Death domain mutagenesis of KILLER/DR5 reveals residues critical for apoptotic signaling.
The Fas/tumor necrosis factor (TNF)/TRAIL receptors signal death through a cytoplasmic death domain (DD) containing six alpha-helices with positively charged helix 2 interacting with negatively charged helix 3 of another DD. DD mutation occurs in head/neck and lung cancer (TRAIL receptor KILLER/DR5) and in lpr mice (Fas). We examined the apoptotic potential of known KILLER/DR5 lung tumor-derived mutants (n = 6) and DD mutants (n = 18) generated based on conservation with DR4, Fas, Fas-associated death domain (FADD), and tumor necrosis factor receptor 1 (TNFR1). With the exception of Arg-330 required in Fas or FADD for aggregation or for TNFR1 cytotoxicity, surprisingly major loss-of-function KILLER/DR5 alleles (W325A, L334A (lpr-like), I339A, and W360A) contained hydrophobic residues. Loss-of-function of I339A (highly conserved) has not been reported in DDs. Charged residue mutagenesis revealed the following points. 1) E326A, conserved in DR4, is dispensable for death; the homologous residue is positively charged in Fas, TNFR1, and FADD and is critical for DD interactions. 2) K331A, D336A, E338A, K340A, K343A, and D351A have partial loss-of-function suggesting multiple charges stabilize receptor-adapter interactions. Analysis of the tumor-derived KILLER/DR5 mutants revealed the following. 1) L334F has partial loss-of-function versus L334A, whereas E338K has major loss-of-function versus E338A, examples where alanine and tumor-specific substitutions have divergent phenotypes. 2) Unexpectedly, S324F, E326K, K386N, and D407Y have no loss-of-function with tumor-specific or alanine substitutions. Loss-of-function KILLER/DR5 mutants were deficient in recruitment of FADD and caspase 8 to TRAIL death-inducing signaling complexes. The results reveal determinants within KILLER/DR5 for death signaling and drug design.
Laboratory of Molecular Oncology and Cell Cycle Regulation, Howard Hughes Medical Institute, Departments of Medicine, Genetics, and Pharmacology, and the Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
E R
ER
McDonald
P C
PC
Chui
P F
PF
Martelli
D T
DT
Dicker
W S
WS
El-Deiry
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000502:physiology*
D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; C505639:TNFRSF10B protein, human