CD95 receptor activation by ligand-induced trimerization is independent of its partial pre-ligand assembly

Abstract

CD95 (Fas, APO-1, TNFRSF6) is a widely expressed single-pass transmembrane protein that is implicated in cell death, inflammatory response, proliferation and cell migration. CD95 ligand (CD95L, FasL, TNFSF6), is a potent apoptotic inducer in the membrane form but not when cleaved into soluble CD95L (sCD95L). Here, we aimed at understanding the relation between ligand-receptor multimerization and receptor activation by correlating the kinetics of ligand binding, receptor oligomerization, FADD (FAS-Associated via Death Domain) recruitment and caspase-8 activation inside living cells. Using single molecule localization microscopy and Forster resonance energy transfer imaging we show that the majority of CD95 receptors on the plasma membrane are monomeric at rest. This was confirmed functionally as the wild-type receptor is not blocked by a receptor mutant that cannot bind ligand. Moreover, using time-resolved fluorescence imaging approaches we demonstrated that receptor multimerization follows instantaneously ligand binding, whereas FADD recruitment is delayed. This process can explain the typical delay time seen with caspase-8 activity reporters. Finally, the low activity of sCD95L, which was caused by inefficient FADD recruitment, was not explained by the low avidity for the receptor but by a receptor clustering mechanism that was different from the one induced by the strong apoptosis inducer IZ-sCD95L. Our results reveal that receptor activation is modulated by the capacity of its ligand to trimerize it.\n\nHighlightsO_LIAt a density of less than 10 receptors per {micro}m2 CD95 exists as monomer (58%) and dimer (42%)\nC_LIO_LIPre-formed dimers do not contribute to ligand-induced CD95 apoptotic signaling\nC_LIO_LIThe PLAD of CD95 attenuates overexpression-induced, ligand-independent cell death\nC_LIO_LIsoluble CD95L can rapidly multimerize CD95 after binding but it is still a poor inducer of apoptosis through inefficient FADD recruitment\nC_LIO_LIFADD recruitment kinetics but not ligand binding kinetics correlates with caspase-8 onset of activity\nC_LI