Limits...
The transmembrane domains of TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 co-regulate apoptotic signaling capacity.

Neumann S, Bidon T, Branschädel M, Krippner-Heidenreich A, Scheurich P, Doszczak M - PLoS ONE (2012)

Bottom Line: Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics.However, the respective TRAILR2-derived molecule more efficiently induced apoptosis.It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.

ABSTRACT
TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) ligand family that exerts its apoptotic activity in human cells by binding to two transmembrane receptors, TRAILR1 and TRAILR2. In cells co-expressing both receptors the particular contribution of either protein to the overall cellular response is not well defined. Here we have investigated whether differences in the signaling capacities of TRAILR1 and TRAILR2 can be attributed to certain functional molecular subdomains. We generated and characterized various chimeric receptors comprising TRAIL receptor domains fused with parts from other members of the TNF death receptor family. This allowed us to compare the contribution of particular domains of the two TRAIL receptors to the overall apoptotic response and to identify elements that regulate apoptotic signaling. Our results show that the TRAIL receptor death domains are weak apoptosis inducers compared to those of CD95/Fas, because TRAILR-derived constructs containing the CD95/Fas death domain possessed strongly enhanced apoptotic capabilities. Importantly, major differences in the signaling strengths of the two TRAIL receptors were linked to their transmembrane domains in combination with the adjacent extracellular stalk regions. This was evident from receptor chimeras comprising the extracellular part of TNFR1 and the intracellular signaling part of CD95/Fas. Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics. However, the respective TRAILR2-derived molecule more efficiently induced apoptosis. It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels. These results suggest that the transmembrane domains together with their adjacent stalk regions can play a major role in control of death receptor activation thereby contributing to cell type specific differences in TRAILR1 and TRAILR2 signaling.

Show MeSH

Related in: MedlinePlus

Receptor chimeras with the transmembrane and stalk regions from TRAILR1 and TRAILR2 show comparable ligand-induced receptor aggregation and internalization.A. MF-TM1, MF-TM2 and MF-TNFR1-Fas cells were transiently transfected with a construct expressing human FADD-eGFP in the presence of 20 µM z-VAD-fmk. The following day cells were treated with Alexa Fluor 546-labeled TNF (100 ng/ml) for the indicated time periods, fixed and examined by confocal laser-scanning microscopy. Shown are optical sections through the center of representative cells. B. Adherent cells were incubated with 125I-TNF (30 ng/ml) for 1 h on ice, followed by incubation at 37°C and 5% CO2 for the indicated times. Subsequently cells were washed with PBS, followed by washing with acidic buffer (pH = 3.0) to disrupt ligand/receptor interactions on the cell surface, or again with PBS (pH = 7.0). For non-specific binding (NSB) a 200-fold excess of unlabeled TNF was added during the first incubation step. Radioactivity of the cell lysates was then quantified in a γ-counter. Data points represent mean values ± SD from three independent experiments each performed in duplicates.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3420232&req=5

pone-0042526-g007: Receptor chimeras with the transmembrane and stalk regions from TRAILR1 and TRAILR2 show comparable ligand-induced receptor aggregation and internalization.A. MF-TM1, MF-TM2 and MF-TNFR1-Fas cells were transiently transfected with a construct expressing human FADD-eGFP in the presence of 20 µM z-VAD-fmk. The following day cells were treated with Alexa Fluor 546-labeled TNF (100 ng/ml) for the indicated time periods, fixed and examined by confocal laser-scanning microscopy. Shown are optical sections through the center of representative cells. B. Adherent cells were incubated with 125I-TNF (30 ng/ml) for 1 h on ice, followed by incubation at 37°C and 5% CO2 for the indicated times. Subsequently cells were washed with PBS, followed by washing with acidic buffer (pH = 3.0) to disrupt ligand/receptor interactions on the cell surface, or again with PBS (pH = 7.0). For non-specific binding (NSB) a 200-fold excess of unlabeled TNF was added during the first incubation step. Radioactivity of the cell lysates was then quantified in a γ-counter. Data points represent mean values ± SD from three independent experiments each performed in duplicates.

Mentions: It is currently accepted that TNFR1 forms two distinct, subsequently assembled signaling complexes of which the secondary, internalized complex is capable to activate caspase-8 [26], [27]. In contrast, ligand activated TRAIL death receptors are believed to signal apoptosis induction both via a membrane-associated primarily formed DISC, and from intracellular ubiquitin-rich foci formed after polyubiquitylation of caspase-8 [9]. We therefore compared receptor cluster formation and internalization of the receptor chimeras after TNF stimulation. Mouse fibroblasts positive for TM1, TM2 or TNFR1-Fas were transiently transfected with a construct expressing human FADD-eGFP. The next day cells were treated with Alexa Fluor 546-labeled TNF (100 ng/ml) for the indicated time periods at 37°C, fixed and examined by confocal laser-scanning microscopy. The examples shown in Fig. 7A are optical sections through the center of the cells and demonstrate comparable cluster formation of the two chimeras comprising the TRAILR1- and TRAILR2-derived transmembrane parts. Additionally, they indicate significant internalization of the chimeras in contrast to TNFR1-Fas chimeras, where internalized clusters were hardly visible.


The transmembrane domains of TNF-related apoptosis-inducing ligand (TRAIL) receptors 1 and 2 co-regulate apoptotic signaling capacity.

Neumann S, Bidon T, Branschädel M, Krippner-Heidenreich A, Scheurich P, Doszczak M - PLoS ONE (2012)

Receptor chimeras with the transmembrane and stalk regions from TRAILR1 and TRAILR2 show comparable ligand-induced receptor aggregation and internalization.A. MF-TM1, MF-TM2 and MF-TNFR1-Fas cells were transiently transfected with a construct expressing human FADD-eGFP in the presence of 20 µM z-VAD-fmk. The following day cells were treated with Alexa Fluor 546-labeled TNF (100 ng/ml) for the indicated time periods, fixed and examined by confocal laser-scanning microscopy. Shown are optical sections through the center of representative cells. B. Adherent cells were incubated with 125I-TNF (30 ng/ml) for 1 h on ice, followed by incubation at 37°C and 5% CO2 for the indicated times. Subsequently cells were washed with PBS, followed by washing with acidic buffer (pH = 3.0) to disrupt ligand/receptor interactions on the cell surface, or again with PBS (pH = 7.0). For non-specific binding (NSB) a 200-fold excess of unlabeled TNF was added during the first incubation step. Radioactivity of the cell lysates was then quantified in a γ-counter. Data points represent mean values ± SD from three independent experiments each performed in duplicates.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3420232&req=5

pone-0042526-g007: Receptor chimeras with the transmembrane and stalk regions from TRAILR1 and TRAILR2 show comparable ligand-induced receptor aggregation and internalization.A. MF-TM1, MF-TM2 and MF-TNFR1-Fas cells were transiently transfected with a construct expressing human FADD-eGFP in the presence of 20 µM z-VAD-fmk. The following day cells were treated with Alexa Fluor 546-labeled TNF (100 ng/ml) for the indicated time periods, fixed and examined by confocal laser-scanning microscopy. Shown are optical sections through the center of representative cells. B. Adherent cells were incubated with 125I-TNF (30 ng/ml) for 1 h on ice, followed by incubation at 37°C and 5% CO2 for the indicated times. Subsequently cells were washed with PBS, followed by washing with acidic buffer (pH = 3.0) to disrupt ligand/receptor interactions on the cell surface, or again with PBS (pH = 7.0). For non-specific binding (NSB) a 200-fold excess of unlabeled TNF was added during the first incubation step. Radioactivity of the cell lysates was then quantified in a γ-counter. Data points represent mean values ± SD from three independent experiments each performed in duplicates.
Mentions: It is currently accepted that TNFR1 forms two distinct, subsequently assembled signaling complexes of which the secondary, internalized complex is capable to activate caspase-8 [26], [27]. In contrast, ligand activated TRAIL death receptors are believed to signal apoptosis induction both via a membrane-associated primarily formed DISC, and from intracellular ubiquitin-rich foci formed after polyubiquitylation of caspase-8 [9]. We therefore compared receptor cluster formation and internalization of the receptor chimeras after TNF stimulation. Mouse fibroblasts positive for TM1, TM2 or TNFR1-Fas were transiently transfected with a construct expressing human FADD-eGFP. The next day cells were treated with Alexa Fluor 546-labeled TNF (100 ng/ml) for the indicated time periods at 37°C, fixed and examined by confocal laser-scanning microscopy. The examples shown in Fig. 7A are optical sections through the center of the cells and demonstrate comparable cluster formation of the two chimeras comprising the TRAILR1- and TRAILR2-derived transmembrane parts. Additionally, they indicate significant internalization of the chimeras in contrast to TNFR1-Fas chimeras, where internalized clusters were hardly visible.

Bottom Line: Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics.However, the respective TRAILR2-derived molecule more efficiently induced apoptosis.It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.

ABSTRACT
TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) ligand family that exerts its apoptotic activity in human cells by binding to two transmembrane receptors, TRAILR1 and TRAILR2. In cells co-expressing both receptors the particular contribution of either protein to the overall cellular response is not well defined. Here we have investigated whether differences in the signaling capacities of TRAILR1 and TRAILR2 can be attributed to certain functional molecular subdomains. We generated and characterized various chimeric receptors comprising TRAIL receptor domains fused with parts from other members of the TNF death receptor family. This allowed us to compare the contribution of particular domains of the two TRAIL receptors to the overall apoptotic response and to identify elements that regulate apoptotic signaling. Our results show that the TRAIL receptor death domains are weak apoptosis inducers compared to those of CD95/Fas, because TRAILR-derived constructs containing the CD95/Fas death domain possessed strongly enhanced apoptotic capabilities. Importantly, major differences in the signaling strengths of the two TRAIL receptors were linked to their transmembrane domains in combination with the adjacent extracellular stalk regions. This was evident from receptor chimeras comprising the extracellular part of TNFR1 and the intracellular signaling part of CD95/Fas. Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics. However, the respective TRAILR2-derived molecule more efficiently induced apoptosis. It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels. These results suggest that the transmembrane domains together with their adjacent stalk regions can play a major role in control of death receptor activation thereby contributing to cell type specific differences in TRAILR1 and TRAILR2 signaling.

Show MeSH
Related in: MedlinePlus