Structural requirements for signal-induced target binding of FADD determined by functional reconstitution of FADD deficiency.
FADD is a key adaptor modulating several signaling pathways such as apoptosis induced by Fas (CD95) and tumor necrosis factor receptor 1, and cell proliferation induced by mitogens. Whereas mutations in Fas disrupt its binding to FADD and cause autoimmune lymphoproliferative (lpr) syndromes, a FADD deficiency blocks embryonic development in mice. To delineate the multifunction of FADD in vivo, we performed functional reconstitution analysis by introducing wild type and mutant FADD into FADD-/- cells or FADD-/- mice lacking the endogenous FADD. An lpr-like FADD mutant, V121N, was reported previously as being defective in Fas binding in vitro. However, we found that in mice V121N can bind to Fas and is functional in signaling apoptosis. Unexpectedly, this lpr-like mutant FADD failed to support mouse development, indicating that the death domain of FADD has an additional function required for embryogenesis, which is independent of that required for receptor-induced apoptosis. Further mutagenesis was targeted at charged residues in the FADD death domain, presumably mediating electrostatic interactions with Fas. We showed that the target binding and apoptosis signaling functions of FADD were not affected when mutations were introduced to a majority of the charged residues. In one exception, replacing arginine 117 with an uncharged residue disrupted target binding and apoptosis signaling, but restoring the positive charge at position 117 failed to reconstitute the FADD function. Therefore, in vivo target binding of FADD involves an additional mechanism distinct from electrostatic interaction.
Imtiyaz
Hongxia Z
HZ
Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Zhang
Yuhang
Y
Zhang
Jianke
J
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D053200:Fas-Associated Death Domain Protein; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000235:genetics; D053199:TNF Receptor-Associated Death Domain Protein; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C505534:Fadd protein, mouse; D053200:Fas-Associated Death Domain Protein; D053199:TNF Receptor-Associated Death Domain Protein; C481226:Tradd protein, mouse; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D019014:fas Receptor