A key property of TNFRs that determines the overall cellular response to TNF is their differential responsiveness to sTNF and mTNF (24
). Accordingly, sTNF and mTNF can exert specific and counteracting functions under normal and pathological conditions (36
), and distinct TNFR-mediated signaling pathways balance specific immune responses and neurobiological functions (4
). Despite the central roles of TNFRs in the regulation of TNF responses, the underlying molecular mechanisms determining receptor responsiveness have long remained unresolved. By using our previously developed unique cellular system and novel TNF variants (10
), we identified 42 aa within the stalk region of TNFR2 that, in contrast to the stalk region of TNFR1, effectively prevent responsiveness (, , and ). An assessment of various coparameters for their impacts on sTNF responsiveness revealed that neither ligand-receptor complex stability, which was previously suggested to be a major determinant of sTNF signaling initiation (24
), nor O-glycosylation controls sTNF responsiveness (; see Fig. S4 and S5 in the supplemental material). O-glycoslylation is a prominent molecular feature of the TNFR2 stalk region, but not of TNFR1, and might be expected to influence sTNF responsiveness in a manner similar to that seen in other signaling systems (35
). However, by using Benzyl-α-GalNAc, as well as a site-directed mutagenesis approach, to eliminate potential glycosylation sites, we could not define a specific role for O-glycosylation in the regulation of sTNF responsiveness (see Fig. S5 and S6 in the supplemental material). After excluding stalk length and any stretch of 10 aa on its own as a determinant of sTNF responsiveness (), our data together suggest that a likely determinant controlling the receptor's unresponsiveness to sTNF is TNFR2 stalk rigidity, which is attributable to its richness in proline residues. In this context, it is salient to note that longer, Pro-rich stalk regions are characteristic of other members of the TNFR superfamily, including Ox40, CD27, 4-1BB, and TACI. These receptors require preoligomerized soluble ligands for efficient activation (8
), suggesting that, like TNFR2, the respective membrane-bound ligands are in fact their relevant activators. Moreover, differential responsiveness to soluble and membrane-bound ligand has been described for DR5, but not DR4 (76
), and it is noteworthy that the stalk region of DR5 (but not of DR4) is comparable in length and proline content to the TNFR2 stalk region.
Another molecular feature with potential relevance for ligand binding and signal initiation of several TNFR superfamily members, including TNFR1, TNFR2, and Fas, is the PLAD located in the N-terminal CRD1 of the receptors. This domain mediates homotypic receptor interactions in the absence of ligand and has been proposed to mediate stable ligand-receptor clusters (9
). Chan et al. have shown that removal of the PLAD-positive CRD1 abolishes ligand binding (13
), although CRD1 of TNFR1 does not appear to be directly involved in ligand-receptor interaction (5
). Our own experiments argued for a scaffold function of CRD1 in the stabilization of CRD2 and supported the hypothesis of Chan et al. that chemical cross-linkers preferentially detect PLAD-mediated homomultimers (9
). However, data presented here demonstrate that the receptor's ability to allow formation of covalently cross-linked homodimers correlates well with sTNF responsiveness, but not with its ligand binding affinity, arguing against a need for PLAD-mediated avidity effects for high-affinity binding (see Fig. S8 in the supplemental material and ). Our data thus support the notion that PLAD-mediated homotypic receptor preassembly is not mandatory for high-affinity sTNF binding but rather is essential for sTNF signaling initiation by the formation of stable ligand-receptor clusters.
Receptor clustering confers several advantages for signal transduction, including increased sensitivity and specificity, simultaneity of response, and the segregation of similar signaling systems (17
). Indeed, ligand-mediated trimerization of TNFR is believed not to be sufficient for signal initiation, but oligomerization of ligand-receptor complexes into larger clusters has been observed in parallel with signaling initiation (9
). Membrane-bound ligands themselves tend to be locally enriched and inevitably result in the oligomerization of receptors (27
). However, other TNFRs, such as TNFR1 and DR4 (76
), can be readily activated by the respective soluble forms of their ligands, which is perhaps surprising given their low cell surface numbers (typically only 300 to 1,000 receptors per cell for TNFR1) (55
). Our data from wild-type TNFR2, the TNFR2-Fas chimera, and their stalk variants indicate that the ability to respond to sTNF is favored by ligand-independent local enrichment of receptors in the plasma membrane (compare , , and with to and with and ). Moreover, we identified the stalk region as the critical determinant controlling spontaneous local enrichment of wild-type TNFR2, as well as TNFR2-Fas chimeras ( to and to ). Our experiments indicate that the ability of receptors to become locally enriched in the absence of ligand is essential, both for efficient signal transduction by sTNF and for the induction of spontaneous, ligand-independent cell death ( to ). Parallel to local enrichment, enhanced formation of cross-linker-reactive homodimers was observed. Thus, we propose that cell surface TNFR distribution, regulated by the stalk regions, ultimately controls homotypic receptor preassembly; in turn, the requirement for soluble or membrane-bound ligand is defined, along with the efficiency of subsequent signal initiation.
What might cause TNF receptors to distribute nonhomogeneously in the cellular membrane and to be enriched in locally confined areas? Receptor preassembly/enrichment in transient small (6 to 12 nm in diameter) cholesterol-dependent nanoclusters may provide an explanation and reportedly promotes efficient signal complex formation (26
). In support of this, TNFR1, but not TNFR2, contains a potential cholesterol-binding motif in its transmembrane domain (LMYRYQR; aa 232 to 238) (18
), which is maintained in TNFR1-Fas, TNFR2-S1T1-Fas, TNFR2-S2T1-Fas, and TNFR2-S1T1-R2 chimeras. Indeed, in contrast to TNFR2, TNFR1 was found to localize to cholesterol- and sphingolipid-enriched membrane microdomains in the plasma membrane (15
). Consistent with this, we have observed a much slower membrane diffusion of TNFR1 than of TNFR2 that is sensitive to cholesterol depletion (23
). Together, these data suggest that the enrichment of the two TNFRs in different membrane microcompartments might control receptor distribution, PLAD-mediated homomultimerization, and, thus, sTNF responsiveness. However, results from our TNFR2-S2T1-Fas chimera experiments demonstrate that, rather than the transmembrane region, the extracellular stalk region is the key orchestrator of these processes: TNFR2-S2T1-Fas, similar to TNFR2-Fas, does not show pronounced levels of cross-linker-sensitive homodimers, whereas TNFR2-S2Δ42
T2-Fas is efficient in the formation of preassembled receptors (; see Fig. S3I in the supplemental material). Indeed, TNFR2-S2T1-Fas is largely unresponsive to sTNF (see Fig. S3H in the supplemental material). In addition, stalk-mediated disruption of receptor localization in nanostructured membrane microdomains might represent a mechanism for downregulating sTNF responsiveness. With this in mind, it is worth noting that in the case of Fas, T-cell receptor (TCR) engagement leads to the redistribution of this death receptor into “lipid rafts,” rendering T cells sensitive to Fas-specific antibodies in the absence of a secondary cross-linker (47
). This rearrangement seems to be regulated by Rac-1-dependent cytoskeletal remodeling, presumably through the dephosphorylation of the ezrin-radixin-moesin (ERM) proteins (52
). Due to sequence homologies between Fas and TNFR2 in the membrane-proximal intracellular region, an interaction of TNFR2 with the cytoskeleton is conceivable (63
). Alternatively, stalk-mediated conformational hindrance, posttranslational modifications, or lipid-protein interactions could antagonize cluster formation. Due to its comparable interactions with both TNF receptors and its competition with ligand binding, the recently identified TNFR-interacting molecule progranulin does not appear to represent a good candidate for such an interaction partner (73
). Overall, the definitive means by which the long, proline-rich stalk region interferes with local enrichment of receptors, resulting in reduced formation of PLAD-mediated homomultimers and sTNF unresponsiveness, remains elusive but highlights another exciting level of complexity of receptor activity regulation.
In conclusion, our data reveal a novel mechanism of TNFR partitioning in the absence of ligand controlling sTNF responsiveness. Our comprehensive analysis identified the stalk regions of TNFR as key determinants for TNFR arrangements and sTNF responsiveness. Complications currently associated with TNF-directed therapeutics, including impaired host defense and the paradoxical triggering of autoimmunity, demand the design of more TNFR-selective therapeutic agents (20
). The stalk regions of TNFR may become intriguing targets for the specific modulation of TNFR responses in vivo