Signaling Active CD95 Receptor Molecules Trigger Co-translocation of Inactive CD95 Molecules into Lipid Rafts

Lang, Isabell; Fick, Andrea; Schäfer, Viktoria; Giner, Tina; Siegmund, Daniela; Wajant, Harald

doi:10.1074/jbc.m111.328211

Cited by 25 publications

(31 citation statements)

References 61 publications

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“…To confirm this by an independent method and also to gain insight into the stability of the CD40L-CD40 interaction, we also performed ligand binding studies. We recently demonstrated for TWEAK and the related ligands TNF and CD95L that these molecules can be labeled by genetic engineering using GpL as a reporter domain (36,37). We therefore fused in a similar approach the GpL domain to CD40L and used the resulting GpL-Flag-CD40L fusion protein for cellular binding studies.…”

Section: Cd40 Cell Surface Expression and Cd40l-cd40 Interaction Occumentioning

confidence: 99%

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

Salzmann

Lang

Rosenthal

et al. 2013

The Journal of Immunology

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We found recently that TNF-like weak inducer of apoptosis (TWEAK) and fibroblast growth factor–inducible-14 (Fn14) by virtue of their strong capability to reduce the freely available cytoplasmic pool of TNFR-associated factor (TRAF)2 and cellular inhibitors of apoptosis (cIAPs) antagonize the functions of these molecules in TNFR1 signaling, resulting in sensitization for apoptosis and inhibition of classical NF-κB signaling. In this study, we demonstrate that priming of cells with TWEAK also interferes with activation of the classical NF-κB pathway by CD40. Likewise, there was strong inhibition of CD40 ligand (CD40L)–induced activation of MAPKs in TWEAK-primed cells. FACS analysis and CD40L binding studies revealed unchanged CD40 expression and normal CD40L–CD40 interaction in TWEAK-primed cells. CD40L immunoprecipitates, however, showed severely reduced amounts of CD40 and CD40-associated proteins, indicating impaired formation or reduced stability of CD40L–CD40 signaling complexes. The previously described inhibitory effect of TWEAK on TNFR1 signaling has been traced back to reduced activity of the TNFR1-associated TRAF2–cIAP1/2 ubiquitinase complex and did not affect the stability of the immunoprecipitable TNFR1 receptor complex. Thus, the inhibitory effect of TWEAK on CD40 signaling must be based at least partly on other mechanisms. In line with this, signaling by the CD40-related TRAF2-interacting receptor TNFR2 was also attenuated but still immunoprecipitable in TWEAK-primed cells. Collectively, we show that Fn14 activation by soluble TWEAK impairs CD40L–CD40 signaling complex formation and inhibits CD40 signaling and thus identify the Fn14-TWEAK system as a potential novel regulator of CD40-related cellular functions.

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Section: Cd40 Cell Surface Expression and Cd40l-cd40 Interaction Occumentioning

confidence: 99%

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

Salzmann

Lang

Rosenthal

et al. 2013

The Journal of Immunology

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“…Furthermore, soluble CD40L, which benefits only moderately from ligand oligomerization, has a relatively poor affinity of 7.1 nM, whereas EDA-A1, which interacts with EDAR with an affinity of 0.05 nM, still gains activity upon oligomerization (33). Indeed, we recently addressed the relevance of ligand oligomerization for affinity by help of GpL fusion proteins for soluble TWEAK, which poorly stimulate Fn14-mediated induction of the classical NFB target IL8, and for soluble CD95L, which fails to trigger robust apoptosis induction (9,10). In these two cases, we noticed no major effect of ligand oligomerization on receptor occupancy and apparent affinity.…”

Section: Discussionmentioning

confidence: 99%

“…In these cases the affinity of the soluble molecule would again be a functional relevant parameter. It is worth mentioning that binding studies with GpL-TNFSF ligand fusion proteins also easily allow the determination of association and dissociation rate constants and the mean lifetime of ligand-receptor complexes (9,10). The systematic evaluation of these parameters may give new insights into the question of how the dynamics/stability of the ligand-receptor complex contributes to the quality and quantity by which a certain TNFRSF receptor type activates intracellular signaling pathways.…”

Section: Discussionmentioning

confidence: 99%

“…Cloning, Production, and Purification of Recombinant Proteins-Cloning of GpL-FLAG-TNC-TNF, GpL-FLAG-TNC-TWEAK, and GpL-fLAG-TNC-CD95L has been described elsewhere (9,10). The expression plasmids encoding the GpL-FLAG-TNC variants of the other TNF ligands used in this study have been obtained by exchange of the TNF-encoding fragment of the pCR3-based GpL-FLAG-TNC-TNF expression plasmid with PCR amplicons encoding THD-encompassing soluble versions of the other TNFSF ligand types ( Table 2) by help of flanking EcoRI (5Ј end) and XbaI (3Ј end) restriction sites.…”

Section: Methodsmentioning

confidence: 99%

“…We recently took advantage of this fact and generated GpL fusion proteins of the TNFSF ligands CD95L, TNF, and TWEAK for cellular binding studies (9,10). These GpL-TNFSF ligand fusion proteins were proved to be highly traceable and functionally not distinguishable from their conventional counterparts.…”

Section: Generation Production and Purification Of A Panel Of Solubmentioning

confidence: 99%

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Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

Lang

Füllsack

Wyzgol

et al. 2016

Journal of Biological Chemistry

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Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle‐Based Protein Signaling

et al. 2022

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Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes, especially those involved in immune signalling. However, the mechanistic understanding concerning the biophysical principles underlying EV-based communication is still incomplete. Towards holistic understanding, particular mechanisms explaining why and when cells apply EV-based communication and how protein-based signalling is promoted by EV surfaces are sought. Here, the authors study vesicle-induced receptor sequestration (VIRS) as a universal mechanism augmenting the signalling potency of proteins presented on EV-membranes. By bottom-up reconstitution of synthetic EVs, the authors show that immobilization of the receptor ligands FasL and RANK on EV-like vesicles, increases their signalling potential by more than 100-fold compared to their soluble forms. Moreover, the authors perform diffusion simulations within immunological synapses to compare receptor activation between soluble and EV-presented proteins. By this the authors propose vesicle-triggered local clustering of membrane receptors as the principle structural mechanism underlying EV-based protein presentation. The authors conclude that EVs act as extracellular templates promoting the local aggregation of membrane receptors at the EV contact site, thereby fostering inter-protein interactions. The results uncover a potentially universal mechanism explaining the unique structural profit of EV-based intercellular signalling.

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Signaling Active CD95 Receptor Molecules Trigger Co-translocation of Inactive CD95 Molecules into Lipid Rafts

Cited by 25 publications

References 61 publications

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

TWEAK Inhibits TRAF2-Mediated CD40 Signaling by Destabilization of CD40 Signaling Complexes

Binding Studies of TNF Receptor Superfamily (TNFRSF) Receptors on Intact Cells

Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle‐Based Protein Signaling

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