The Transmembrane Domains of TNF-Related Apoptosis-Inducing Ligand (TRAIL) Receptors 1 and 2 Co-Regulate Apoptotic Signaling Capacity

<div><p>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.</p> </div

Wild type mouse-derived immortalized fibroblasts, as well as TRAILR1- and TRAILR2-positive transfectants lack sensitivity to TRAIL-induced cell death.

<p><b>A.</b> Mouse fibroblasts are nonresponsive to the cytotoxic effects of TRAIL. Cells were treated with FLAG-TRAIL (400 ng/ml) in the absence or presence of the crosslinking anti-FLAG antibody M2 (2 µg/ml; 1 h 37°C) and/or 0.5 µg/ml cycloheximide, as indicated. Cell viability was determined by crystal violet staining the next day. One representative experiment (n = 3, shown are the mean values ± standard deviation (SD)) out of three is shown. <b>B and C.</b> Immortalized mouse fibroblasts were stably transfected with wild-type human TRAILR1 (B) or TRAILR2 (C) expression plasmids. Cell viability was determined by crystal violet staining following 16 hour incubation with cross-linked FLAG-TRAIL in the absence (open symbols) or presence (closed symbols) of cycloheximide (0.5 µg/ml). One representative experiment (n = 3, shown are the mean values ± SD) out of three is shown. Caspase-8 (dashed bars) and caspase-3 (black bars) enzymatic activity following triggering of indicated receptors with FLAG-TRAIL (previously cross-linked with anti-FLAG M2 antibody, 1 h 37°C) was quantified using specific fluorogenic substrates (Ac-IEPD-AMC and Ac-DMQD-AMC, respectively). The insets show the expression pattern of the human TRAIL receptors as analyzed by flow cytometry. One representative experiment (out of three) is shown.</p

The transmembrane and stalk regions of TRAILR1 and TRAILR2 regulate apoptotic signaling.

<p><b>A.</b> Cytotoxic effects of TNF in TM1, TM2 and TNFR1-Fas expressing mouse fibroblasts. MF-TM1 (closed squares), MF-TM2 (closed triangles) or MF-TNFR1-Fas (open circles) cells were treated with serial dilutions of TNF and cell viability was determined by crystal violet staining after 6 hours of stimulation. All experimental groups shown were performed in parallel: one representative experiment out of three is shown. <b>B.</b> Caspase-8 (left panel) and caspase-3 (right panel) enzymatic activities following triggering of TM1, TM2 and TNFR1-Fas (closed squares, closed triangles and open circles, respectively) positive cells with TNF were determined using specific fluorogenic substrates (Ac-IEPD-AMC and Ac-DMQD-AMC, respectively). Data from a representative experiment out of three are shown. The graphs depict normalized caspase activities (maximum relative activity = 1), as absolute activity values are likely to be strongly influenced by the differential expression levels of the chimeric receptors. <b>C.</b> Western blot analyses performed using procaspase-8- and cleaved caspase-3-specific antibodies. Cells stably expressing chimeric receptors were treated with TNF (100 ng/ml) for the indicated times, followed by cell lysis.</p

Receptor chimeras with the transmembrane and stalk regions from TRAILR1 and TRAILR2 show comparable ligand-induced receptor aggregation and internalization.

<p><b>A.</b> 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>B.</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.</p

TRAILR1-Fas and TRAILR2-Fas chimeric receptors show ligand binding affinities comparable to the respective wild-type TRAIL receptors.

<p><b>A.</b> Schematic representation of TRAILR1-Fas and TRAILR2-Fas chimeric proteins. The cytoplasmic domain of Fas (Fas cyt), amino acids 191–335, was fused to the C-terminus of the potential transmembrane region of TRAILR1 (amino acid 262) or TRAILR2 (amino acid 231). S = stalk region, TM = transmembrane domain. <b>B.</b> Immortalized mouse fibroblasts were stably transfected with TRAILR1-Fas and TRAILR2-Fas expression plasmids. Cell surface expression was analyzed by flow cytometry using TRAILR1- and TRAILR2-specific antibodies. Isotype controls are shown in grey. <b>C.</b> Representative curves from ligand binding competition experiments on wild-type (wt) TRAILR positive cells and TRAILR-Fas chimera expressing cells using ¹²⁵I-labeled sTRAIL. IC₅₀-values determined from ligand binding competition studies indicate differential affinities of the ligand towards TRAILR1(-Fas) <i>versus</i> TRAILR2(-Fas) chimeric receptors.</p

Ligand binding affinities of receptor chimeras differing only in their transmembrane and stalk regions.

<p><b>A-C.</b> 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 K_D-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.</p