The p55 tumour necrosis factor receptor TNFR1 contains a ... - NCBI

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Biochem. J. (2002) 366, 15–22 (Printed in Great Britain)

The p55 tumour necrosis factor receptor TNFR1 contains a trans-Golgi network localization signal in the C-terminal region of its cytoplasmic tail Helen STOREY*, Abigail STEWART*, Peter VANDENABEELE† and J. Paul LUZIO*1 *Department of Clinical Biochemistry and Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2XY, U.K., and †Molecular Signalling and Cell Death Unit, Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Gent, Ledeganckstraat 35, B-9000 Gent, Belgium

It has been reported in some human cells that, in addition to a plasma membrane localization, members of the tumour necrosis factor receptor superfamily may be localized to the Golgi complex. We have shown by immunofluorescence and immunoelectron microscopy that the p55 tumour necrosis factor receptor, TNFR1, is principally localized to the trans-Golgi network in the human breast carcinoma cell line, MCF7. Chimaeras consisting of the extracellular and transmembrane domains of CD8 together with the cytoplasmic tail of TNFR1 were targeted to the transGolgi network in stably transfected rat fibroblastic cells. Deletions in the cytoplasmic tails of these chimaeras demonstrated

the requirement for the C-terminal sequence of 23 amino acids for this targeting. The 23 amino acid sequence is mostly outside the death domain and contains both an acid patch and a dileucine motif. Interaction of this sequence with membrane traffic adaptor proteins may play an important role in controlling the responses of cells to tumour necrosis factor, since binding of signalling adaptor proteins has only been demonstrated for plasma membrane, and not Golgi-localized, TNFR1.

INTRODUCTION

Within the Golgi complex, TNFR1 has been shown to be mainly localized to the trans-Golgi network (TGN) [9] by investigating cells in which brefeldin A treatment causes collapse of the TGN into a cluster of vesicles near the microtubule organizing centre, giving a dot-like appearance by immunofluorescence microscopy. This behaviour of the TGN occurs in many cells and differs from that of the Golgi cisternal membranes which tubulate and fuse with the endoplasmic reticulum (ER) [11–13]. However, in some cells TGN membrane proteins also redistribute to the ER following brefeldin A treatment [14]. In the present study, we have examined the localization of intracellular TNFR1 in the TNF-sensitive cell line, MCF7 [15], where the TGN cannot be distinguished from the Golgi cisternae by the effects of brefeldin A treatment. Targeting information for localization of TNFR1 to the TGN is present in the cytoplasmic tail of TNFR1. Gaeta et al. [10] showed that deletion of the tail from an epitope tagged TNFR1 results in expression on the surface of transiently transfected cells. However, the localization of a chimaera of the mainly plasma-membrane localized, full-length TNFR2 with the death domain of TNFR1 added to its C-terminus, was similar to TNFR2 alone. A small proportion of TNFR2 was observed in the Golgi region but this was assumed to represent newly synthesized molecules. The data obtained by Gaeta et al. [10] suggested that the cytoplasmic tail was necessary but not sufficient for TGN localization of TNFR1. This conclusion is at odds with studies on the localization of other type I membrane proteins to the TGN. Chimaeras of the ecto- and trans-membrane domains of several different type I plasma-membrane proteins together with the cytoplasmic tail of TGN38 were localized to the TGN [16–19]. Similarly, the cytoplasmic tail of furin is both necessary and sufficient to localize chimaeric proteins to the TGN

Members of the tumour necrosis factor receptor (TNFR) superfamily have unique structural attributes that couple them directly to signalling pathways for cell proliferation, cell death and differentiation [1]. They are type I membrane proteins which signal through two main classes of cytoplasmic adaptor proteins : TNFR-associated factors and death-domain molecules. Death domains in the cytoplasmic tails of many members of the family consist of 60–80 amino acids in a globular bundle of six antiparallel conserved α helices [1– 4]. Two members of the family, Fas (CD95) and TNFR1 (CD120a) have been shown to be localized to the Golgi complex as well as the plasma membrane in at least some cell types. In human vascular smooth muscle cells, Fas shows a Golgi localization from which it can be recruited to the plasma membrane following p53 activation [5] or the addition of proteosome inhibitors [6]. Similarly, TNFR1 is primarily a Golgi-associated protein in human umbilical vein endothelial cells, in the human monocyte cell line U937, the bladder carcinoma cell line ECV , the human neuroblastoma $!% cell line SK-N-BE and in transfected bovine aortic endothelial cells [7–10]. Despite this major site of localization, addition of exogenous tumour necrosis factor (TNF) results in rapid binding of TNF receptor-associated death-domain protein (TRADD) to TNFR1 at the plasma membrane, but there is no association between TNFR1 and TRADD in the Golgi complex [9]. Therefore, alteration of the amounts of TNFR1 at the cell surface relative to the Golgi, or vice versa, may have an important effect on the ability of a cell to respond to exogenous TNF. For this reason, it is important to understand the mechanisms by which TNFR1 is localized to the Golgi complex and its membrane traffic itinerary.

Key words : dileucine signal, Golgi complex membrane traffic, protein targeting.

Abbreviations used : DMEM, Dulbecco’s Modified Eagle’s Medium ; ER, endoplasmic reticulum ; FITC, fluorescein isothiocyanate ; NRK, normal rat kidney ; TGN, trans-Golgi network ; TNF, tumour necrosis factor α ; TNFR, TNF receptor ; TNFR1, p55 TNF receptor ; TNFR2, p75 TNF receptor ; TRADD, TNF receptor-associated death-domain protein. 1 To whom correspondence should be addressed (e-mail JPL10!CAM.AC.UK). # 2002 Biochemical Society

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H. Storey and others

[20]. To obtain a clearer picture of the role of the cytoplasmic tail of TNFR1 in Golgi localization, we have constructed and localized chimaeras of the plasma membrane protein CD8, a reporter that we have used previously [19,21], together with full-length and truncated cytoplasmic tails of TNFR1. Because the cytoplasmic tail contains a death domain, we have when appropriate, used constructs in which a single leucine to alanine mutation in the death domain has been shown to prevent activation of any cell death programme in transiently transfected cells [22]. We have also used the inducible expression vector, ∆pMEP4, to minimize expression until stable cell lines were established and protein expression induced [21,23].

EXPERIMENTAL Antibodies The mouse monoclonal antibody to human TNFR1 used for immunofluorescence was from Genzyme (Boston, MA, U.S.A.) and for immunoelectron microscopy from R&D Systems, (Oxford, U.K.). The sheep anti-human TGN46 [24], a gift from Dr S. Ponnambalam (University of Leeds, U.K.), the mouse monoclonal antibody to rat TGN38, designated 2F7.1 [25] and the rat monoclonal antibody to the α-chain of human CD8 (Campath 8c ; [26]), a gift from Dr G. Hale (School of Pathology, University of Oxford, U.K.), were as described previously. Rabbit anti-CD8 was from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Texas Red-labelled donkey anti-mouse IgG, fluorescein isothiocyanate (FITC)-labelled donkey anti-mouse IgG and FITC-labelled donkey anti-sheep IgG were from Jackson Immunoresearch Laboratories (West Grove, PA, U.S.A.). Texas Red-labelled goat anti-rat IgG was from Molecular Probes (Eugene, OR, U.S.A.). Goat anti-mouse IgG conjugated to 5 nm colloidal gold and donkey anti-sheep IgG conjugated to 10 nm colloidal gold were from British BioCell (Cardiff, U.K.). Horseradish peroxidase-labelled donkey antirabbit IgG, was from Amersham (Little Chalfont, Bucks, U.K.) and was used to detect CD8\TNFR1 chimaeras, separated by SDS\PAGE, on immunoblots with the Amersham ECL2 system.

Cell culture and transfection Human breast carcinoma MCF7 cells (from the European Collection of Cell Cultures, Salisbury, U.K.) and fibroblastic normal rat kidney (NRK) cells were grown in Dulbecco’s modified Eagles medium (DMEM), supplemented with 10 % (v\v) fetal-calf serum, 100 units\ml penicillin, 100 µg\ ml streptomycin, 4.5 g\l glucose and 2 mM -glutamine [27]. Cells were grown in 25 cm# tissue culture flasks or on glass coverslips in a 5 % CO incubator at 37 mC [27]. NRK cells were transfected # with ∆pMEP4 constructs using FuGENE 6TM transfection reagent (Roche Molecular Biochemicals, Sussex, U.K.) and stable cell lines selected as previously described [21]. Protein expression was induced by addition of 10 µM CdCl for 16 h unless # otherwise stated.

Indirect immunofluorescence microscopy Cells grown on glass coverslips were fixed and permeabilized for 10 min in methanol at k20 mC, rehydrated with PBS and then labelled as previously described [21,27]. Cells were treated with brefeldin A, chloroquine, nocodazole and cycloheximide (Sigma), prior to fixation and labelling, as described previously [16,27]. Following incubation with antibodies and mounting, the cells were examined using a Nikon Optiphot II epi-fluorescence # 2002 Biochemical Society

microscope equipped with a Biorad MRC 1024 confocal laser scanning attachment and a 60i objective.

Immunoelectron microscopy Cells were fixed with 4 % (v\v) paraformaldehyde\0.1 % glutaraldehyde in 250 mM Hepes, pH 7.2, at 20 mC for 1 h, infused with 1.7 M sucrose\15 % (w\v) polyvinyl pyrolidone and prepared as described previously [28]. Ultrathin frozen sections were collected from the knife edge with (50 : 50, v\v) 2 % methyl cellulose\2.3 M sucrose [29] and immunolabelled, contrasted, dried and observed in a Philips CM100 [28].

Constructs All recombinant DNA procedures were carried out essentially as described by Sambrook and Russell [30] unless stated otherwise. Pfu DNA polymerase (Stratagene, Cambridge, U.K.) was used in all PCR reactions and sequences of all final constructs were verified by DNA sequencing using the Big Dye ‘ ABI PRISM ’2 Terminator4 Cycle Sequencing kit (ABI-PerkinElmer, Boston, MA, U.S.A.) and the service provided by the Department of Genetics, University of Cambridge, U.K. Oligonucleotides for PCR were synthesized by Genosys (Cambridge, U.K. ; sequences are available from J. P. L. on request) and human TNFR1 cytoplasmic tail templates were as described by Boone et al. [22]. The ‘ full- length ’ tail template and the template encoding a tail with a major deletion, both encoded an epitope tag replacing the C-terminal five amino acids of the tail. PCR reactions were designed to remove nucleotides encoding this tag and replace them with nucleotides encoding the missing PSLLR sequence (Figure 1A) followed by a stop codon. An Af l II site was engineered at the 5h end of each tail construct and either a Not I site or a blunt site at the 3h end (see construct diagrams in Figure 1B). ∆pMEP4 plasmids containing inserts encoding the lumenal and transmembrane domains of CD8 and the mutated cytoplasmic tails of TNFR1 were constructed as described previously, such that the Af l II site was at the transmembrane\cytoplasmic domain boundary [21]. The presence of the sequence encoding the transmembrane domain of CD8 and the Af l II site converted the first four amino acids of the cytoplasmic tail of TNFR1 from RYQR to KRLK in the CD8\TNFR1 chimaeras. A diagram of the sequence of the cytoplasmic tail of TNFR1 is shown in Figure 1(A) and indicates sequence missing from the various chimaeric constructs prepared. Numbering of amino acids is based on the notation provided with the sequence in GenBank2 Accession number P19438. This numbering starts from the beginning of the predicted signal sequence and differs from that used by Boone et al. [22] such that the L351A mutation reported in [22] becomes L380A here.

SDS/PAGE and Western blotting Cells were pelleted in PBS and lysed in cold PBS containing 1 % (v\v) Nonidet P40 plus CompleteTM protease inhibitors (Roche Molecular Biochemicals, Sussex, U.K.), for 30 min on ice. Media samples were removed directly from the tissue culture flasks and centrifuged to remove cell debris. Samples were electrophoresed on 7.5–20 % gradient polyacrylamide\SDS gels (according to the discontinuous buffer system of Laemmli [31]) and proteins electrophoretically transferred to nitrocellulose membranes for 3 h at 40 V. Transferred proteins were stained with 0.2 % (w\v) Ponceau S\3 % (w\v) trichloroacetic acid for 5–10 min, followed by washing with PBS\0.1 % (v\v) Tween-20. Membranes were then blocked for 1 h at 20 mC in blocking buffer

Tumour necrosis factor receptor in the Golgi

Figure 1

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Cytoplasmic tail of TNFR1

(A) Amino acid sequence of the cytoplasmic tail. Boxed sequence is the death domain. Highlighted sequence (grey) is missing from ∆ constructs. Dashed underlined sequence is missing from R constructs. (B) The cytoplasmic tails of CD8-TNFR1 constructs. Death domains are shown in grey highlight. The acidic region (435– 459) is shown as a black vertical bar and the PSLLR sequence as a grey vertical bar. Position of mutation L380A is shown with an asterisk. CCT indicates the lumenal and transmembrane domains of CD8 (CC) followed by the full length tail of TNFR1 containing the L380A mutation (T). Similarly CCTR, CCT∆ and CCT∆R indicate CD8/TNFR1 chimaeras with deletions in the tail as indicated.

[PBS, 5 % (w\v) non-fat dry milk powder, 0.1 % (v\v) Tween-20], followed by washes in PBS\0.1 % (v\v) Tween-20. Polyclonal rabbit anti-human CD8 was diluted (1 : 500) in blocking buffer and incubated with the membranes overnight at 4 mC on a continuous shaker. Membranes were then washed with PBS\0.1 % Tween-20 (v\v) at 4 mC. Horseradish peroxidase-labelled secondary antibody was diluted in blocking buffer and incubated with membranes for 30 min at room temperature. Membranes were washed in PBS\0.1 % (v\v) Tween-20, and then developed using the ECL2 method of Amersham.

RESULTS The localization of TNFR1 in MCF7 cells was examined by immunofluorescence and immunoelectron microscopy. Colocalization with the human TGN protein TGN46 [32] was observed by immunofluorescence, with little TNFR1 seen on the cell surface (Figures 2A and 2B). Treatment of the cells with

Figure 2

Immunomicroscopy of TNFR1 and TGN46 in MCF7 cells

Cells were labelled with monoclonal mouse anti-TNFR1 (A, C, E and G) and polyclonal sheep anti-TGN46 (B, D, F and H), followed by Texas Red anti-mouse Ig (A, C, E, and G) and FITC anti-sheep Ig (B, D, F and H). Scale bar, 10 µm. Cells were incubated at 37 mC, prior to fixation and processing for immunofluorescence, as follows : (C, D) 2 h, 5 µg/ml brefeldin A (BFA) ; (E, F) 2 h, 100 µM chloroquine (CQ) ; (G, H) 2 h, 20 µg/ml nocodazole (Noc). (I) Immunoelectron micrograph of a section through an MCF7 cell, labelled with mouse anti-TNFR1 and sheep anti-TGN46, followed by 5 nm gold conjugated anti-mouse Ig and 10 nm gold conjugated anti-sheep Ig. Scale bar, 100 nm. (J) Quantification of immunogold labelling. The histogram shows the distance from the nearest 10 nm particle (TGN46) to each 5 nm particle (TNFR1). The number of 5 nm particles used for these measurements was 210, from a total of 28 separate fields.

brefeldin A resulted in redistribution of both TNFR1 and TGN46 throughout the cell (Figures 2C and 2D). This pattern is characteristic of the redistribution of Golgi cisternal markers to the ER and has been observed previously for the redistribution of Golgi complex localized polysialic acid in MCF7 cells [33] in experiments in which conventional TGN markers, such as TGN46, were not examined. However, similar redistribution of TGN markers to the ER has also been observed in rat exocrine pancreas cells [14]. Further evidence for the Golgi # 2002 Biochemical Society

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Figure 3


Expression of CD8/TNFR1 chimaeras in transfected NRK cells

Stably transfected NRK cell lines containing ∆pMEP constructs encoding CD8 chimaeras were incubated with 0–10 µM CdCl2 for 16 h prior to solubilization and analysis by SDS/PAGE followed by ECL2. (A) CD8 (CCC) ; predicted size of polypeptide chain, 26.6 kDa. The position of the 31 kDa marker is shown. (B) CCT ; predicted size of polypeptide chain, 45.2 kDa. The position of the 45 kDa marker is shown. (C) CCT∆ ; predicted size of polypeptide chain, 31.2 kDa. The position of the 31 kDa marker is shown. (D) CCT∆R ; predicted size of polypeptide chain, 29.0 kDa. The position of the 31 kDa marker is shown.

localization of TNFR1 was obtained by treatment with chloroquine and nocodazole. Chloroquine causes redistribution of TGN38 family members including TGN46 to swollen early endosomes [34,35], whereas nocodazole treatment results in the break up of the Golgi complex into many mini-Golgis [11,13]. After both treatments, TNFR1 remained co-localized with TGN46, implying that it started in the same intracellular compartment as this protein and was able to follow the same membrane traffic routes (Figures 2E–2H). To confirm that both TGN46 and TNFR1 were in the TGN of MCF7 cells, immunoelectron microscopy was carried out. As expected, TGN46 was localized to one side of the stack of Golgi cisternae, consistent with it being in the TGN (Figure 2I). TNFR1 was observed within the same membrane-bound compartment (Figure 2I). The localization of TNFR1 relative to TGN46 was quantified by measuring the distance between gold particles, labelling antibodies reacting with the two molecules. Over 57 % of gold particles marking TNFR1 were within 50 nm of those marking TGN46, confirming the major site of TNFR1 localization as the TGN. To study the role of the cytoplasmic tail of TNFR1 in targeting the molecule to the Golgi complex, plasmids encoding chimaeras of the ecto- and trans-membrane domains of human CD8 together with full-length and truncated cytoplasmic tails of TNFR1 were constructed and used to transfect NRK cells. These cells have a TGN which collapses into a brefeldin ‘ dot ’ near the microtubule organizing centre following treatment with brefeldin A [12,13]. Stably transfected cell lines were selected over several weeks. Despite using a plasmid, ∆pMEP4, with an inducible promoter and the presence of a mutation in the death domain (L380A) that allows cell survival during the time course of transient transfections [22], it was found that cells containing plasmids encoding chimaeras containing the tail death domain (CCT and CCTR) grew more slowly than those with a major deletion in the death domain (CCT∆ and CCT∆R). Nevertheless, # 2002 Biochemical Society

Figure 4 cells

Localization of CD8/TNFR1 chimaeras in transfected NRK

Stably transfected NRK cells were double labelled with monoclonal rat anti-CD8 (A, C, E, G and I) and monoclonal mouse anti-TGN38 (B, D, F, H and J), followed by Texas Red anti-rat Ig (A, C, E, G and I) and FITC anti-mouse Ig (B, D, F, H and J). Arrows indicate colocalization. Scale bar, 10 µm.

stably transfected cell lines containing all four constructs were isolated. Examination of representative lines by SDS\PAGE following incubation with CdCl (Figure 3) revealed that the # expression of each chimaera varied such that relative levels of expression were CCT∆ l CCC (the full length reporter molecule CD8) CCT∆R CCT CCTR. The CCTR chimaera was barely visible following SDS\PAGE (results not shown, although immunofluorescence experiments confirmed that synthesis of CCTR was induced by CdCl ). The metallothionein II # promoter was leaky in all the cell lines tested so that some protein was synthesized during cell-line selection (Figure 3 ; results not shown for CCT and CCTR). Attempts to achieve significantly higher levels of expression by induction with 20 µM CdCl # plus 80 µM ZnCl for 16 h [23] were unsuccessful. # Immunofluorescence analysis of the stably transfected NRK cell lines was carried out following induction of protein synthesis, and the localization of CD8\TNFR1 chimaeras was compared with that of the full-length reporter molecule CD8 (CCC) which was located entirely at the cell surface (Figure 4A). In contrast, in those cells expressing CCT, an intracellular pool was observed co-localizing with the rat TGN marker, TGN38, in addition to a cell surface pool (Figures 4C and 4D). The chimaera in which the C-terminal 23 amino acids of the TNFR1 tail was missing, CCTR, showed no colocalization with TGN38 (Figures 4E and 4F), whereas some colocalization was observed when there was a major deletion in the death domain but the C-terminal end of the tail was maintained (CCT∆ ; Figures 4G and 4H). When removal of the C-terminal 23 amino acids was carried out in addition to the major deletion (CCT∆R), no colocalization with TGN38 was seen (Figures 4I and 4J). The immunofluorescence

Tumour necrosis factor receptor in the Golgi

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Figure 5 Cycloheximide treatment of MCF7 cells and transfected NRK cells expressing CD8/TNFR1 chimaeras MCF7 cells (A, B) and stably transfected NRK cells expressing either CCT (C, D) or CCT∆ (E, F), were incubated for 6 h at 37 mC, with 20 µg/ml cycloheximide, prior to processing for immunofluorescence. Cells were labelled with monoclonal mouse anti-TNFR1 (A), polyclonal sheep anti-TGN46 (B), monoclonal rat anti-CD8 (C, E) and monoclonal mouse anti-TGN38 (D, F), followed by Texas Red anti-mouse Ig (A), FITC anti-sheep Ig (B), Texas Red anti-rat Ig (C, E) and FITC anti-mouse Ig (D, F). Scale bar, 10 µm.

experiments shown in Figure 4 were carried out on cells fixed with methanol, which allows antibody access to both the cell surface and intracellular pools of antigen. We also showed the presence of all four CD8\TNFR1 chimaeras on the cell surface of cells fixed with paraformaldehyde (results not shown), thus confirming that each of the chimaeras was able to pass along the secretory pathway through the Golgi complex to the plasma membrane. Taken together, these data suggested that the 23 amino acids at the C-terminus of TNFR1 are responsible for targeting to the Golgi complex. One possible explanation for localization of a protein to the Golgi complex is that it is simply present in a pool on the secretory pathway to the cell surface. This can be tested by adding cycloheximide which inhibits protein synthesis and has been used to chase proteins through the secretory pathway whilst not affecting the distribution of resident proteins of the Golgi complex [16]. Treatment of MCF7 cells for 6 h at 37 mC with 20 µg\ml cycloheximide, prior to processing for immunofluorescence, had no effect on the Golgi localization of TNFR1 (Figures 5A and 5B). Similarly it had no effect on the partial Golgi localization of the CD8\TNFR1 chimaeras CCT and CCT∆ in stably transfected NRK cells (Figures 5C–5F). After brefeldin A treatment of stably transfected NRK cells, the Golgi localized pool of CCT relocated to the brefeldin ‘ dot ’ labelled with antibodies to TGN38 (Figures 6A and 6B). Treatment with chloroquine and nocodazole caused redistribution of TGN38, as expected, and the Golgi localized pool of CCT redistributed to the same structures (Figures 6C–6F). Similar results were obtained after treatment of stably transfected NRK cells expressing CCT∆ (Figures 7A–7F). These results are consistent with the Golgi-localized pool of CCT and CCT∆ being

Figure 6

Immunofluorescence of NRK cells expressing CCT

Stably transfected NRK cells expressing CCT were incubated for 2 h at 37 mC, prior to processing for immunofluorescence, with : (A, B) 5 µg/ml brefeldin A (BFA) ; (C, D) 100 µM chloroquine (CQ) ; (E, F) 20 µg/ml nocodazole (Noc). Cells were double labelled with monoclonal rat anti-CD8 (A, C, E) and monoclonal mouse anti-TGN38 (B, D, F), followed by Texas Red anti-rat Ig (A, C, E) and FITC anti-mouse Ig (B, D, F). Arrows indicate colocalization. Scale bar, 10 µm.

in the TGN and the 23 amino acids at the C-terminus of TNFR1 containing a signal for TGN localization.

DISCUSSION It has been reported previously that the cytoplasmic tail, and more particularly the death domain, of TNFR1 is necessary but not sufficient for targeting TNFR1 to the TGN [10]. This conclusion was based on the results of experiments where no difference in the localization between TNFR2\TNFR1 chimaeras and TNFR2 was observed in transiently transfected cells. Why do the results of our experiments, using CD8\TNFR1 constructs in stably transfected cell lines, apparently contradict these findings ? In both sets of experiments substantial proportions of the chimaeras were observed on the cell surface. It is our contention that, under these circumstances, any Golgi-localized pool can only be properly investigated by establishing that it is not simply a pool of protein present in the secretory pathway en route to the cell surface. In the present work this was shown by investigating the effect of cycloheximide, and also by adding agents which cause relocalization of Golgi marker proteins, allowing us to show that the chimaera(s) moves with them. Using these criteria, we have established firstly, that the cytoplasmic tail of TNFR1 is able to direct a reporter molecule, CD8, to the TGN. Secondly, we have shown that a major deletion in the death domain (amino acids 273– 413) has no effect on the localization of CD8\TNFR1 chimaeras, demonstrating that the death domain as a whole is not required for TGN localization. Finally, we have established that the 23 amino acids at the C-terminus # 2002 Biochemical Society

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A – BFA

CCTD

B

TGN38

Figure 8 CCTD

C –CQ

CCTD

E – Noc

Figure 7

D

F

TGN38

TGN38

Immunofluorescence of NRK cells expressing CCT∆

Stably transfected NRK cells expressing CCT∆ were incubated for 2 h at 37 mC, prior to processing for immunofluorescence, with : (A, B) 5 µg/ml brefeldin A (BFA) ; (C, D) 100 µM chloroquine (CQ) ; (E, F) 20 µg/ml nocodazole (Noc). Cells were double-labelled with monoclonal rat anti-CD8 (A, C, E) and monoclonal mouse anti-TGN38 (B, D, F), followed by Texas Red anti-rat Ig (A, C, E) and FITC anti-mouse Ig (B, D, F). Arrows indicate colocalization. Scale bar, 10 µm.

of TNFR1 contain a signal for TGN localization and that these amino acids are necessary and sufficient for such localization. The C-terminal 23 amino acid sequence of TNFR1 is CLEDIEEALCGPAALPPAPSLLR which contains both an acidic cluster EDIEE as well as a putative dileucine targeting motif. The acidic cluster is located towards the C-terminus of the predicted sixth conserved α helix of the death domain. However, NMR studies have shown that this region is not necessary for receptor self-association or interaction with the TNFR1associated death-domain protein TRADD [4]. Several other type I integral membrane proteins, which at steady state are almost fully or partially localized to the TGN, have been shown to achieve this localization as a result of acidic clusters with or without dileucine motifs in their cytoplasmic tails. The best studied of these proteins are the endoprotease furin [36–38] and the mannose6-phosphate receptors [39,40]. In addition, in the integral membrane proteins, glucose transporter GLUT4 [41] and insulin-regulated aminopeptidase [42], acidic cluster and dileucine motifs in cytoplasmic tails are required for the regulation of sorting in endosomal compartments and transport to the cell surface. How acidic clusters and\or dileucine motifs mediate targeting within membrane traffic pathways is not fully understood # 2002 Biochemical Society

The C-terminal amino acids of TNFR1 in different mammals

The amino acid sequence of the C-terminal 23 amino acids of human TNFR1 (amino acids 423– 445) is aligned with the C-terminal sequence from TNFR1 of other mammals. Positions of complete identity are starred. GenBank accession numbers are human, P19438 ; pig, P50555 ; cow, O19131 ; rat, P22934 ; mouse, P25118. The 10 amino acids predicted to be in the C-terminal α-helix of the death domain are underlined. –, alignment gap.

since, to date, no crystal structure of a membrane traffic adaptor protein together with a peptide containing a dileucine motif and\or acidic cluster has been solved. Nevertheless, binding of cytoplasmic tails of membrane proteins through acidic clusters and\or dileucine motifs to membrane traffic adaptor proteins has been established, including binding to the Golgi-associated, γ-adaptin homologous, ADP ribosylation factor interacting proteins or GGAs [43– 45], the adaptor proteins AP-1, AP-2 and AP-3 (reviewed in [46]) and to phosphofurin acidic cluster sorting protein, PACS [47], which interacts with AP-1. Phosphorylation of the acidic cluster may activate the dileucine motif and modulate such binding [48]. In TNFR1, the acidic cluster towards the C-terminus of the cytoplasmic tail is well conserved across different mammalian species, for example, cow, pig and man. In rodents the acidic cluster is clearly less acidic and the dileucine motif close to the C-terminus is replaced by the amino acids LP (Figure 8), raising the question of whether these sequences in the rodent TNFR1 cytoplasmic tail can target the protein to the TGN. Whether functioning as an internalization signal from the cell surface or as a lysosomal targeting sequence, the dileucine motif has been known for many years to retain function even when one of the leucines is mutagenized or naturally replaced by another amino acid. Indeed, in the paper reporting the discovery of the motif, it was noted that in the chimaeric protein examined, lysosomal targeting was reduced, not abolished, when the second leucine was replaced by alanine, but that the first leucine was less resilient to change [49]. Further analysis of the specific requirements within the C-terminal 23 amino acid sequence of TNFR1 to obtain TGN localization may not be easy in the present experimental system. The difficulty of obtaining stable clones expressing chimaeras containing the TNFR1 death-domain, even that with the L380A mutation, was described above, with reference to the leakiness of the metallothionein II promoter. This leakiness has been observed by others, for example in a study of the expression of grenn fluorescent protein-tagged TGN38 in NRK cells where the level of expression of recombinant protein in stable transfectants incubated in the absence of CdCl was 7.5i that of the endo# genous protein [23]. A plasmid vector allowing better control of protein expression, together with use of chimaeric constructs containing TNFR1 tails with major deletions of the death domain (as in CCT∆), may provide a way to allow the production of clones expressing tails mutagenized to substitute individual amino acids. A further problem is that in the transfected NRK cells none of our chimaeric constructs was solely localized to the TGN. This may reflect a requirement for a signal elsewhere in the

Tumour necrosis factor receptor in the Golgi molecule, e.g. the transmembrane domain, which in TGN38 contains a TGN retention signal that functions together with a tail retrieval signal to ensure that most of the protein is present in the TGN at steady state [19]. We also cannot rule out the possibility that there are competing signals within the TNFR1 tail that function to give different intracellular targeting, the overall localization being the result of the sum of these signals. In this respect it is of interest that phosphorylation of the membrane proximal domain of the cytoplasmic tail of TNFR1 has been shown to alter localization of the receptor, resulting in loss of expression at the plasma membrane and Golgi complex, but accumulation in intracellular tubular structures associated with the ER [50]. Finally, the TGN localization signal in the cytoplasmic tail of TNFR1 may function with different efficiency in different cell types, and NRK fibroblasts may not be the best experimental host for the analysis of TNFR1 tail chimaeras. What is the functional importance of the intracellular localization of TNFR1 at the TGN if the TGN is not a site for signalling ? One possibility suggested by the presence of a TGN localization signal in the cytoplasmic tail is that there may be regulation of a recycling pathway between the TGN and cell surface, allowing control of the amount of TNFR1 on the cell surface, or at other sites on the pathway, thus influencing signalling. There is already good evidence that after TNF binding and TNFR1 internalization, TRADD dissociates and signalling terminates [9]. A close relationship between membrane traffic and signalling has also been established for several other cell surface receptors [51]. In the case of Fas, a member of the TNFR superfamily, it has been shown that in vascular smooth muscle cells activation of p53 causes a redistribution of Fas to the cell surface from the Golgi complex [5]. A relationship between cell sensitivity to TNF and p53 activity has also been reported [15,52], and resistance of MCF7 cells to TNF-induced cell death has been associated with loss of p53 function [15]. Thus, it is tempting to speculate that there may be a similar relationship between p53 and TNFR1 localization\function in these cells as there is between p53 and Fas in vascular smooth muscle cells.

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H. S. held a Medical Research Council collaborative studentship. We thank the Medical Research Council for financial support. Cambridge Institute for Medical Research is in receipt of financial support from the Wellcome Trust. P. V. was supported by the Interuniversitaire Attractiepolen IV/26, the Fonds voor Wetenschappelijk OnderzoekVlaanderen (grant 3G.0006.01) and the Bijzonder Onderzoeksfonds. We thank Dr John Bradley, Dr Barbara Reaves, Dr Elizabeth Ledgerwood and Dr John Prins for many helpful discussions.

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# 2002 Biochemical Society

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