Molecular characterization of CD40 signaling intermediates.
Signal transduction through the CD40 receptor is initiated by binding of its trimeric ligand and propagated by interactions of tumor necrosis factor receptor-associated factor (TRAF) proteins with the multimerized CD40 cytoplasmic domain. Using defined multimeric constructs of the CD40 cytoplasmic domain expressed as either soluble or myristoylated proteins, we have addressed the extent of receptor multimerization needed to initiate signal transduction and identified components of CD40 signaling complexes. Signal transduction in human embryonic kidney 293 cells, measured by nuclear factor kappaB activation, was observed in cells expressing soluble trimeric CD40 cytoplasmic domain and to a lesser extent in cells expressing dimeric CD40 cytoplasmic domain. Nuclear factor kappaB activation was strongest in cells expressing myristoylated trimeric CD40 cytoplasmic domain. Signal transduction through trimeric CD40 cytoplasmic domains with various point mutations in the TRAF binding sites was similar to signal transduction through analogous full-length receptors. Transiently expressed soluble trimeric CD40 cytoplasmic domain was isolated as complexes that contained TRAF2, TRAF3, TRAF5, TRAF6, and the inhibitor of apoptosis protein (c-IAP1). Association of c-IAP1 with the CD40 cytoplasmic domain complex was indirect and dependent on the presence of an intact TRAF1/2/3 binding site. These results suggest that extracellular ligation of CD40 can be bypassed and that soluble trimerized CD40 complexes can be isolated and used to identify components that link CD40 with signaling pathways.
Werneburg
B G
BG
Department of Biology, Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, Connecticut 06877, USA.
Zoog
S J
SJ
Dang
T T
TT
Kehry
M R
MR
Crute
J J
JJ
D001665:Binding Sites; D019013:CD40 Antigens / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D009227:Myristic Acids / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014162:Transfection
D019013:CD40 Antigens; D008024:Ligands; D009227:Myristic Acids; D016328:NF-kappa B; D008156:Luciferases