Ectodermal Dysplasia Molecular Pathways: Functional Interaction of EDARADD and TRAFs

Objectives: The interaction of Edar-associated death domain (Edaradd) with Ectodysplasin receptor (Edar), is essential for the development of exocrine glands, teeth, hair follicles, and other ectodermal derived tissues. Dysfunction in Edar signaling causes ectodermal dysplasia, a disorder characterized by lack of sweat glands, hair, and teeth. Edaradd has been shown to interact with various tumor necrosis factor (TNF) receptor-associated factor (TRAF) family members, and some data suggests it could activate NF-kB and JNK pathways. In this study, we determine which TRAFs interact with ERARADD, and through which specific binding domains. Also, we show which TRAFs are utilized to propagate certain signaling cascades.

Methods: We performed a yeast two-hybrid and gst-pulldown to identify which TRAFs interact with EDARADD. We then generated point mutations in the TRAF2 (Edaradd ΔT2BS) and TRAF6 (Edaradd ΔT6BS) binding consensus sites of Edaradd. . We also used a NF-ĸB luciferase reporter system and an in vitro kinase assay for JNK to determine which TRAFs play roles in the signaling pathways.

Results: The yeast two-hybrid screen and GST-pull revealed that TRAF2 and TRAF6 interact with EDARADD. TRAF2 – Edaradd and TRAF6 – Edaradd interactions were abolished when we used Edaradd ΔT2BS and Edaradd ΔT6BS, respectively; However, mutating one TRAF binding site did not eliminate the interaction between Edaradd and the other TRAF. Finally, using the NF-ĸB luciferase reporter system, as well as the in vitro kinase assay for JNK, we demonstrated that mutations within the TRAF6 binding site impaired Edaradd-induced activation of NF-ĸB and JNK signaling pathways to a greater extent than mutations inside the TRAF2 binding site.

Conclusion: TRAF2 and TRAF6 interact with EDARADD at specific binding consensus sites. TRAF6-EDARADD interaction results in the activation of NF-ĸB and JNK signaling pathways. These results suggest TRAF6 plays a role EDAR signaling, and further elucidates the molecular pathway involved in ectodermal dysplasia.