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Short Communication |
1 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Forchheimer Building, Room 411, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Division of Infectious Diseases, Department of Pediatrics, Albert Einstein College of Medicine, Forchheimer Building, Room 411, 1300 Morris Park Avenue, Bronx, NY 10461, USA
Correspondence
Y. Rebecca Chin
rchin1{at}bidmc.harvard.edu
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ABSTRACT |
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B activation. Total cellular content of TNFR1 is also reduced in the presence of RID, which can be inhibited by treatment with lysosomotropic agents. In this report, surface biotinylation experiments revealed that, although RID and TNFR1 were able to form a complex on the cell surface, the rate of TNFR1 endocytosis was not affected by RID. However, the degradation of internalized TNFR1 was enhanced significantly in the presence of RID. Therefore, these data suggest that RID downregulates TNFR1 levels by altering the fate of internalized TNFR1 that becomes associated with RID at the plasma membrane, probably by promoting its sorting into endosomal/lysosomal degradation compartments.
Deceased. This paper is dedicated to his memory. 
Present address: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, Research North 216, Boston, MA 02215, USA.
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; Horwitz, 2004). The E3 receptor internalization and degradation (RID) complex, composed of two RID
and one RID
subunits, downregulates a specific set of plasma-membrane receptors, including FAS (Shisler et al., 1997; Elsing & Burgert, 1998; Tollefson et al., 1998), tumour necrosis factor (TNF)-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) (Benedict et al., 2001; Tollefson et al., 2001) and epidermal growth factor receptor (EGFR) (Carlin et al., 1989; Tollefson et al., 1991). Together with another E3 protein, E3/6.7K, RID also reduces the surface expression of TRAIL-R2 (Lichtenstein et al., 2004). Whilst both of the RID subunits are critical for the downregulation of FAS and TRAIL receptors, it appears that the RID subunit is sufficient to downregulate EGFR under certain experimental conditions (Hoffman et al., 1990). Recently, we demonstrated that RID also downregulates TNFR1, thereby inhibiting TNF-induced NF-B activation, which mediates the transcription of a large number of genes involved in inflammation and immune responses such as chemokine expression (Fessler et al., 2004a; Delgado-Lopez & Horwitz, 2006).
In vivo experiments have shown that RID is an essential component of the Ad E3 cassette, which facilitates transplantation of allogeneic pancreatic cells (Efrat et al., 1995) and decreases autoimmune type 1 diabetes incidence (von Herrath et al., 1997; Efrat et al., 2001; Pierce et al., 2003). To potentially use RID as a therapeutic immunomodulator, it is important to elucidate the molecular mechanism of RID-mediated downregulation of receptors. We recently showed that the tyrosine sorting motifs of RID and clathrin play important roles in the downregulation of TNFR1, and that RID-mediated TNFR1 degradation occurs via an endosomal/lysosomal pathway (Chin & Horwitz, 2005). In this report, we further examined the effect of RID on TNFR1 trafficking. Specifically, we tested the ability of RID to interact with TNFR1 on the cell surface and investigated whether RID enhances TNFR1 endocyotosis, or rather promotes the degradation of internalized TNFR1.
We have shown previously that mutation in the tyrosine sorting motif of RID [122tyrosine (Y) mutated to phenylalanine (F); RID(YF)] not only abolishes the downregulation of surface TNFR1, but paradoxically increases surface TNFR1 expression, suggesting that RID and TNFR1 may form a complex on the cell surface (Chin & Horwitz, 2005). To investigate this directly, cell-surface biotinylation was performed followed by co-immunoprecipitation and NeutrAvidin pull-down (Fig. 1a). HeLa cells were infected with a total m.o.i. of 5000 particles per cell (1 p.f.u. is equivalent to 20 virus particles) of Ad vectors (Chin & Horwitz, 2005) for 14 h. Cells were washed twice with ice-cold PBS and incubated with 300 µg EZ-link Sulfo-NHS-biotin ml–1 (Pierce) in PBS for 30 min at 4 °C. The biotinylation reaction was quenched by rinsing cells three times with Tris-buffered saline. Whole-cell lysates were prepared and co-immunoprecipitation was performed by using a Seize primary mammalian immunoprecipitation kit (Pierce), where affinity-purified rabbit anti-RID antibody (Genemed Synthesis Inc.; Chin & Horwitz, 2005) or non-immune rabbit IgG (Santa Cruz Biotechnology) was cross-linked to immobilized protein G before immunoprecipitation. The immunoprecipitated complex was eluted in an acidic elution buffer and the eluant was neutralized by adding 2.5 µl 1 M Tris (pH 9.5) per 50 µl of sample. The immunoprecipitated complex was then dissociated in 1 % SDS by heating at 95 °C for 7 min and then diluted 20-fold with lysis buffer [1 % Triton X-100, 150 mM NaCl, 1 mM EDTA, 2 mM Na2P2O7, 30 mM NaF, 20 mM Tris/HCl (pH 7.5) and 1x complete proteinase inhibitor cocktail (Roche Molecular Biochemicals)]. Biotinylated proteins were then precipitated with 30 µl immobilized NeutrAvidin (Pierce) for 2 h at 4 °C. The beads were washed four times with lysis buffer and once with PBS. Proteins precipitated by NeutrAvidin beads were analysed by Western blotting [cell-surface proteins, left panel of Fig. 1(b)] with rabbit anti-RID antiserum (Genemed Synthesis Inc.), mouse anti-TNFR1 antibody (Santa Cruz Biotechnology) and mouse anti-transferrin receptor antibody (Zymed). The non-biotinylated proteins remaining in the supernatant after NeutrAvidin precipitation were also collected, concentrated with 10 % trichloroacetic acid and analysed by Western blot [intracellular proteins, middle panel of Fig. 1(b)]. Equal amounts of whole-cell lysates from different samples were also immunoblotted [right panel of Fig. 1(b)] to determine expression levels of RID, TNFR1 and transferrin receptor.
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from cells infected with Ad/RID and Ad/RID [left panel of Fig. 1(b)
], suggesting that RID and TNFR1 are able to interact on the cell surface, either directly or through an intermediate bridging molecule. On the other hand, transferrin receptor, which was shown previously not to be downregulated by RID (Carlin et al., 1989; Tollefson et al., 1998), did not co-immunoprecipitate with RID, thus providing a specificity control for the Western blot. RID(YF), which itself has a higher level of surface expression, co-immunoprecipitated more plasma-membrane TNFR1 than did wild-type (WT) RID [left panel of Fig. 1(b)]. These results agree with previous observations that the tyrosine sorting motif of RID plays an important role in determining surface levels of RID (Hilgendorf et al., 2003) and TNFR1 (Chin & Horwitz, 2005). Interestingly, although there was less RID(YF) mutant present intracellularly than WT RID, similar amounts of intracellular TNFR1 were co-immunoprecipitated [middle panel of Fig. 1(b)]. It is possible that TNFR1, or the molecule that mediates the interaction between TNFR1 and RID, has a higher affinity for the RID(YF) mutant than the WT RID. Taken together, although we cannot completely rule out the possibility that part of the surface TNFR1 signal may be derived from internal labelling, the increased signal detected for RID(YF) supports the view that labelling indeed occurred at the cell surface. Therefore, these data suggest that RID is able to associate with TNFR1 on the cell surface. It is also noteworthy that, although TNFR1 appears to interact with RID on the cell surface, the interaction is much more pronounced in the intracellular fraction.
We next investigated whether RID downregulates surface TNFR1 by enhancing its endocytosis rate, or rather by promoting degradation of internalized TNFR1. In order to find the most appropriate time to carry out the assays, we first performed a kinetic study of RID-mediated TNFR1 downregulation. 293 cells (5x105) were infected with 1000 particles Ad/null per cell for 13 h or 1000 particles Ad/RID per cell for various durations (0–13 h). Downregulation of surface TNFR1 by RID was then assayed by flow cytometry as described previously (Chin & Horwitz, 2005). As shown in Fig. 2(a), the maximal TNFR1 downregulation activity by RID was between 5 and 7 h post-infection (the steepest slope of the curve). Therefore, the endocytosis assay was performed at 5.5 h post-infection, when RID had a significant activity towards downregulation of surface TNFR1, yet sufficient amounts of surface TNFR1 were still present to enable accurate quantification.
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Fig. 2(b) shows that the kinetics of TNFR1 endocytosis were very similar in Ad/null- and Ad/RID-infected cells, with approximately 10 and 20 % of surface TNFR1 internalized in 4 and 6 min, respectively (Fig. 2c). TNFR1 endocytosis was also examined up to 15 min incubation at 37 °C, with no difference observed between Ad/null- and Ad/RID-infected cells (data not shown). RID was detectable on the cell surface as well as being endocytosed (Fig. 2b), suggesting that the lack of an effect in TNFR1 internalization rate was not due to the lack of RID expression. To control for potential leakage of biotin across the plasma membrane, the amount of biotinylated -tubulin was quantified on the same blot. The abundant intracellular protein -tubulin was not biotinylated (Fig. 2b), suggesting that only cell-surface proteins were biotinylated and cell integrity was maintained during biotinylation. For comparison, cells were also stimulated with TNF, which has been shown to induce TNFR1 internalization (Higuchi & Aggarwal, 1994; Porteu & Hieblot, 1994). As expected, TNFR1 endocytosis was increased significantly in the presence of TNF (Fig. 2b). These results are in accord with the notion that TNFR1 internalizes continuously (Yoshie et al., 1986). Importantly, the data showed that RID did not downregulate TNFR1 by increasing its endocyotsis rate. Therefore, we hypothesized that RID downregulates surface TNFR1 by altering the fate of internalized TNFR1.
We have shown previously that RID decreases the total levels of TNFR1, which can be reversed by inhibitors of endosomal acidification (Chin & Horwitz, 2005). However, these studies assessed total cellular TNFR1 levels. In order to focus specifically on the fate of internalized TNFR1 and determine whether its degradation was enhanced in the presence of RID, a modified surface biotinylation assay was used. 293 cells were infected with 1000 particles Ad/null or Ad/RID per cell. At 4 h post-infection, when RID had no effect on the cell-surface level of TNFR1 (Fig. 2a), cells were rinsed twice with ice-cold PBS. Surface proteins were biotinylated with 300 µg EZ-link Sulfo-NHS-biotin ml–1 (Pierce) as described in the legend to Fig. 1. Cells were then lysed immediately to determine the initial amount of surface TNFR1 or incubated in DMEM supplemented with 2 % FBS for various times (2, 3 or 4 h) at 37 °C to allow trafficking and degradation of biotinylated TNFR1. NeutrAvidin precipitation was then performed to recover biotinylated TNFR1 remaining on and inside the cells. Detection of biotinylated TNFR1 and transferrin receptor on the Western blot was done as described above.
As shown in Fig. 3(a), there were similar amounts of biotinylated TNFR1 in Ad/null- and Ad/RID-infected cells at 4 h post-infection. After incubating cells at 37 °C for 2–4 h, there was less biotinylated TNFR1 recovered from the Ad/RID-infected cells than from the Ad/null-infected cells at all time points examined, indicating that internalized TNFR1 was degraded more rapidly in the presence of RID. By fitting a second-order polynomial curve to the data, the half-life of degradation of surface TNFR1 in Ad/null-infected cells was approximately 99 min, whereas in the presence of RID, the half-life of TNFR1 was shortened to 63 min (Fig. 3b). For comparison, the amount of transferrin receptor was also quantified on the same blot (Fig. 3a). Biotinylated transferrin receptors exhibited little or no degradation at all time points examined, agreeing with previous observations that, once being internalized, transferrin receptors were recycled back to the plasma membrane instead of being degraded in lysosomes (Mayor et al., 1993). The constant levels of biotinylated transferrin receptor also control for the possible non-specific effects of viral infection on cell viability and function. In addition, the lack of effect on transferrin receptor by RID suggests that the effect of RID on TNFR1 degradation was specific. As a control, biotinylation of the intracellular protein -tubulin was not observed (data not shown), confirming that no biotin leaked across the plasma membrane during the biotinylation procedure. These results indicated that RID enhanced the degradation of internalized TNFR1.
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), a number of viral proteins target the TNF receptor and components of its associated signalling pathways. In this study, we have demonstrated that RID downregulates the surface levels of TNFR1 without accelerating its rate of endocytosis. To our knowledge, this is the first report showing that a viral protein promotes the degradation of endocytosed TNFR1. The mechanisms by which RID enhances TNFR1 degradation remain to be elucidated. It is possible that, by associating with TNFR1 on the cell surface, RID stimulates mono-ubiquitination of TNFR1 or causes the exposure of a cryptic degradation or sorting signal on TNFR1, thereby rerouting the recycling TNFR1 to the endosomal/lysosomal pathway. Interestingly, RID also downregulates EGFR by enhancing its degradation, without affecting its endocytosis rate (Hoffman & Carlin, 1994); this agrees with the finding that RID associates with EGFR in early endosomes (Crooks et al., 2000). For TNFR1, we showed here that RID could associate with TNFR1 on the cell surface. In addition, downregulation of TNFR1 by RID is decreased in the presence of small interfering RNAs against the µ2 subunit of AP-2, a complex that plays an important role in clathrin-mediated endocytosis (Chin & Horwitz, 2005). However, RID has no effect on the TNFR1 endocytosis rate. These data suggested that, instead of accelerating the internalization of TNFR1, RID and clathrin play an important role in mediating delivery of TNFR1 to intracellular sites that accelerate its degradation, probably late endosomes or lysosomes, as lysosomotropic agents prevent degradation of TNFR1 by RID (Chin & Horwitz, 2005).
Experiments in this study did not address whether RID was degraded together with TNFR1 in the lysosomes. However, previous observations showed limited co-localization of RID and FAS in lysosomes (Hilgendorf et al., 2003). In addition, whereas EGFR was degraded in lysosomes, RID was retained on the limiting membrane of multivesicular bodies (Crooks et al., 2000). Thus, it is possible that RID escapes to the recycling pathway for another round of internalization. Indeed, it would be advantageous for the virus to use the same protein repeatedly for enhancing the efficiency of receptor downregulation.
Although experiments of the current study were performed with CMV promoter-driven RID, previous flow-cytometry experiments from our laboratory have shown that infection of 293 cells with WT adenovirus led to a modest (
17 %) downregulation of TNFR1 (data not shown), suggesting that TNFR1 downregulation by RID is not restricted to an artificial system. We believe that our current mechanistic studies with the potentially overexpressed CMV promoter-driven RID are relevant for natural adenovirus infection, as expression levels and effects of RID in vivo are likely to be dependent on the cell type, as well as many other parameters. Nonetheless, the potential of expressing and utilizing E3 proteins, including RID, out of the context of adenovirus to facilitate allogeneic cell transplantation and decrease the incidence of type I autoimmune diabetes prompts further studies aimed at dissecting precise mechanisms of RID-mediated TNFR1 degradation.
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ACKNOWLEDGEMENTS |
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Received 3 March 2006;
accepted 7 July 2006.
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