Molecular analysis of transport and oligomerization of the Yersinia enterocolitica adhesin YadA

The Yersinia adhesin YadA is the prototype of a novel class of bacterial adhesins which form oligomeric lollipop-like structures and are anchored in the outer membrane by the C terminus. For YadA, six different regions (R) or domains (D) are predicted from the amino acid sequence: the N-terminal leader sequence, head-D, neck-D, stalk-D, linking-R, and a C-terminal transmembrane region consisting of four beta-strands. To identify structural and functional features of these domains, we performed in-frame deletion mutagenesis and constructed N-terminally tagged YadA variants. Diverse YadA variants were analyzed for outer membrane localization, surface exposure, oligomerization adhesion properties, and ability to protect against complement-mediated lysis. We demonstrated that (i) the C-terminal region (amino acids [aa] 353 to 422) is sufficient for outer membrane insertion and formation of trimers in the outer membrane; (ii) the head, neck, and stalk domains (aa 26 to 330) are surface exposed, forming a passenger domain; and (iii) the linking region (aa 331 to 369) is responsible for outer membrane translocation of the passenger domain. Thus, YadA meets all the criteria of an autotransporter. The same may be true for all other members of the YadA family, forming a subfamily of surface-attached oligomeric autotransporters. Moreover, in-frame truncation mutagenesis suggested that the head and neck domains together form the YadA-binding module which is located on the top of the stalk. However, the YadA-binding module did not confer serum resistance. Mutants lacking the head and neck domain were resistant to complement-mediated lysis. In-frame truncation of the stalk domain did not result in significant attenuation of the mutant in an orogastric mouse infection model.     

Structural determinants for membrane trafficking and G protein selectivity of a mouse olfactory receptor

The G protein-coupled olfactory receptor (OR) superfamily plays a critical role in recognizing a broad range of odorants. Each OR appears to recognize odorants based on similarities in molecular structures such that mOR-EG, a mouse OR, binds eugenol, vanillin, and some other structurally related odorants. Only a few ORs, however, have been characterized functionally due to the difficulties in expressing ORs in heterologous cells. In this report, we demonstrate roles of the N- and C-terminal domains as key elements in the functional expression and signal transducing activity of an OR. Disruption of the N-terminal glycosylation site of the mOR-EG completely impaired its membrane trafficking to the cell surface. Functional expression of the mOR-EG was greatly enhanced by addition of extra N-terminal glycosylation sequences. Addition of a C-terminal epitope-tag or C-terminal truncation significantly reduced the odorant-response activity, although the receptors were properly targeted to the plasma membrane. Analysis of a series of truncated ORs revealed a region in the C-terminus that was crucial for the receptor activity. Replacement of the C-terminal portion of the mOR-EG with that of rhodopsin disrupted the coupling to G(alphas) but not to G(alpha15), demonstrating that the C-terminus is involved in regulating G protein specificity. These results suggest that glycosylation of the N-terminal portion is critical for OR expression and membrane trafficking, while the C-terminal portion plays a role in defining proper conformation, which, in turn, specifies the G protein selectivity of the OR. This information helps clarify the mechanisms that regulate membrane trafficking and G protein interaction of the OR superfamily.     

Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34

Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-ΔUL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.     

Mechanism for removal of tumor necrosis factor receptor 1 from the cell surface by the adenovirus RIDalpha/beta complex

Proteins encoded in adenovirus early region 3 have important immunoregulatory properties. We have recently shown that the E3-10.4K/14.5K (RIDalpha/beta) complex downregulates tumor necrosis factor receptor 1 (TNFR1) expression at the plasma membrane. To study the role of the RIDbeta tyrosine sorting motif in the removal of surface TNFR1, tyrosine 122 on RIDbeta was mutated to alanine or phenylalanine. Both RIDbeta mutations not only abolished the downregulation of surface TNFR1 but paradoxically increased surface TNFR1 levels. RID also downregulates other death receptors, such as FAS; however, surface FAS expression was not increased by RIDbeta mutants, suggesting that regulation of TNFR1 and that of FAS by RID are mechanistically different. In the mixing experiments, the wild-type (WT) RID-mediated TNFR1 downregulation was partially inhibited in the presence of RIDbeta mutants, indicating that the mutants compete for TNFR1 access. Indeed, an association between RIDbeta and TNFR1 was shown by coimmunoprecipitation. In contrast, the mutants did not affect the WT RID-induced downregulation of FAS. These differential effects support a model in which RID associates with TNFR1 on the plasma membrane, whereas RID probably associates with FAS in a cytoplasmic compartment. By using small interfering RNA against the mu2 subunit of adaptor protein 2, dominant negative dynamin construct K44A, and the lysosomotropic agents bafilomycin A1 and ammonium chloride, we also demonstrated that surface TNFR1 was internalized by RID by a clathrin-dependent process involving mu2 and dynamin, followed by degradation of TNFR1 via an endosomal/lysosomal pathway.     

Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor

The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys¹⁷⁴, Cys²²⁶, Cys²⁹⁶ and Cys⁴⁰³ are important for the GLP-1-mediated response, whereas Cys²³⁶, Cys³²⁹, Cys³⁴¹, Cys³⁴⁷, Cys⁴³⁸, Cys⁴⁵⁸ and Cys⁴⁶² are not. Extensive deletions were made in the C-terminal tail of GLP-1R in order to determine the limit for truncation. As for other family B GPCRs, we observed a direct correlation between the length of the C-terminal tail and specific binding of ¹²⁵I-GLP-1, indicating that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function.     

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca²⁺-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca²⁺/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca²⁺/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca²⁺/NCS-1·D2R peptide complex, the C-terminal region adopts a 3₁₀ helix-turn-3₁₀ helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.     

C-terminal processing of GABARAP is not required for trafficking of the angiotensin II type 1A receptor

ObjectiveGABARAP, a small (117 aa) trafficking protein, binds to the C-terminal, cytoplasmic domain of rat angiotensin type-1A receptor (AT₁R), the predominant effector of the octapeptide angiotensin II (Ang II) (Cook et al., Circ. Res. 2008;102:1539–47). The objectives of this study were to map the interaction domains of GABARAP and AT₁R, to determine the effect of GABARAP association on AT₁R signaling activity, and to determine the importance of post-translational processing of GABARAP on accumulation of AT₁R on the plasma membrane and its signaling function.ResultsDeletion analysis identified two regions within GABARAP necessary for interaction with AT₁R in yeast two-hybrid assays: 1) a domain comprised of residues 32–51 that is nearly identical to that involved in binding and intracellular trafficking of the GABA_A receptor and 2) a domain encompassing the C-terminal 21 aa. The GABARAP interaction domain of AT₁R was delimited to the 15 aa immediately downstream of the last membrane spanning region. Overexpression of GABARAP in rat adrenal pheochromocytoma PC-12 cells increased the cell-surface expression of AT₁R and Ang II-dependent activation of the cAMP signaling pathway. Residues within AT₁R necessary for these responses were identified by mutational analysis. In PC-12 cells, GABARAP was constitutively and quantitatively cleaved at the C-terminus peptide bond and this cleavage was prevented by mutation of Gly¹¹⁶. Wild-type GABARAP and the G116A mutant were, however, equally effective in stimulating AT₁R surface expression and signaling activity.ConclusionsGABARAP and AT₁R interact through discrete domains and this association regulates the cell-surface accumulation and, consequently, ligand-induced function of the receptor. Unlike that observed with the GABA_A receptor, this regulation is not dependent on C-terminal processing and modification of GABARAP.     

Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of beta-arrestins

β-Arrestins play a role in AT₁ endocytosis by binding the cytoplasmic, C-terminus region T332–S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting β-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT₁ to investigate two possibilities: activated AT₁ induces global relocation of β-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract β-arrestins. Results obtained using high osmolarity and dominant-negative β-arrestin confirmed that internalization of AT₁ in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving β-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT₁ internalization. Confocal microscopy revealed that wild-type AT₁ induced a time-dependent translocation of GFP-tagged β-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic β-arrestin 1 at all, and only trafficked β-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both β-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and β-arrestin 1 less so than β-arrestin 2. In conclusion, this study shows that the high affinity binding of β-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced β-arrestin recruitment to the cell surface and AT₁ receptor.     

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.     

LMP1 signal transduction differs substantially from TNF receptor 1 signaling in the molecular functions of TRADD and TRAF2

The Epstein-Barr virus latent membrane protein 1 (LMP1) binds tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) and the TNFR-associated death domain protein (TRADD). Moreover, it induces NF-kappaB and the c-Jun N-terminal kinase 1 (JNK1) pathway. Thus, LMP1 appears to mimick the molecular functions of TNFR1. However, TNFR1 elicits a wide range of cellular responses including apoptosis, whereas LMP1 constitutes a transforming protein. Here we mapped the JNK1 activator region (JAR) of the LMP1 molecule. JAR overlaps with the TRADD-binding domain of LMP1. In contrast to TNFR1, LMP1 recruits TRADD via the TRADD N-terminus but not the TRADD death domain. Consequently, the molecular function of TRADD in LMP1 signaling differs from its role in TNFR1 signal transduction. Whereas NF-kappaB activation by LMP1 was blocked by a dominant-negative TRADD mutant, LMP1 induces JNK1 independently of the TRADD death domain and TRAF2, which binds to TRADD. Further downstream, JNK1 activation by TNFR1 involves Cdc42, whereas LMP1 signaling to JNK1 is independent of p21 Rho-like GTPases. Although both LMP1 and TNFR1 interact with TRADD and TRAF2, the different topologies of the signaling complexes correlate with substantial differences between LMP1 and TNFR1 signal transduction to JNK1.     

Characterization of the stomatin domain involved in homo-oligomerization and lipid raft association

The cytoplasmically oriented monotopic integral membrane protein stomatin forms high-order oligomers and associates with lipid rafts. To characterize the domains that are involved in oligomerization and detergent-resistant membrane (DRM) association, we expressed truncation and point mutants of stomatin and analyzed their size and buoyancy by ultracentrifugation methods. A small C-terminal region of stomatin that is largely hydrophobic, Ser-Thr-Ile-Val-Phe-Pro-Leu-Pro-Ile (residues 264-272), proved to be crucial for oligomerization, whereas the N-terminal domain (residues 1-20) and the last 12 C-terminal amino acids (residues 276-287) were not essential. The introduction of alanine substitutions in the region 264-272 resulted in the appearance of monomers. Remarkably, only three of these residues, Ile-Val-Phe (residues 266-268), were found to be indispensable for the DRM association. Interestingly, the exchange of Pro-269 and to some extent the residues 270-272, which are essential for oligomerization, did not affect the DRM association of stomatin. This suggests that the formation of oligomers is not necessary for the association of stomatin with DRMs. Internal deletions near the membrane anchoring domain resulted in the formation of intermediate size oligomers suggesting a conformational interdependence of large parts of the C-terminal region. Fluorescence recovery after photobleaching analysis of the tagged, monomeric, non-DRM mutant ST-(1-262)-green fluorescent protein and wild type stomatin StomGFP showed a significantly higher lateral mobility of the truncation mutant in the plasma membrane suggesting a membrane interaction of the respective C-terminal region also in vivo.     

Expression of HBcAg mutant with long internal deletion in Saccharomyces cerevisiae and observation of its self-assembly particles by atomic force microscopy (AFM)

An internally truncated C gene of adr hepatitis B virus core antigen with long internal deletion (aa81–aa116) (ΔHBcAg with 36aa truncation) was expressed in Saccharomyces cerevisiae and the products (ΔrHBcAg) were purified from a crude lysate of the yeast by three steps: Sephrose CL-4B chromatography, sucrose step-gradient ultracentrifugation and CsCl-isopycnic ultracentrifugation. Results of ELISA test and density analysis of CsCl-isopycnic ultracentrifugation indicated that the purified products (ΔrHBcAg protein) with HBeAg antigenicity mainly located at the densities of 1.23 g ml⁻¹. Observation and analysis of the purified ΔrHBcAg products by AFM indicated that the ΔrHBcAg (core) protein produced in S. cerevisiae could self-assemble into three or more size classes of core particles which exhibited a polymorphous distribution of ΔrHBcAg (core) particles. These different size classes of core particles mainly centred on the range whose mean diameter was from 10 nm to 48 nm, especially on the position of 11 nm, 15.6 nm and the range from 27 nm to 41 nm, respectively. Furthermore, the most number of core particles mainly centred on the range whose mean diameter was from 27 nm to 41 nm. These results above indicated that the truncated internal long fragment (aa81–aa116) probably had no effect on self-assembly of the HBcAg core particles which implied the internal length fragment (aa81–aa116) was not the sole domain for self-assembly of HBcAg dimer or the truncated HBcAg protein subunit formed the fresh interactive domain with each other. These initial results above by AFM analysis were very important for further research on the self-assembly, ultrastructure, subunit interaction and core internal deletion mutant (CIDM) function of HBcAg core particles.     

A membrane-proximal basic domain and cysteine cluster in the C-terminal tail of CCR5 constitute a bipartite motif critical for cell surface expression

We examined the structural requirements for cell surface expression, signaling, and human immunodeficiency virus co-receptor activity for the chemokine receptor, CCR5. Serial C-terminal truncation of CCR5 resulted in progressive loss of cell surface expression; mutants truncated at the 317th position and shorter were not detected at the cell surface. Alanine substitution of basic residues in the membrane-proximal domain (residues 314-322) in the context of a full-length C-tail resulted in severe reduction in surface expression. C-terminal truncation that excised the three cysteines in this domain reduced surface expression, but further truncation of upstream basic residue(s) abolished surface expression. Substituting the carboxyl-terminal domain of CXCR4 for that of CCR5 failed to rectify the trafficking defect of the tailless CCR5. In contrast, tailless CXCR4 or a CXCR4 chimera that exchanged the native cytoplasmic domain for that of wild type CCR5 was expressed at the cell surface. Deletion mutants that expressed at the cell surface responded to chemokine stimulation and mediated human immunodeficiency virus entry. Substitution of all serine and threonine residues in the C-terminal tail of CCR5 abolished chemokine-mediated receptor phosphorylation but preserved downstream signaling (Ca(2+) flux), while substitutions of tyrosine residues in the C-tail affected neither phenotype. CCR5 mutants that failed to traffic to the plasma membrane did not exhibit obvious changes in metabolic turnover and were retained in the Golgi or pre-Golgi compartments(s). Thus, the basic domain (-KHIAKRF-) and the cysteine cluster (-CKCC-) in the C-terminal tail of CCR5 function cooperatively for optimal surface expression.     

Functional characterization of mutations in melanocortin-4 receptor associated with human obesity

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.     

Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.     

C-terminal deletion analogs of a crustacean pigment-dispersing hormone

This study deals with the effect of deamidation and C-terminal truncation on the potency of an octadecapeptide pigment-dispersing hormone (PDH: Asn- Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala-NH₂), first described as light-adapting distal retinal pigment hormone (DRPH) from Pandalus borealis. Bioassay of synthetic analogs for melanophore pigment dispersion in destalked fiddler crabs (Uca pugilator) showed that deamidation causes a 300-fold decrease in potency. The analogs 1–17-NH₂ and 1–16-NH₂ were about 3 times more potent than 1–18-OH. Further truncation led to decreases in potency, with the peptide 1–9-NH₂ being the smallest C-terminal deletion analog to display activity (0.001% potency). Smaller analogs (1–8-NH₂, 1–6-NH₂ and 1–4-NH₂) were inactive when tested in doses as high as 500 nmoles/crab. On the basis of our earlier work on N-terminal deletion analogs and the present findings the residues 6 to 9 seem to be important for PDH action.     

Inhibition of tumor necrosis factor-induced phenotypes by short intracellular versions of latent membrane protein-1

Tumor necrosis factor (TNF) is a potent multi-functional cytokine with a homeostatic role in host defence. In case of deregulation, TNF is implicated in numerous pathologies. The latent membrane protein-1 (LMP1) is expressed by Epstein–Barr virus during viral latency and displaying properties of a constitutively activated member of the TNF receptor family. Both TNFR1 and LMP1 share a similar set of proximal adapters and signalling pathways although they display different biological responses. We previously demonstrated that the intracellular part of LMP1, LMP1-CT, a dominant-negative form of LMP1, inhibits LMP1 signalling.Here, we developed shorter versions derived from C-terminal part of LMP1 to investigate their roles on LMP1 and TNF signalling. We constructed several mutants of LMP1 containing a part of cytoplasmic signalling region fused to the green fluorescent protein. These mutants selectively impair signalling by LMP1 and TNF but not by IL-1β which uses other adapters. Dominant-negative effect was due to binding and sequestration of LMP1 adapters RIP, TRAF2 and TRADD as assessed by coimmunoprecipitation experiments and confocal analysis. Expression of these mutants impairs the recruitment of these adapters by TNFR1 and TNF-associated phenotypes. These mutants did not display cytostatic properties but were able to modulate TNF-induced phenotypes, apoptosis or cell survival, depending on the cell context. Interestingly, these mutants are able to inhibit a pro-inflammatory response in endothelial cells. These data demonstrate that LMP1 derived molecules can be used to design compounds with potential therapeutic roles in diseases due to TNF overactivation.