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. 2013 May;35(5):450-5.
doi: 10.1007/s10059-013-0072-3. Epub 2013 May 3.

EphrinA5-EphA7 complex induces apoptotic cell death via TNFR1

Haeryung Lee  1 , Eunjeong Park, Yujin Kim, Soochul Park
Affiliations

EphrinA5-EphA7 complex induces apoptotic cell death via TNFR1

Haeryung Lee et al. Mol Cells. 2013 May.

Abstract

A previous study showed that the EphA7 receptor regulates apoptotic cell death during early brain development. In this study, we provide evidence that the EphA7 receptor interacts with death receptors such as tumor necrosis factor receptor 1 (TNFR1) to decrease cell viability. We showed that ephrinA5 stimulates EphA7 to activate the TNFR1-mediated apoptotic signaling pathway. In addition, a pull-down assay using biotinylated ephrinA5-Fc revealed that ephrinA5-EphA7 complexes recruit TNFR1 to form a multi-protein complex. Immunocytochemical staining analysis showed that EphA7 was co-localized with TNFR1 on the cell surface when cells were incubated with ephrinA5 at low temperatures. Finally, both the internalization motif and death domain of TNFR1 was important for interacting with an intracytoplasmic region of EphA7; this interaction was essential for inducing the apoptotic signaling cascade. This result suggests that a distinct multi-protein complex comprising ephrinA5, EphA7, and TNFR1 may constitute a platform for inducing caspase-dependent apoptotic cell death.

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Figures

Fig. 1.
Fig. 1.
Screening of death receptors interacting with EphA7 using cell viability assay. (A) The indicated death receptor expression constructs were co-transfected with EphA7 expression vector into HEK293 cells; at 30 h post-transfection, dead cells were briefly washed away with culture medium before attached cells were photographed as DIC images. Staurosporine (2 μM) was used as a positive control for inducing apoptotic cell death. (B, C) Transfected cells were subjected to an XTT assay, which measures absorbance at 450 nm to detect cell viability. Data represent the means ± standard error (SE). *p < 0.001.
Fig. 2.
Fig. 2.
The activated EphA7 receptor interacts with TNFR1 to enhance apoptotic signaling. (A) HEK 293 cells were transfected as described in Fig. 1; at 14 h post-transfection, cells were treated with Fc or ephrinA5-Fc for 1 h at 37°C. For clustered ephrinA5-Fc, ephrinA5-Fc (1 μg) was incubated with goat anti-human IgG antibody for 30 min on ice. The cells were washed, fixed, and subjected to immunocytochemical staining using anti-cleaved caspase-3 antibody to detect apoptotic cells (red). Nuclear staining was performed using DAPI (blue). (B) Apoptotic cells in each microscopic field (× 200) were counted; cell counting was repeated for five different fields per dish for quantification. Z-VAD was used as a caspase inhibitor. u, unclustered; c, clustered. Data were obtained from three independent experiments and shown as the means ± SE. *p < 0.001.
Fig. 3.
Fig. 3.
EphrinA5 induces formation of multi-protein complex containing EphA7 and TNFR1. (A) HEK 293 cells were transfected as described in Fig. 1; at 14 h post-transfection, cells were treated with biotinylated ephrinA5-Fc on ice for 1 h. Cell lysates were further incubated with streptavidin-agarose beads prior to precipitation. Protein complexes were resolved by SDS-PAGE and subjected to Western blot analysis using anti-TNFR1 antibody (top panel) or EphA7 antibody (middle panel). Whole cell lysates were also analyzed by Western blot using anti-TNFR1 antibody (bottom panel). (B) Transfected cells were incubated with ephrinA5-Fc on ice for 1 h. Cells were transferred into a 37°C incubator. At the indicated time, cells were briefly washed, fixed, and subjected to immunocytochemical staining using goat anti-human IgG antibody (conjugated with rhodamine) and anti-TNFR1 antibody (pre-incubated with FITC-conjugated secondary antibody). Note that anti-human IgG antibody specifically stains ephrinA5-Fc bound to EphA7.
Fig. 4.
Fig. 4.
Internalization motif or death domain of TNFR1 is critical for interacting with the cytoplasmic region of EphA7. (A) Schematic diagrams showing EphA7 or TNFR1 mutants used for expression in HEK293 cells. EphA7-T1 comprises a unique peptide sequence (SLVTMEHLSVL) at its carboxyl-terminus, which replaces the intracytoplasmic region spanning amino acids 599 to 994 of mouse EphA7. The TNFR1 internalization motif (amino acids 237 to 240) is critical for receptor endocytosis. DD represents death domain as marked by the black bar. The hatched bar indicates the transmembrane domain for each protein. (B, C) Experiments were performed essentially as described in the legends for Figs. 2 and 3. (D) Data represent the means ± SE. *p < 0.001.
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