The extracellular domains of FasL and Fas are sufficient for the formation of supramolecular FasL-Fas clusters of high stability.
Using fluorescent variants of Fas and FasL, we show that membrane FasL and Fas form supramolecular clusters that are of flexible shape, but nevertheless stable and persistent. Membrane FasL-induced Fas clusters were formed in caspase-8- or FADD-deficient cells or when a cytoplasmic deletion mutant of Fas was used suggesting that cluster formation is independent of the assembly of the cytoplasmic Fas signaling complex and downstream activated signaling pathways. In contrast, cross-linked soluble FasL failed to aggregate the cytoplasmic deletion mutant of Fas, but still induced aggregation of signaling competent full-length Fas. Moreover, membrane FasL-induced Fas cluster formation occurred in the presence of the lipid raft destabilizing component methyl-beta-cyclodextrin, whereas Fas aggregation by soluble FasL was blocked. Together, these data suggest that the extracellular domains of Fas and FasL alone are sufficient to drive membrane FasL-induced formation of supramolecular Fas-FasL complexes, whereas soluble FasL-induced Fas aggregation is dependent on lipid rafts and mechanisms associated with the intracellular domain of Fas.
Henkler
Frank
F
Department of Molecular Internal Medicine, Medical Polyclinic, University of Wuerzburg, 97070 Wuerzburg, Germany.
Behrle
Eva
E
Dennehy
Kevin M
KM
Wicovsky
Andreas
A
Peters
Nathalie
N
Warnke
Clemens
C
Pfizenmaier
Klaus
K
Wajant
Harald
H
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics; D017209:Apoptosis / Q000502:physiology*; D053181:Caspase 8; D020169:Caspases / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D009154:Mutation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012995:Solubility / Q000187:drug effects; D047392:beta-Cyclodextrins / Q000494:pharmacology; D019014:fas Receptor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C505533:FADD protein, human; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D047392:beta-Cyclodextrins; D019014:fas Receptor; C108732:methyl-beta-cyclodextrin; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases