The Ectodomain of the Toll-like Receptor 4 Prevents Constitutive Receptor Activation

Gabriela Panter,Roman Jerala

doi:10.1074/jbc.m110.205419

Abstract

Toll-like receptor 4 (TLR4) is involved in activation of the innate immune response in a large number of different diseases. Despite numerous studies, the role of separate domains of TLR4 in the regulation of receptor activation is poorly understood. Replacement of the TLR4 ectodomain with LPS-binding proteins MD-2 or CD14 resulted in a robust ligand-independent constitutive activation comparable with the maximal stimulation of the receptor with LPS. The same effect was achieved by the replacement of the ectodomain with a monomeric fluorescent protein or a 24-kDa gyrase B fragment. This demonstrates an intrinsic dimerization propensity of the transmembrane and cytoplasmic domains of TLR4 and reveals a previously unknown function of the ectodomain in inhibiting spontaneous receptor dimerization. Constitutive activation was abolished by the replacement of the ectodomain by a bulkier protein ovalbumin. N-terminal deletion variants of TLR4 revealed that the smallest segment of the ectodomain that already prevents constitutive activity comprises only 90 residues (542 to 631) of the total 608 residues. We conclude that TLR4 represents a receptor with a low threshold of activation that can be rapidly activated by the release of inhibition exerted by its ectodomain. This is important for the sensitivity of TLR4 to activation by different agonists. The TLR4 ectodomain has multiple roles in enabling ligand regulated activation, providing proper localization while serving as an inhibitor to prevent spontaneous, ligand-independent dimerization.

Highlights

The Toll-like receptor 4 (TLR4) Ectodomain Has Additional Roles Besides Providing a Scaffold for the Coreceptor and Ligand Binding—To investigate additional roles of the TLR4 ectodomain, we have examined the effect of replacement of the ectodomain of TLR4 with either of the two LPS-binding proteins, myeloid differentiation protein-2 (MD-2) or cluster of differentiation 14 (CD14), generating MD-2-TM-TLR4 and CD14-TM-TLR4 chimeric constructs (Fig. 1A)
Chimeric proteins were expressed in the cells (Fig. 1C) and at the cell surface (D), and the latter were capable of binding LPS (E). They all signaled through the same signaling pathways as the native receptor pair MD-2/TLR4 because their signaling could effectively be inhibited by dominant negative versions of the downstream adapter proteins MyD88 and TRIF
We have investigated the additional roles of the TLR4 ectodomain in receptor activation and subsequent signal transduction by its replacement and deletions

Summary

To whom correspondence should be addressed

TLRs have to be able to mount an immediate immune response upon binding of the agonist, whereas in the absence of the ligand it is imperative that they remain in an inactive state to prevent unwanted activation that may lead to excessive inflammation and autoimmune disease [5] This is especially important for TLR4, the cellular receptor for LPS, a molecular signature of the outer cell membrane of Gram-negative bacteria [6]. MD-2 is a secreted glycoprotein that binds to the N terminus of the ectodomain of TLR4 [21] and is the actual LPS-binding protein of the receptor complex and indispensable for signaling [20, 22, 23] Another molecule, cluster of differentiation 14 (CD14), facilitates signaling by TLR4 [24, 25]. We show that the transmembrane and cytoplasmic TIR domain of TLR4 contribute the major interaction energy for receptor dimerization, the ectodomain plays an important role in providing controlled responsiveness to LPS, primarily by inhibiting spontaneous dimerization of the transmembrane and cytoplasmic domains, thereby preventing constitutive, ligand-independent receptor signaling

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION