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Differential Regulation of TNF-R1 Signaling: Lipid Raft Dependency of p42^mapk/erk2 Activation, but Not NF-B Activation¹

Joyce E. S. Doan^*, David A. Windmiller^*, and David W. H. Riches^2,*,,

^* Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206; and Department of Immunology and Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The TNFR, TNF-R1, is localized to lipid raft and nonraft regions of the plasma membrane. Ligand binding sets in motion signaling cascades that promote the activation of p42^mapk/erk2 and NF-B. However, the role of receptor localization in the activation of downstream signaling events is poorly understood. In this study, we investigated the dynamics of TNF-R1 localization to lipid rafts and the consequences of raft localization on the activation of p42^mapk/erk2 and NF-B in primary cultures of mouse macrophages. Using sucrose density gradient ultracentrifugation and a sensitive ELISA to detect TNF-R1, we show that TNF-R1 is rapidly and transiently recruited to lipid rafts in response to TNF-. Disruption of lipid rafts by cholesterol depletion prevented the TNF--dependent recruitment of TNF-R1 to lipid rafts and inhibited the activation of p42^mapk/erk2, while the activation of NF-B was unaffected. In addition, phosphorylated p42^mapk/erk2, but not receptor interacting protein, I-B kinase-, or I-B was detected in raft-containing fractions following TNF- stimulation. These findings suggest that TNF-R1 is localized to both lipid raft and nonraft regions of the plasma membrane and that each compartment is capable of initiating different signaling responses. We propose that segregation of TNF-R1 to raft and nonraft regions of the plasma membrane contributes to the diversity of signaling responses initiated by TNF-R1.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The 55-kDa TNF- receptor, TNF-R1 (CD120a), plays a key role in TNF--dependent responses that include proliferation, differentiation, and apoptosis. Clustering of TNF-R1 following ligand binding promotes the recruitment of the adaptor protein, TNFR-associated death domain protein (1). TNFR-associated death domain protein subsequently serves to recruit TNFR-associated factor 2 and receptor interacting protein (RIP),³ which facilitate the recruitment and activation of the I-B kinase (IKK) complex resulting in the activation of NF-B (2, 3, 4). Recruitment of TNFR-associated factor 2 is also required for the activation of c-Jun NH₂-terminal kinase and p38^mapk (5), while the initiation of apoptosis by TNF-R1 requires Fas-associated death domain protein and procaspase-8 (6, 7). Less is known about the mechanism of activation of p42^mapk/erk2 following TNF-R1 ligation, though this event may involve the recruitment of MADD to the death domain of the receptor (8). These studies emphasize the complexity of the signaling cascades that are initiated by TNF-R1, but provide few insights into how these signaling cascades are differentially regulated.

Studies originating over a decade ago support the view that certain receptors and signaling proteins become associated with organized regions of the plasma membrane called lipid rafts (9, 10). These liquid-ordered domains are enriched with sphingolipids and cholesterol, and are physically characterized by their insolubility at 4°C in Triton X-100 (9, 11). The integrity of lipid rafts has been shown to be essential for the initiation of signaling responses by multichain immune recognition receptors, including the Ag-receptor complexes of B and T cells and FcR1 (12). In the case of signaling by the TCR, the CD4-associated Src-kinase, Lck, and the adaptor protein linker for activation of T cells have been shown to be constitutively associated with lipid rafts (13, 14). Ligation of the TCR/CD3 complex promotes the initial recruitment of the receptor complex to lipid rafts thereby bringing the receptor into proximity with coreceptor-associated signaling molecules (15). Thus, one function of lipid rafts is to segregate signal transduction molecules in the absence of ligand, while facilitating signaling in the presence of ligand. Lipid rafts thereby provide an environment in which spatially segregated signaling modules can be assembled into active signaling complexes (10).

Recent studies have shown that members of the TNFR superfamily, including CD40 and TNF-R1, also become associated with lipid rafts (16, 17) though in the case of TNF-R1, a significant amount of the receptor is also localized to "nonraft" fractions (18). Given that TNF-R1 exhibits heterogeneity in signal transduction (19), we hypothesized that the distribution of the receptor to raft and nonraft compartments may play a role in shaping the activation of different signal transduction cascades. In the work reported herein, we show that ligation of TNF-R1 augments receptor accumulation in lipid rafts. In addition, we show that the integrity of lipid rafts is essential for the activation of p42^mapk/erk2, but is dispensable for the activation of NF-B.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Recombinant mouse and human TNF- were purchased from R&D Systems (Minneapolis, MN). TNF-R1 ELISA kits were provided by ELISA Tech (Aurora, CO). Anti-phospho-extracellular signal-regulated kinase Ab was from Promega (Madison, WI). Mouse monoclonal anti-RIP Ab was from BD Biosciences Transduction Laboratories (San Diego, CA). All other Abs were provided by Santa Cruz Biotechnology (Santa Cruz, CA).

Macrophage isolation and culture

Mouse bone marrow-derived macrophages were prepared and cultured in DMEM containing 10% (v/v) FBS and 10% (v/v) L cell-conditioned medium as a source of M-CSF for 5 days as previously described (20). We also cultured macrophages under nonadherent conditions on 100-mm diameter bacteriologic grade petri dishes as described (21) for use in the subcellular fractionation experiments. Macrophage monolayers were depleted of cholesterol by washing twice in DMEM followed by incubation with 2-hydroxypropyl--cyclodextrin (20 mg/ml) at 37°C for 30–60 min (22). The cells were then washed and incubated overnight in 1% (v/v) lipid-depleted serum (16).

Isolation of lipid rafts

Lipid rafts were isolated from mouse macrophages by sucrose density gradient ultracentrifugation as described (18, 23) or by differential centrifugation. Sucrose density gradient centrifugation was conducted using lysates prepared by sonication of 10⁸ cells in 1 ml of ice-cold lysis buffer (25 mM MES, pH 6.5, containing 150 mM NaCl, 0.25% (v/v) Triton X-100, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 mM Na₃VO₄, and 1 mM NaF) for 1 min on ice. The lysates were centrifuged at 1000 x g for 10 min at 4°C, and the postnuclear supernatants were combined with an equal volume of 85% (w/v) sucrose and placed at the bottom of 12.5 ml ultracentrifuge tubes. The lysates were overlaid with 8 ml of 30% (w/v) sucrose, followed by 2 ml of 5% (w/v) sucrose, and centrifuged at 170,000 x g for 18–22 h. One-milliliter fractions were collected from the bottom of the gradients and were analyzed for raft markers (alkaline phosphatase, cholesterol, and GM₁) as described (18, 24). Based on the distribution of these markers, the data from fractions 1–4 were combined to obtain a total raft pool, while data from fractions 9–12 were combined to yield a total nonraft pool as indicated in Results. We also prepared lipid raft and nonraft fractions by differential centrifugation. Approximately 10⁷ macrophages were stimulated with TNF- (20 ng/ml) for 10 min under nonadherent conditions at 37°C, washed in ice-cold PBS, and resuspended in lysis buffer lacking Triton X-100. The cells were disrupted by sonication with 70–80 pulses at setting 2.5 on a Fisher Sonic Dismembrator Model 100 (Fisher Scientific, Pittsburgh, PA) ensuring minimal organelle disruption as determined empirically by light microscopy. The nuclei and unbroken cells were removed as described above and the postnuclear supernatant was centrifuged at 86,000 x g for 15 min in a Beckman TL-100 ultracentrifuge (Beckman Coulter, Fullerton, CA). The supernatant, comprising the cytosolic fraction, was removed and saved and the membrane pellet was solubilized in ice-cold lysis buffer containing 0.25% (v/v) Triton X-100 and re-centrifuged at 86,000 x g for 15 min. The Triton X-100 soluble membrane fraction was removed and saved, and the Triton X-100 insoluble fraction containing the lipid rafts was solubilized by sonication for 4 s at setting 3 in lysis buffer containing 1% (v/v) Nonidet P-40 and 60 mM octylglucoside. Insoluble material was removed by centrifugation at 20,800 x g in an Eppendorf 5417R microcentrifuge (Eppendorf Scientific, Westbury, NY). Approximately 5 µg of protein from each fraction were analyzed by SDS-PAGE and Western blotting as described below.

Biochemical analyses and procedures

Activity of p42^mapk/erk2 was determined by immunoprecipitation and in vitro kinase assay with recombinant activating transcription factor-2 and [³²P]-ATP as described (25). Activation of NF-B was determined by EMSA. Nuclear proteins were prepared from 3 x 10⁷ macrophages per condition as described (26) and used in EMSAs with a commercially available ³²P-labeled NF-B consensus sequence probe (Promega, Madison, WI). Levels of TNF-R1 were quantified by ELISA using recombinant mouse TNF-R1 extracellular domain as a standard. The sensitivity of the assay was 4 pg/ml. Duplicate samples of each raft fraction were adjusted to contain 0.1% (w/v) SDS to disperse the lipid rafts and were diluted to place the values within the linear portion of the standard curve. The concentration of each sample was then calculated by comparison with the values of the standard curve. The standards were also diluted to contain 0.1% (w/v) SDS.

PAGE and immunoblotting

SDS-PAGE was conducted using 10 or 12% denaturing polyacrylamide gels. Following electrophoresis, samples were transferred to nitrocellulose membranes as described (27). Membranes were then subjected to immunoblotting using the primary Abs indicated in Results. The ganglioside, GM₁, was detected using HRP-conjugated cholera toxin B subunit (Sigma-Aldrich, St. Louis, MO) as described (18). All immunoblots were developed by ECL (Amersham Pharmacia Biotech, Piscataway, NJ).

Measurement of cholesterol

Unesterified cholesterol was measured by a stable isotope dilution assay using gas chromatograph/mass spectrometry (GC/MS) as previously described (24). Briefly, two volumes of methanol were added to aliquots of the sucrose density gradient samples and stable isotope-labeled cholesterol (500 ng of 2,2,3,4,4,6-[²H]-cholesterol; Cambridge Isotope Laboratories, Andover, MA) was added as an internal standard. Cholesterol was extracted with isooctane and converted to the trimethylsilyl derivative by heating at 60°C for 1 h with 25 µl of bis-trimethylsilyl-trifluoracetamide (Supelco, Bellefonte, PA) in 25 µl of acetonitrile. The derivatized cholesterol was then analyzed with a Trace GC/MS System (ThermoFinnigan, San Jose, CA) by selected ion monitoring of the ions at m/z 368 and 372 [M-90] for the endogenous and internal standard cholesterol, respectively. The abundance of these ions was ratioed, then compared with a standard curve prepared with each sample set to determine the quantity of cholesterol in the original sample.

Unless indicated, the results are representative of at least three independent experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Localization of endogenous CD120a to lipid rafts

We initially assessed the distribution of endogenous TNF-R1 in lipid raft and nonraft compartments in primary cultures of unstimulated mouse macrophages. Monolayers of 10⁸ macrophages were solubilized in lysis buffer containing 0.25% (v/v) Triton X-100 and subjected to sucrose density gradient ultracentrifugation to separate raft and nonraft fractions. Raft fractions were identified by their low buoyant density and by enrichment with raft markers (unesterified cholesterol, alkaline phosphatase, and GM₁). As shown in Fig. 1A, the majority of the TNF-R1 was detected in the nonraft fractions located at the bottom of the gradient (fractions 9–12). However, using a sensitive ELISA to quantify TNF-R1 levels, small amounts of the receptor, comprising between 2 and 6% of the total receptor pool, were also detected in raft-containing fractions that were enriched with cholesterol, alkaline phosphatase, and GM₁ (Fig. 1, A–C; fractions 1–4). As a control to verify the specificity of the TNF-R1 immunoblots and ELISA, we also separated lipid raft and nonraft fractions from TNF-R1 null mice. TNF-R1 was not detected in fractions from these gradients by Western blotting or ELISA (Fig. 1D). These data indicate that in the absence of stimulation, a small proportion of endogenous TNF-R1 is localized to lipid rafts but the majority of the receptor is localized to a nonraft pool.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Localization of endogenous TNF-R1 in mouse macrophages. Approximately 108 macrophages were lysed and separated by sucrose density gradient ultracentrifugation. Gradient fractions were analyzed for TNF-R1 by ELISA and Western blotting (A and D). Lipid raft-containing fractions were identified by enrichment with cholesterol (B), alkaline phosphatase, and GM1 (C). A, Sucrose density was measured by refractometry. B, Total protein is shown. D, The specificity of the ELISA and Western blots were confirmed using macrophages from TNF-R1 null mice. Results are representative of six independent experiments.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. TNF- induced a rapid, transient, and selective increase in lipid raft-associated TNF-R1 in mouse macrophages. Approximately 108 macrophages were stimulated with TNF- for the indicated times, lysed, and separated by density gradient ultracentrifugation. Fractions were analyzed for TNF-R1, alkaline phosphatase, and cholesterol. The data from fractions 1–4 were pooled to yield a total raft compartment and the data from fractions 9–12 were pooled to obtain the nonraft compartment. A, Time course of changes in the level of TNF-R1 in lipid rafts following stimulation with TNF-. B, Time course of changes in TNF-R1 in the nonraft compartment in response to TNF-. C, Effect of TNF- on lipid raft levels of cholesterol and alkaline phosphatase. Results are representative of three independent experiments.

Raft requirements for TNF--induced p42^mapk/erk2 and NF-B activation

Cholesterol is essential for maintaining the functional integrity of lipid rafts (9, 10). To determine the role of lipid raft integrity in TNF-R1 signaling, we investigated the effects of cholesterol depletion on the TNF--induced activation of p42^mapk/erk2 and NF-B. Macrophage monolayers were depleted of cholesterol as described in Materials and Methods. Quantification of the cellular level of free cholesterol by GC/MS showed that cholesterol levels were reduced by 70% from an average of 2.29 µg per 10⁶ cells to 0.68 µg per 10⁶ cells, consistent with previous studies using methyl--cyclodextrin (22). As can be seen in Fig. 3A, incubation with TNF- alone for 10 min resulted in a rapid increase in p42^mapk/erk2 kinase activity as previously described (29). However, the activation of p42^mapk/erk2 by TNF- was completely blocked in cholesterol-depleted cells (Fig. 3A). Similar results were also seen at time points up to 30 min (data not shown) indicating a loss, rather than a delay, in signaling activity. These effects were specific to signaling by TNF-R1 because: 1) macrophages from TNF-R2 null mice yielded identical results as those from wild-type mice and 2) human TNF-, a specific ligand for mouse TNF-R1, produced the same pattern of p42^mapk/erk2 activation as mouse TNF- (data not shown). In addition, we have previously shown that ligation of TNF-R2 in mouse macrophages does not induce activation of p42^mapk/erk2 (29). As a control, we also investigated the effect of cholesterol depletion on p42^mapk/erk2 phosphorylation induced by PMA, a raft-independent stimulus. In contrast to the inhibitory effect of cholesterol depletion on the activation of p42^mapk/erk2 by TNF-, the phosphorylation of p42^mapk/erk2 in response to PMA stimulation was not inhibited (Fig. 3A). In fact, cholesterol depletion appeared to modestly increase p42^mapk/erk2 activation by PMA (Fig. 3A).

View larger version (42K): [in this window] [in a new window]   FIGURE 3. Cholesterol depletion inhibits TNF--induced activation of p42mapk/erk2, but not NF-B. Macrophage monolayers were depleted of cholesterol as described in Materials and Methods. A, Effect of cholesterol depletion on the activation of p42mapk/erk2 by TNF- (10 ng/ml, 10 min) as detected by immunoprecipitation and in vitro kinase assay using activating transcription factor-2 as substrate (i) and effect of cholesterol depletion on the activation of p42mapk/erk2 by PMA (10 ng/ml, 15 min) as detected by immunoblotting with anti-phospho-p42/p44 extracellular signal-regulated kinase Ab (ii). The lower panels in i) and ii) are immunoblots showing equal loading of p42mapk/erk2. B, Effect of cholesterol depletion on the activation of NF-B as determined by EMSA (upper panel) and IKK as determined by degradation of I-B (lower panel). Results are representative of three independent experiments.

Active p42^mapk/erk2 is associated with lipid rafts, but IKK signaling molecules are not

We next investigated the hypothesis that the differential dependence of p42^mapk/erk2 and NF-B activation on lipid raft integrity was accompanied by differences in the localization of signaling molecules associated with each pathway in the raft and nonraft fractions. Accordingly, macrophage monolayers were stimulated with TNF- for 10 min, lysed, and subjected to sucrose density gradient or differential ultracentrifugation. We then measured the distribution of phosphorylated p42^mapk/erk2, total p42^mapk/erk2, RIP, and IKK in each fraction. As can be seen in Fig. 4A, phosphorylated p42^mapk/erk2 was detected in raft and nonraft fractions in response to TNF- stimulation, but was absent in unstimulated cells (Fig. 4A) or following stimulation for 15 min (data not shown). Small amounts of p42^mapk/erk2 were also detected in raft fractions (fractions 1–4) in both the absence and presence of TNF-, though, as expected, the majority was localized in the nonraft fraction, which also contains the cytosol (Fig. 4A). In contrast, neither RIP nor IKK were detected in the raft-containing fractions (fractions 1–4) in either unstimulated or TNF--stimulated cells at any time point, but both proteins were detected in the nonraft fractions (Fig. 4B).

View larger version (53K): [in this window] [in a new window]   FIGURE 4. Localization of p42mapk/erk2 and active phosphorylated p42mapk/erk2, but not RIP or IKK, to lipid rafts. Approximately 108 macrophages were incubated in medium alone or stimulated with TNF- for 5 min, lysed, and subjected to sucrose density gradient ultracentrifugation as described in Materials and Methods. Raft markers were detected in fractions 2–4. A, Localization of p42mapk/erk2 and active phosphorylated p42mapk/erk2. B, Localization of RIP and IKK. Results are representative of four independent experiments.

View larger version (46K): [in this window] [in a new window]   FIGURE 5. Distribution of phosphorylated p42mapk/erk2, p42mapk/erk2, RIP, and I-B- in mouse macrophages. Approximately 107 macrophages were incubated in medium alone or stimulated with TNF- for 10 min, lysed, and fractionated by differential centrifugation as described in Materials and Methods. A, Distribution of raft markers (cholesterol, ; alkaline phosphatase, ) and nonraft markers (total protein, ; and blots for -actin and total p42mapk/erk). B, Distribution of phosphorylated p42mapk/erk, p42mapk/erk2, I-B, and RIP. Results are representative of three independent experiments.

Because TNF-R1 is recruited to lipid rafts in response to TNF-, and lipid raft integrity is required for the TNF--dependent activation of p42^mapk/erk2, we next investigated the role of lipid raft integrity on the recruitment of TNF-R1 to lipid rafts in response to stimulation with TNF-. Macrophages were depleted of cholesterol as described in Materials and Methods and were incubated in the presence or absence of TNF- for 10 min before lysis and separation of the raft (fractions 1–4) and nonraft (fractions 9–12) compartments by sucrose density gradient ultracentrifugation. As can be seen in Fig. 6, cholesterol depletion substantially inhibited the TNF--stimulated recruitment of TNF-R1 to lipid rafts, but did not affect basal levels. These findings suggest that the ligand-dependent recruitment of TNF-R1 to lipid rafts is necessary for the initiation of p42^mapk/erk2 activation.

View larger version (34K): [in this window] [in a new window]   FIGURE 6. Cholesterol depletion blocks the TNF--induced recruitment of TNF-R1 to lipid rafts. Approximately 108 macrophages were depleted of cholesterol as described in Materials and Methods or were incubated in medium alone. The cells were then stimulated with TNF- for 10 min, lysed, and subjected to sucrose density gradient ultracentrifugation. Fractions containing lipid rafts (fractions 1–4) or the nonraft fractions at the bottom of the gradient (fractions 9–12) were pooled and TNF-R1 levels were determined by ELISA. Shown are the mean ± SD of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Quantification of the amount of TNF-R1 associated with lipid rafts by ELISA afforded novel insights into the dynamics of receptor trafficking in primary cultures of mouse macrophages. Small amounts of TNF-R1 were associated with lipid rafts in unstimulated cells but the amount of receptor associated with the rafts rapidly and transiently increased in response to stimulation with TNF-, suggesting recruitment of additional TNF-R1 from the nonraft compartment. Although palmitoylation and myristoylation play a role in the targeting of some signaling molecules and receptors to lipid rafts (10), the mechanism underlying the recruitment of unmodified transmembrane proteins to lipid rafts appears to rely primarily on amino acid sequence and composition. Coffin et al. (31) have shown that residues present in transmembrane regions 1 and 2 of LMP1 are necessary for raft localization, while Scheiffele et al. (32) concluded that hydrophobic residues in the transmembrane domain of influenza virus hemagglutinin contact with lipids in the outlet leaflet of the plasma membrane to promote raft localization. In contrast, sequences located in the extracellular domain of the epidermal growth factor receptor contribute to the localization of this receptor to rafts (33). Previously, we showed that the death domain is necessary for TNF-R1 raft clustering (18), although it is possible that other regions of TNF-R1 may be required for basal localization of the receptor to rafts.

In contrast to the TNF--dependent transient recruitment of TNF-R1 to lipid rafts, the amount of TNF-R1 in the nonraft compartment progressively declined until levels stabilized 15 min after stimulation. Although some of this loss is likely accounted for by translocation to lipid rafts at early time points, this is unlikely to account for the decline in receptor abundance in the nonraft compartment at later time points, which amounted to 50% of the nonraft pool of TNF-R1. Previous studies have shown that TNF-R1 is endocytosed and degraded in response to ligand binding (28, 34, 35). Thus, we propose that following ligand binding, TNF-R1, rapidly translocates from the nonraft compartment to the lipid raft compartment, but at later times the receptor is removed from both raft and nonraft compartments, most likely via endocytosis.

The functional significance of the localization of TNF-R1 to lipid rafts and its consequence on downstream signaling events are poorly understood. We therefore used cholesterol depletion to assign the functional role of lipid rafts in the TNF-R1-initiated activation of p42^mapk/erk2 and NF-B. Strikingly, the activation of p42^mapk/erk2 was completely inhibited by cholesterol depletion. Other studies have also suggested that the activation of p42^mapk/erk2 is dependent on lipid raft integrity. For example, Park et al. (36) showed that the cholesterol-binding agents, nystatin and digitonin, inhibit the activation of p42^mapk/erk2 induced by exposure of bovine endothelial cells to shear stress. Rizzo et al. (37) reported a similar dependence on lipid rafts during insulin-induced activation of p42^mapk/erk2 in fibroblasts. In addition, p42^mapk/erk2-associated signaling molecules such as Raf, p21^ras oncoprotein, and MEK1, are enriched in lipid rafts (38). Because the results from the present study also show that the TNF--induced recruitment of TNF-R1 to lipid rafts is dependent on raft integrity, we propose that TNF-R1 recruitment to lipid rafts is intimately linked to the activation of p42^mapk/erk2.

In contrast to the dependency of p42^mapk/erk2 activation on lipid rafts, NF-B activation was not inhibited by cholesterol depletion. In addition, signaling molecules involved in NF-B activation were largely excluded from lipid rafts. These findings differ from the results of a recent study in the fibrosarcoma cell line, HT-1080, in which the TNF--induced activation of NF-B was shown to be blocked by cholesterol depletion (30). Furthermore, depletion of cholesterol in HT-1080 cells augmented TNF--induced apoptosis (30). This latter finding also differs from previous studies by Ko et al. (16) which showed that TNF--induced apoptosis in the promonocytic cell line, U937, is initiated in lipid rafts. Further studies are clearly needed to resolve these differences. However, an intriguing possibility is that different cell types (i.e., fibroblasts vs monocytes/macrophages) use lipid rafts in different ways to regulate TNF-R1 signaling.

It will also be important in future studies to determine the consequences of TNF-R1-induced p42^mapk/erk2 signaling in lipid rafts. Previous studies from our laboratory have shown that TNF-R1 is rapidly and transiently phosphorylated by p42^mapk/erk2 in response to stimulation with TNF- and other stimuli (25, 39). In addition, we have shown that the phosphorylation of TNF-R1 by p42^mapk/erk2 inhibits the ability of TNF-R1 to induce apoptosis, though the ability of the receptor to signal NF-B activation is preserved (40). Thus, based on the findings of the present study, it is conceivable that the phosphorylation of TNF-R1 also takes place in lipid rafts (40). In summary, our findings support the conclusion that TNF-R1 is: 1) localized to both lipid raft and nonraft plasma membranecompartments, 2) competent to initiate signaling in both compartments, and 3) able to initiate distinct signaling events within each compartment. Thus, the dynamic composition of the raft and nonraft regions of the plasma membrane may play an important role in specifying signaling responses induced by TNF-R1.

	Acknowledgments

 
At National Jewish Medical and Research Center, we are indebted to Linda Remigio for providing outstanding technical support, Jeff Colbert, Jenny Terry, and Dr. Vincent Cottin for helpful discussions, and Dr. Robert Murphy and Chris Johnson for conducting the GC/MS cholesterol analyses. We also thank Dr. Jay Westcott at ELISA Tech for developing the TNF-R1 ELISA.

	Footnotes

 
¹ This work was supported by Public Health Service Grants HL55549, HL68628, and HL65326 from the National Heart, Lung, and Blood Institute of the National Institutes of Health. J.D. was supported in part by Individual F32 Grant HL67581, Institutional T32 Grant AI07405, and by an Arnold and Sheila Aronsen Fellowship in Pediatric Pulmonary Medicine.

² Address correspondence and reprint requests to Dr. David W. H. Riches, Program in Cell Biology, 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: RIP, receptor interacting protein; IKK, I-B kinase; GC/MS, gas chromatograph/mass spectrometry.

Received for publication August 21, 2003. Accepted for publication March 22, 2004.

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