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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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Ligand binding affinities of receptor chimeras differing only in their transmembrane and stalk regions.A-C. Mouse fibroblasts expressing the indicated chimeric receptors (2×105 cells per sample) were incubated with radioiodinated TNF at different concentrations for 2 hours on ice. Cells were separated from unbound label by centrifugation through phthalate oil and cell bound protein was quantified. Free radioactive TNF was plotted against bound label, a one site binding hyperbola was fitted through the data points and KD-values were determined by using the Graphpad Prism software. Data points represent mean values out of duplicates. The insets show Scatchard plots of the corresponding data.
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pone-0042526-g006: Ligand binding affinities of receptor chimeras differing only in their transmembrane and stalk regions.A-C. Mouse fibroblasts expressing the indicated chimeric receptors (2×105 cells per sample) were incubated with radioiodinated TNF at different concentrations for 2 hours on ice. Cells were separated from unbound label by centrifugation through phthalate oil and cell bound protein was quantified. Free radioactive TNF was plotted against bound label, a one site binding hyperbola was fitted through the data points and KD-values were determined by using the Graphpad Prism software. Data points represent mean values out of duplicates. The insets show Scatchard plots of the corresponding data.

Mentions: TNF and TRAIL both form homotrimeric molecules capable of binding up to three receptors [11], [13]. In addition all four TRAIL membrane receptors are able to homomultimerize in the absence of ligand via the aforementioned PLAD [7], [25]. It is therefore feasible that in binding studies, performed under conditions of reduced membrane fluidity, ligand/receptor interactions occur with different stoichiometries resulting in distinct effective affinities caused by avidity effects. We therefore performed equilibrium binding studies with radioiodinated TNF revealing comparable ligand binding affinities for the two receptor chimeras (225±17.2 nM for TM1 and 71±4.3 nM for TM2; mean KD-values ± SD of 3 experiments each) as well as for TNFR1-Fas (112±10.8 nM; n = 3). This finding argues against major differential avidity effects. Consistently, however, MF-TM2 cells showed an about threefold lower value for the apparent dissociation constant compared to MF-TM1 cells (see also the example shown in Fig. 6). Binding data had been fitted to a one site binding hyperbola and the linearity of the respective Scatchard analyses (Fig. 6A–C, insets) indicates good agreement with this assumption. No indications are visible in these diagrams for the existence of e.g. two distinct affinities. It is therefore likely that minor conformational changes within the chimeric molecules cause the slight affinity differences observed. In any case the observed differences in ligand binding affinities appear too small to explain the differential apoptotic capabilities observed. Additional constraint(s) in TM1 and TM2 molecules must exist, most likely located within the TM regions and/or the adjacent stalk regions, which efficiently regulate ligand sensitivity.


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)

Ligand binding affinities of receptor chimeras differing only in their transmembrane and stalk regions.A-C. Mouse fibroblasts expressing the indicated chimeric receptors (2×105 cells per sample) were incubated with radioiodinated TNF at different concentrations for 2 hours on ice. Cells were separated from unbound label by centrifugation through phthalate oil and cell bound protein was quantified. Free radioactive TNF was plotted against bound label, a one site binding hyperbola was fitted through the data points and KD-values were determined by using the Graphpad Prism software. Data points represent mean values out of duplicates. The insets show Scatchard plots of the corresponding data.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3420232&req=5

pone-0042526-g006: Ligand binding affinities of receptor chimeras differing only in their transmembrane and stalk regions.A-C. Mouse fibroblasts expressing the indicated chimeric receptors (2×105 cells per sample) were incubated with radioiodinated TNF at different concentrations for 2 hours on ice. Cells were separated from unbound label by centrifugation through phthalate oil and cell bound protein was quantified. Free radioactive TNF was plotted against bound label, a one site binding hyperbola was fitted through the data points and KD-values were determined by using the Graphpad Prism software. Data points represent mean values out of duplicates. The insets show Scatchard plots of the corresponding data.
Mentions: TNF and TRAIL both form homotrimeric molecules capable of binding up to three receptors [11], [13]. In addition all four TRAIL membrane receptors are able to homomultimerize in the absence of ligand via the aforementioned PLAD [7], [25]. It is therefore feasible that in binding studies, performed under conditions of reduced membrane fluidity, ligand/receptor interactions occur with different stoichiometries resulting in distinct effective affinities caused by avidity effects. We therefore performed equilibrium binding studies with radioiodinated TNF revealing comparable ligand binding affinities for the two receptor chimeras (225±17.2 nM for TM1 and 71±4.3 nM for TM2; mean KD-values ± SD of 3 experiments each) as well as for TNFR1-Fas (112±10.8 nM; n = 3). This finding argues against major differential avidity effects. Consistently, however, MF-TM2 cells showed an about threefold lower value for the apparent dissociation constant compared to MF-TM1 cells (see also the example shown in Fig. 6). Binding data had been fitted to a one site binding hyperbola and the linearity of the respective Scatchard analyses (Fig. 6A–C, insets) indicates good agreement with this assumption. No indications are visible in these diagrams for the existence of e.g. two distinct affinities. It is therefore likely that minor conformational changes within the chimeric molecules cause the slight affinity differences observed. In any case the observed differences in ligand binding affinities appear too small to explain the differential apoptotic capabilities observed. Additional constraint(s) in TM1 and TM2 molecules must exist, most likely located within the TM regions and/or the adjacent stalk regions, which efficiently regulate ligand sensitivity.

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