent as recommended by the manufacturer (Life Technologies). Eighteen hours after transfection, the cells were incubated for 15 min with 1 µg/ml of hamster monoclonal anti-CD120a Ab, or monoclonal hamster Ab anti-TCR H57 (generously provided by Dr. J. Freed, National Jewish Medical and Research Center) as a control, washed, and incubated for 15 min on ice in HBSS (without Mg2+, Ca2+, and phenol red, pH 7.2) containing 5 mM EGTA. Cells were lifted from the plates, washed, resuspended in 1 ml of cold PBS, and fixed for 15 min at room temperature by the addition of 1 ml of 2x fixative (6% (w/v) paraformaldehyde and 6% (w/v) sucrose in PBS, pH 7.5). Cells were washed three times with PBS, incubated for 20 min at 4°C in 100 µl blocking buffer (HBSS containing 5% of normal goat serum), then incubated for 30 min with fluorescein-conjugated goat anti-hamster IgG (1/200 in blocking buffer). After washing with PBS, cells were resuspended in 100 µl of cold sample buffer (2% FBS in PBS containing 0.02% sodium azide). Cells (104) were analyzed on a FACSCaliber (BD Biosciences, Mountain View, CA) and data were processed by using the CellQuest software (BD Biosciences). Data are presented as a histogram of cell number vs fluorescence intensity.
TUNEL assay
HeLa cells grown on coverslips and transfected 18 h earlier with the appropriate expression vectors were washed with PBS, fixed and permeabilized as above, and incubated with terminal transferase reaction solution containing fluorescein-conjugated dUTP for 1 h at 37°C as recommended by the manufacturer (Roche Diagnostics, Indianapolis, IN). The cells were washed three times with 0.03 M sodium citrate (pH 7.4) containing 0.3 M sodium chloride to remove unbound nucleotides, then washed with PBS. Cells were then blocked and incubated with Abs as above. The percentage of TUNEL-positive cells among transfected cells was determined by counting at least 200 cells with a confocal microscope.
Isolation of lipid rafts
Lipid rafts were isolated by sucrose density gradient
centrifugation of Triton X-100 lysates using a modification of the
method of Cheng et al. (18). Approximately 1.2 x
107 HeLa cells or 8 x
106 COS-7 cells were transfected as described
above. Twelve to 16 h posttransfection, the cells were washed once
with either ice-cold PBS or MES-buffered saline (25 mM MES buffer,
pH 6.5, containing 150 mM NaCl) and then harvested by
scraping into PBS. The cells were collected by centrifugation and then
lysed in 1 ml TMBS (25 mM MES (pH 6.5) containing 150 mM NaCl,
1% (v/v) Triton X-100, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mM
PMSF, 0.1 mM Na3VO4, and 1
mM NaF). The cell lysates were sonicated at full strength for 1 min at
4°C and the high-density insoluble debris was removed by
centrifugation at 1,000 x g for 10 min at 4°C.
Clarified lysates were combined with an equal volume of 80% (w/v)
sucrose, transferred to 12.5 ml ultracentrifuge tubes, and overlaid
with a discontinuous sucrose gradient comprised of 8 ml 35% sucrose,
followed by 2 ml of 5% sucrose. Separation of the low-density lipid
rafts was achieved by centrifugation at 37,000 rpm in an SW41 rotor for
18–22 h at 4°C. Following centrifugation, 1 ml fractions were
harvested from the bottom of the gradients. Fractions were analyzed for
the raft markers alkaline phosphatase activity and ganglioside
GM1 by catalytic assay using 5 mM
p-nitrophenyl phosphate in 0.1 M 3-amino-2-methyl-1-propanol
(pH 10.0) as buffer, and Western blotting with cholera toxin
B-conjugated HRP, respectively (19, 20). Fractions were
also blotted with anti-CD120a and anti-CD120b Abs, as indicated
in Results.
Results shown are representative of at least three separate experiments unless indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In previously reported studies, we showed that CD120a is
expressed at the cell surface at focal points, rather than being
uniformly distributed in the plasma membrane (14). In
addition, based on the insolubility of the receptor at low temperature
in Triton X-100 and its low buoyant density in sucrose density
gradients, Ko et al. (16) provided evidence to suggest
that these focal sites represent lipid rafts. To investigate the
structural requirements necessary for the localization of CD120a to
rafts, we transfected wild-type CD120a into HeLa cells and analyzed the
subcellular localization of the receptor by immunofluorescent staining
with a hamster anti-mouse CD120a antagonistic mAb and confocal
microscopy. As can be seen in Fig. 1
, a and b, the wild-type receptor was consistently
expressed at focal sites within the plasma membrane. The receptor was
also detected in a juxtanuclear position that colocalized with the
Golgi marker adaptin-1 (Fig. 1, i–k), as previously
reported (8, 14, 21). A similar pattern was also observed
when endogenous CD120a was stained in HeLa cells (14),
ruling out the possibility of a transfection-associated artifact. We
also conducted experiments in which CD120a was stained in the absence
of detergent permeabilization to evaluate the proportion of receptor
associated with the cell surface. As can be seen in Fig. 1, e and f, while the staining of the
Golgi-associated receptor was lost, the observed focal staining pattern
of CD120a was preserved, indicating that the punctate staining pattern
represented the plasma membrane receptor pool. In addition, when
CD120a-transfected HeLa cells were costained with anti-CD120a Ab
and with fluorescein-conjugated wheat germ agglutinin in the absence of
detergent permeabilization, the focal staining pattern of the receptor
was found to be colocalized with areas of the plasma membrane that were
stained by wheat germ agglutinin (Fig. 1, o–q). These data
suggest that the focal pattern of expression of CD120a represents
receptor localization to discrete areas of the plasma membrane, a
finding consistent with its localization to lipid rafts
(16). To confirm this conclusion, we also isolated low
density Triton X-100 insoluble lipid rafts by sucrose density gradient
ultracentrifugation and investigated the distribution of CD120a and
markers of lipid rafts (GM1 and alkaline
phosphatase) by Western blotting and catalytic assays. As can be seen
in Fig. 1u, CD120a was detected in fractions containing low
density lipid rafts as reflected by its colocalization with alkaline
phosphatase and GM1, as well as in fractions
containing Triton X-100 soluble membrane material located at the bottom
of the gradients. Thus, these findings support the conclusion that
CD120a is present at the cell surface in structures consistent with the
properties of lipid rafts.
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321-425)
resulted in a complete loss of the focal staining pattern (Fig. 1
, c and d). Instead, the receptor was consistently
detected as a diffuse continuous band that circumnavigated the cell
periphery both in the presence and absence of detergent
permeabilization (Fig. 1, g and h). The
continuous staining pattern of the truncated receptor was also observed
to be colocalized with FITC-labeled wheat germ agglutinin (Fig. 1, r–t) confirming its localization to the peripheral plasma
membrane. In addition, juxtanuclear staining of the receptor
corresponding to the presence of the mutant CD120a in the Golgi complex
was also observed (Fig. 1, c, d, and
l–n), suggesting that the DD is not required for the
localization of CD120a to this organelle. We also subjected
CD120a.321-425-transfected HeLa cells to an analysis of the
partitioning of the mutant receptor to lipid rafts by sucrose density
gradient ultracentrifugation. As can be seen in Fig. 2c, the CD120a.321-425
mutant receptor was present in the Triton X-100 soluble membrane
fractions at the bottom of the sucrose density gradients, but was
absent from fractions containing the lipid rafts. In contrast, the
mutant CD120a.208-308, in which the membrane proximal region was
deleted but the DD was preserved, was localized to lipid rafts in a
fashion similar to the wild-type receptor (Fig. 2, a and
b). Therefore, these results indicate that the DD is
required for the appropriate expression of CD120a in lipid rafts.
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321-425 were transfected into HeLa cells, stained for CD120a
in the absence of detergent permeabilization, and analyzed by flow
cytometry. Membrane expression of the endogenous receptor was not
detectable under the conditions used. As expected, transfection of
wild-type CD120a was associated with an increase in membrane expression
of the receptor (Fig. 1v). As compared with wild-type
CD120a, membrane expression of CD120a.321-425 was markedly increased
indicating that removal of the DD may lead to increased receptor
expression at the cell surface. To confirm these data, we performed a
quantitative analysis of the membrane expression of CD120a using
immunofluorescence confocal microscopy. Cells were transfected with
wild-type CD120a or the deletion mutant CD120a.321-425 and stained
for CD120a in the absence of detergent permeabilization. Stacks of 25
images were acquired, deconvolved, and the integrated intensity of
fluorescence was determined. The intensity of fluorescence was
significantly higher (17-fold) in cells transfected with
CD120a.321-425 than in cells transfected with wild-type CD120a
(Mann-Whitney U test = 2, p < 0.0001),
consistent with the flow cytometry results. Thus, the DD is involved in
the localization of CD120a to membrane rafts, and its deletion is
associated with a striking up-regulation of receptor expression at the
cell surface. An intact CD120a DD is required for localization to rafts and for signaling death
Previous studies on the structural requirements for CD120a
signaling have emphasized the importance of specific residues and
sequences within the DD. Therefore, we investigated whether the
requirement of the DD for CD120a trafficking and raft localization was
attributable to a particular region within the DD. The nuclear magnetic
resonance structure of the DDs of FADD, Fas, the p75 neurotrophin
receptor, and CD120a have shown a conserved three-dimensional structure
of the DD comprising six well-conserved -helices
(22, 23, 24, 25, 26). Based on these findings and as shown in Fig. 3a, we
designed and created a series of deletion mutants based on homologies
between the published amino acid sequences of the various DDs
(22, 27). The mutants were then transfected into HeLa
cells and their localization was determined by immunofluorescence
confocal microscopy in nonpermeabilized cells. As expected, deletion of
the entire cytoplasmic domain (CD120a.211-425) prevented the
receptor from being focally expressed but promoted its diffuse
expression at the plasma membrane (Fig. 3c). Removal of two
(CD120a.386-425) or four (CD120a.353-425) of the predicted
-helices within the DD of CD120a also resulted in a complete loss of
the focal staining pattern and in the redistribution of the receptor to
the bulk plasma membrane (Fig. 3c). Similarly, partial
deletion of the two C-terminal DD -helices (constructs
CD120a.391-425, CD120a.399-425, and CD120a.405-425)
resulted in the diffuse membrane expression of CD120a and a complete
loss of the focal expression pattern. In contrast, a short deletion of
the C terminus of the receptor (CD120a.413-425) in which the DD was
preserved did not alter the expression of the receptor as compared with
the wild-type CD120a (Fig. 3c).
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and cycloheximide (50 ng/ml and 10
µg/ml, respectively) for 4 h, fixed, permeabilized, and the
percentage of apoptotic cells among CD120a-transfected cells was
quantified by confocal fluorescence microscopy. As expected,
transfection of wild-type CD120a induced a robust increase in the
proportion of apoptotic cells, both in the presence and in the absence
of TNF- and cycloheximide (Fig. 3b). In contrast,
transfection of any of the DD deletion mutants completely abolished
apoptosis as compared with that induced by the transfected wild-type
receptor, consistent with previous reports (27). Induction
of apoptosis was not affected in the short C-terminal deletion mutant
in which the DD was still present (CD120a.413-425; Fig. 3b). Collectively, these results indicate that the
appropriate expression of CD120a in lipid rafts requires the presence
of the entire DD, and that this property of the DD parallels its
ability to signal apoptosis. The selective localization of CD120a to lipid rafts is contingent on a functional DD
Several point mutations in the DD of Fas or CD120a have been shown
to inactivate the ability of these death receptors to initiate
apoptosis. The lymphoproliferation (lpr) mutation of
mouse Fas (V238N) (28) has been shown to cause a
structural alteration of the molecule, locally unfolding the protein in
the region corresponding to the 3 helix of the wild-type protein
(29), a region that has been shown to be important for the
self association of Fas and for binding to FADD. The lpr
mutant is more soluble than wild-type Fas (29), but its
phenotype of membrane expression has not been studied. L351 in murine
CD120a corresponds to the lpr mutation in Fas, and point
mutagenesis of L351 to either Asn or Ala inhibits the cytotoxic signal
of CD120a (27). To test the hypothesis that the
localization of CD120a to rafts was altered by mutations that
inactivate signaling of the DD of CD120a, HeLa cells were transfected
with the CD120a.L351N point mutant, stained for CD120a, and analyzed by
confocal microscopy. As expected, the L351N mutation abolished the
apoptosis signal induced by CD120a (Fig. 3b). As compared
with wild-type CD120a, CD120a.L351N was more diffusely expressed on the
cell membrane and the focal pattern was lost in >50% of transfected
cells (Fig. 4, a–d). The
continuous staining pattern of the receptor was colocalized with the
staining of the plasma membrane with wheat germ agglutinin (Fig. 4, h–j). However, the CD120a.L351N mutant was still present in
the Golgi apparatus, as shown by the colocalization with adaptin-1
(Fig. 4, e–g). These data thus show that the L351N point
mutation is sufficient to abolish death signaling and to alter the
pattern of CD120a membrane expression, redistributing the receptor from
rafts to a more diffuse membranous localization. This suggests that
proper folding of the DD is required for the adequate targeting of
CD120a to rafts as well as for the initiation of CD120a-mediated
apoptosis.
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We next questioned if the DD was sufficient for the targeting of
CD120a to lipid rafts. The intracellular domain of TNFR CD120b is
unrelated to that of CD120a and does not contain a DD. Reported studies
have shown that CD120b is expressed throughout the plasma membrane
(21). To further investigate the role of the DD in
targeting receptors to rafts, we created a chimeric protein,
CD120b-(1-462)-CD120a-(308-425), or "CD120b-DD" in which the DD of
mouse CD120a (aa 308–425) was fused to the C terminus of CD120b.
Transfection of CD120b-DD into COS-7 cells resulted in the
expression of a protein of the expected size as detected by Western
blotting of whole cell lysates (data not shown) confirming that the
chimeric receptor was appropriately expressed. HeLa cells were then
transfected with vectors encoding wild-type CD120b or CD120b-DD,
stained using Abs specific for the extracellular domain of CD120b or
the C terminus of CD120a, and examined by confocal microscopy. As shown
in Fig. 5a, wild-type CD120b
was expressed at the cell surface in a predominately peripheral but
somewhat punctate fashion, consistent with previous reports (8, 21). In contrast, the CD120b-DD chimeric protein was not
uniformly expressed in the plasma membrane and the staining pattern was
replaced by a more focal membranous staining pattern similar to that
seen with wild-type CD120a (Fig. 5b). In addition, CD120b-DD
formed intracytoplasmic tubular structures, similar to those seen
following phosphorylation of CD120a by
p42mapk/erk2 (14). We also examined
the localization of wild-type CD120b and the CD120b-DD chimera to lipid
rafts by sucrose density gradient ultracentrifugation of Triton X-100
lysates obtained from transfected COS-7 cells. As shown in Fig. 5c, the majority of CD120b was present in the Triton X-100
soluble membrane fractions at the bottom of the gradient with a smaller
proportion present in the lipid rafts. In contrast, the majority of the
CD120b-DD chimera was localized to fractions containing the lipid rafts
with markedly reduced amounts being found in the Triton X-100 soluble
fractions at the bottom of the gradients (Fig. 5c). Thus,
the localized expression of CD120b to lipid rafts was greatly enhanced
by the presence of the CD120a DD.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of some members of the TNFR superfamily, including CD120a,
CD40, and the p75 neurotrophin receptor, to be localized to rafts has
only recently been recognized (13, 14, 15). Studies by Ko et
al. (16) were the first to suggest that TNF--induced
apoptosis was reduced in cells grown under lipoprotein-deficient
conditions, indicating that lipid rafts may be necessary for
CD120a-induced apoptosis. It has been suggested that once clustered,
receptors may associate more effectively with rafts, thereby enhancing
their interaction with raft-associated signaling molecules (12, 30). Thus, the recruitment and/or localization of CD120a to
rafts may facilitate its interaction with adaptor molecules and the
recruitment of other signaling molecules. Indeed, several components of
the CD120a-stimulated signaling pathways have been shown to be
activated in lipid rafts, including Ras, c-Raf-1, and
p42mapk/erk2 (31). Elegant studies
conducted by Liu and Anderson (32) have also revealed that
the production of ceramide in response to IL-1
occurs in lipid
rafts. In addition, the low-affinity p75 neurotrophin receptor, which
also contains a DD, was recently shown to be enriched in
caveolin-containing rafts and was coimmunoprecipitated with caveolin
(32). Furthermore, neurotrophin-induced hydrolysis of
sphingomyelin has been shown to be localized to caveolin-enriched
rafts, suggesting that the ligand-induced production of ceramide might
also occur in caveolae-like rafts (13). Taken together,
these observations suggest that the localization of CD120a to lipid
rafts may be important in receptor signaling.
The region of the DD that was necessary for the targeting of the
receptor to rafts could not be distinguished from the sequences and
individual amino acid residues that account for the induction of cell
death. The results of the original studies by Tartaglia et al.
(27) suggested that continuous amino acid sequences were
not involved in the initiation of signaling by the DD of CD120a, but
that individual amino acids became spatially clustered in the
three-dimensional structure to provide a "patch" capable of
mediating signaling. Recent nuclear magnetic resonance studies
have revealed the importance of the electrostatic charge conferred by
solvent-exposed basic residues in mediating the interactions between
the DDs of CD120a and TRADD (26). Thus, it is tempting to
speculate that the intact three-dimensional structure of the DD is also
necessary to enable CD120a to be appropriately localized to lipid
rafts. This interpretation is consistent with the data reported herein
showing that deletions within the six -helices of the DD of CD120a
inhibited the receptor both from associating with rafts and from
inducing apoptosis. Similarly, Hsu et al. (4) found that a
C-terminal deletion that removed part of the 6 helix, or an internal
deletion that removed the 1 helix and part of the loop between the
1 and 2 helices at the N-terminal end of the DD likewise
inhibited TRADD binding, implying that small deletions in the DD can
have a major effect on signaling. In addition, we have shown that the
introduction of the lpr mutation into the DD of CD120a
similarly blocked both the induction of apoptosis and localization of
the receptor to rafts. Nuclear magnetic resonance studies of the V238N
(lpr) mutation of Fas have shown that the 3 helix
of the DD becomes unfolded, thereby abolishing the interaction of Fas
with FADD (23). Other studies have shown the
lpr mutation of Fas also renders the receptor susceptible to
solubilization in nonionic detergents in contrast to the relative
insolubility of the wild-type Fas protein (29).
Interestingly, and unlike the situation in HeLa cells, CD120a is
principally localized to the trans Golgi network and is only
poorly expressed at the plasma membrane in vascular endothelial cells
(8). Recent studies by Gaeta et al. (33) have
also shown that the DD is necessary but not sufficient to localize
CD120a to the Golgi complex. Our results differ from those of Gaeta et
al. (33) in that we found that deletions of or within the
DD did not affect the accumulation of CD120a in the Golgi complex.
Although seemingly at odds, these divergent findings may suggest that
sequences necessary for the spatial localization of CD120a to different
cell compartments may differ in divergent cell types, and therefore,
may further contribute to the observed heterogeneity of TNF signaling
in different cell populations.
The mechanism through which the DD of CD120a promotes raft localization remains to be determined. Studies in B cells have begun to suggest that the recruitment of B cell receptors (BCR) to rafts is initiated by BCR aggregation but occurs in a fashion that is independent of the cytoskeleton (34), possibly occurring as a result of the increased affinity of the aggregated hydrophobic BCR transmembrane regions for the cholesterol/sphingolipid-rich rafts (35). In contrast, the subsequent aggregation of small receptor-containing rafts into larger aggregates has been proposed to require an intact actin-based cytoskeleton (34). A similar view has also been proposed to explain the initial recruitment of the TCR complex into rafts and the subsequent formation of the so-called "immunological synapse" (36, 37). Recent studies with CD120b have suggested that caveolin forms a complex with TNFR-associated factor 2 which in turn interacts with CD120b (38). However, it remains to be determined how the DD of CD120a promotes raft localization of CD120a and what role, if any, is played by the cytoskeleton.
Recent reports have suggested that the signaling of apoptosis by death
receptors may be regulated in part by the subcellular localization of
these receptors. Studies by Bennett et al. (9) have
suggested that Fas traffics between the Golgi complex and the plasma
membrane in a p53-dependent fashion, but that signaling competence is
only initiated through cell surface Fas. The TNF-related
apoptosis-inducing ligand receptors DR4 and DR5 have also been
found on the cell surface and in the trans Golgi network,
and following exposure to ligand, the receptors traffic to endosomes
(10). In addition, TRADD has been shown to rapidly
translocate from the Golgi complex to the plasma membrane in response
to stimulation with TNF- (21). In the case of CD120a,
we have demonstrated that phosphorylation of the intracellular domain
of CD120a by p42mapk/erk2 induces a
redistribution of the receptor from the plasma membrane and the Golgi
complex to tubular structures located within the endoplasmic reticulum
(14). In addition, the recruitment of phosphorylated
CD120a to the tubular structures results in the corecruitment of Bcl-2
(39). Phosphorylation of CD120a by
p42mapk/erk2 occurs within the membrane proximal
region (40), indicating that different regions of the
receptor affect its intracellular trafficking and localization. Of
note, caveolin has been shown to directly traffic from the plasma
membrane to the endoplasmic reticulum in response to cholesterol
oxidation in a microtubule-dependent manner (41).
Conceivably, the presence of CD120a within lipid rafts might account
for the yet unexplained retrograde trafficking of phosphorylated CD120a
from the plasma membrane and Golgi complex to the endoplasmic
reticulum.
In conclusion, the results of the present study indicate that CD120a is expressed in lipid rafts as well as in the Golgi complex and we have shown that an intact DD is both necessary and sufficient for the restricted expression of CD120a within rafts. Therefore, these findings suggest that: 1) an appropriately folded DD is necessary for raft localization and signaling, and 2) that the previously observed residues defined as being necessary for apoptosis also participate in the localization of the receptor to lipid rafts. Recent studies have also shown that the sequences necessary for the initiation of programmed cell death are indistinguishable from those required for DD self association and TRADD binding (26). Thus, it is conceivable that these signaling components are assembled in lipid rafts following ligand binding (4, 42, 43), and that these structures represent the site of signaling at least for the induction of apoptosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David W. H. Riches, Department of Pediatrics, National Jewish Medical and Research Center, Neustadt Room D405, 1400 Jackson Street, Denver, CO 80206. E-mail address: richesd{at}njc.org
3 Abbreviations used in this paper: DD, death domain; TRADD, TNFR-associated DD protein; FADD, Fas-associated DD protein; MAPK, mitogen-activated protein kinases; BCR, B cell receptor.
Received for publication June 20, 2001. Accepted for publication February 4, 2002.
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B activation. Cell 81:495.[Medline]
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