Regulatory Roles for MD-2 and TLR4 in Ligand-Induced Receptor Clustering

Abstract

LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe119 to Lys132 (Arg132 in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly59 was found to be a novel critical amino acid for LPS binding outside the region 119–132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe126 or Gly129 impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition.

TLR4/MD-2 Monoclonal Antibody Therapy Affords Protection in Experimental Models of Septic Shock

“…1, E and F) and there was no evidence of 5E3-mediated apoptosis induction in mouse whole blood or on mouse endothelial cells following prolonged incubation at 37°C (data not shown). Most likely, 5E3 inhibits TLR4 signaling by blocking LPS-TLR4 binding or by inhibiting TLR4/MD-2 receptor clustering/aggregation, known to be essential for subsequent signal transduction events (19).…”

Increased expression and internalization of the endotoxin coreceptor CD14 in enterocytes occur as an early event in the development of experimental necrotizing enterocolitis

“…LPS must rely on co-receptors such as LBP, MD2 and CD14 to activate the transmembrane TLR4 receptor and initiate signaling. LPS is first bound to soluble LBP and then transferred to CD14 (25)(26)(27), which then acts to shuttle LPS to the TLR4-bound MD2. This direct interaction between MD2 and LPS serves to activate TLR4 and stimulate intracellular signaling.…”

Identification of Interaction Sites for Dimerization and Adapter Recruitment in Toll/Interleukin-1 Receptor (TIR) Domain of Toll-like Receptor 4

“…A new NgoMIV (compatible with AgeI) site was inserted with primers 11 and 12 just before Gly-663 of TLR4 to allow exchange of TLR4ic mutants from the pCLG-TLR4ic MAPPIT bait vector. The pMX-mMD-2 and pMX-mCD14 plasmids were the kind gifts from Dr. Shinichiroh Saitoh (31). The pNFconluc reporter was a gift from Dr. Alain Israel.…”