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The ectodermal dysplasia receptor (EDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to play a key role in the process of ectodermal differentiation. We present evidence that EDAR is capable of activating the nuclear factor-B, JNK, and caspase-independent cell death pathways and that these activities are impaired in mutants lacking its death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death domain, it did not interact with the death domain-containing adaptor proteins TRADD and FADD. EDAR successfully interacted with various TRAF family members; however, a dominant-negative mutant of TRAF2 was incapable of blocking EDAR-induced nuclear factor-B or JNK activation. Collectively, the above results suggest that EDAR utilizes a novel signal transduction pathway. Finally, ectodysplasin A can physically interact with the extracellular domain of EDAR and thus represents its biological ligand.Anhidrotic (or hypohidrotic) ectodermal dysplasia is a disorder of ectodermal differentiation characterized by a triad of signs consisting of sparse hair, abnormal or missing teeth, and an inability to sweat (1). A similar phenotype is seen in mice with mutations involving the downless locus, suggesting the existence of a common underlying genetic defect. Recently, mutations in EDAR, 1 a novel receptor of the TNFR family, were found in several families with autosomal dominant and recessive forms of anhidrotic ectodermal dysplasia and in downless mice (2, 3). Although the above studies established the role of EDAR in ectodermal differentiation, the signaling pathways activated by this receptor remain to be elucidated.Death domain-containing receptors of the TNFR family are believed to activate two main signaling cascades: a kinase cascade leading to NF-B and JNK activation and a caspase cascade leading to cell death (4). As the cytoplasmic domain of EDAR was reported to contain a region with partial homology to the death domain, we decided to test its abilities to activate the above signaling cascades (2, 3). In this report, we present evidence that, like the classical death domain-containing receptors, EDAR is capable of activating the NF-B, JNK, and cell death pathways. However, EDAR does not interact with the death domain-containing adaptor proteins TRADD or FADD and does not activate the caspase cascade. Our results suggest the existence of a novel signaling pathway utilized by EDAR. MATERIALS AND METHODS Cell Lines and Reagents-293Tand MCF7 cells were obtained from Dr. David Han (University of Washington, Seattle, WA). 293 EBNA cells were obtained from Invitrogen. Rabbit polyclonal antibodies against FLAG, Myc, and hemagglutinin tags were obtained from Santa Cruz Biotechnology. FLAG beads and control beads were obtained from Sigma. The pull-down kinase assay kit for JNK was obtained from New England Biolabs Inc., and the constructs for the Pathdetect luciferase reporter assay were purchased from Stratagene....

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to be highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2). By using a subclone of 293F cells with stable expression of XEDAR, we report that XEDAR activates the NF-B and JNK pathways in an EDA-A2-dependent fashion. Treatment with EDA-A2 leads to the recruitment of TRAF3 and -6 to the aggregated XEDAR complex, suggesting a central role of these adaptors in the proximal aspect of XEDAR signaling. Whereas TRAF3 and -6, IKK1/IKK␣, IKK2/IKK␤, and NEMO/IKK␥ are involved in XEDARinduced NF-B activation, XEDAR-induced JNK activation seems to be mediated via a pathway dependent on TRAF3, TRAF6, and ASK1. Deletion and point mutagenesis studies delineate two distinct regions in the cytoplasmic domain of XEDAR, which are involved in binding to TRAF3 and -6, respectively, and play a major role in the activation of the NF-B and JNK pathways. Taken together, our results establish a major role of TRAF3 and -6 in XEDAR signaling and in the process of ectodermal differentiation.

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that is highly expressed during embryonic development and binds to ectodysplasin-A2 (EDA-A2). In this report, we demonstrate that although XE-DAR lacks a death domain, it nevertheless induces apoptosis in an EDA-A2-dependent fashion. The apoptosis-inducing ability of XEDAR is dependent on the activation of caspase 8 and can be blocked by its genetic and pharmacological inhibitors. Although XEDARinduced apoptosis can be blocked by dominant-negative Fas-associated death domain (FADD) protein and FADD small interfering RNA, XEDAR does not directly bind to FADD, tumor necrosis factor receptor-associated death domain (TRADD) protein, or RIP1. Instead, XEDAR signaling leads to the formation of a secondary complex containing FADD, caspase 8, and caspase 10, which results in caspase activation. Thus, XEDAR belongs to a novel class of death receptors that lack a discernible death domain but are capable of activating apoptosis in a caspase 8-and FADD-dependent fashion. XEDAR may represent an early stage in the evolution of death receptors prior to the emergence of the death domain and may play a role in the induction of apoptosis during embryonic development and adult life.