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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.

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Receptor chimeras with identical ligand binding sites and intracellular signaling domains.A. Schematic representation of TNFR1-TRAILR1-Fas (TM1), TNFR1-TRAILR2-Fas (TM2) and TNFR1-Fas chimeric proteins. TNFR1-Fas receptor was established through the fusion of the cytoplasmic domain of Fas, amino acids 191–335, to the C-terminus of the potential transmembrane region of TNFR1 (amino acid 236). In TM1 and TM2 receptor chimeras the stalk (S) and transmembrane (TM) regions of TNFR1 (aa 197–234) were then substituted with the corresponding regions of TRAILR1 (amino acids 230–262) or TRAILR2 (amino acids 179–231). B. Immortalized mouse fibroblasts were stably transfected with TM1, TM2 and TNFR1-Fas expression plasmids. Expression of the chimeras was analyzed by flow cytometry using TNFR1-specific antibodies. Percentage of positive cells is indicated.
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pone-0042526-g004: Receptor chimeras with identical ligand binding sites and intracellular signaling domains.A. Schematic representation of TNFR1-TRAILR1-Fas (TM1), TNFR1-TRAILR2-Fas (TM2) and TNFR1-Fas chimeric proteins. TNFR1-Fas receptor was established through the fusion of the cytoplasmic domain of Fas, amino acids 191–335, to the C-terminus of the potential transmembrane region of TNFR1 (amino acid 236). In TM1 and TM2 receptor chimeras the stalk (S) and transmembrane (TM) regions of TNFR1 (aa 197–234) were then substituted with the corresponding regions of TRAILR1 (amino acids 230–262) or TRAILR2 (amino acids 179–231). B. Immortalized mouse fibroblasts were stably transfected with TM1, TM2 and TNFR1-Fas expression plasmids. Expression of the chimeras was analyzed by flow cytometry using TNFR1-specific antibodies. Percentage of positive cells is indicated.

Mentions: The apoptotic activity of TRAIL receptors has been associated with their localization in cholesterol-rich microdomains, formerly often called lipid rafts [22], [23]. Targeting of signaling molecules into microdomains is likely to be regulated, among others, by the transmembrane part (TM) of the receptor itself and adjacent sequences. For TRAILR1 (cysteine residue(s) 161–163; [22]) and the Fas molecule [24], but not TRAILR2, palmitoylation has been demonstrated as a driving factor for partitioning within cellular membranes. To investigate if the TMs of TRAIL receptors influence their differential strengths in apoptosis signaling, new chimeric molecules were generated: The extracellular ligand binding domains were derived from TNFR1 and their cytoplasmic signaling domains were Fas-derived. However, transmembrane domains and the extracellular stalk regions were derived from TRAIL receptors 1 or 2 (see schematic drawing Fig. 4A). The stalk regions are different in length comprising about 10 amino acids (aa) for TRAILR1 and about 34 aa for TRAILR2.


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 identical ligand binding sites and intracellular signaling domains.A. Schematic representation of TNFR1-TRAILR1-Fas (TM1), TNFR1-TRAILR2-Fas (TM2) and TNFR1-Fas chimeric proteins. TNFR1-Fas receptor was established through the fusion of the cytoplasmic domain of Fas, amino acids 191–335, to the C-terminus of the potential transmembrane region of TNFR1 (amino acid 236). In TM1 and TM2 receptor chimeras the stalk (S) and transmembrane (TM) regions of TNFR1 (aa 197–234) were then substituted with the corresponding regions of TRAILR1 (amino acids 230–262) or TRAILR2 (amino acids 179–231). B. Immortalized mouse fibroblasts were stably transfected with TM1, TM2 and TNFR1-Fas expression plasmids. Expression of the chimeras was analyzed by flow cytometry using TNFR1-specific antibodies. Percentage of positive cells is indicated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3420232&req=5

pone-0042526-g004: Receptor chimeras with identical ligand binding sites and intracellular signaling domains.A. Schematic representation of TNFR1-TRAILR1-Fas (TM1), TNFR1-TRAILR2-Fas (TM2) and TNFR1-Fas chimeric proteins. TNFR1-Fas receptor was established through the fusion of the cytoplasmic domain of Fas, amino acids 191–335, to the C-terminus of the potential transmembrane region of TNFR1 (amino acid 236). In TM1 and TM2 receptor chimeras the stalk (S) and transmembrane (TM) regions of TNFR1 (aa 197–234) were then substituted with the corresponding regions of TRAILR1 (amino acids 230–262) or TRAILR2 (amino acids 179–231). B. Immortalized mouse fibroblasts were stably transfected with TM1, TM2 and TNFR1-Fas expression plasmids. Expression of the chimeras was analyzed by flow cytometry using TNFR1-specific antibodies. Percentage of positive cells is indicated.
Mentions: The apoptotic activity of TRAIL receptors has been associated with their localization in cholesterol-rich microdomains, formerly often called lipid rafts [22], [23]. Targeting of signaling molecules into microdomains is likely to be regulated, among others, by the transmembrane part (TM) of the receptor itself and adjacent sequences. For TRAILR1 (cysteine residue(s) 161–163; [22]) and the Fas molecule [24], but not TRAILR2, palmitoylation has been demonstrated as a driving factor for partitioning within cellular membranes. To investigate if the TMs of TRAIL receptors influence their differential strengths in apoptosis signaling, new chimeric molecules were generated: The extracellular ligand binding domains were derived from TNFR1 and their cytoplasmic signaling domains were Fas-derived. However, transmembrane domains and the extracellular stalk regions were derived from TRAIL receptors 1 or 2 (see schematic drawing Fig. 4A). The stalk regions are different in length comprising about 10 amino acids (aa) for TRAILR1 and about 34 aa for TRAILR2.

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