FUNCTION: This gene belongs to the evolutionarily-conserved Toll-like receptor family, whose members are type-1 transmembrane proteins that are involved in innate immunity. Toll-like receptors are characterized by an extracellular leucine-rich repeat domain that functions in ligand recognition and an intracellular toll/interleukin-1 receptor-like domain that is crucial for signal transduction. The receptor encoded by this gene mediates the innate immune response to bacterial lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria, through synthesis of pro-inflammatory cytokines and chemokines. In addition, this protein can recognize other pathogens from Gram-negative and Gram-positive bacteria as well as viral components. Mice deficient in this gene display a number of immune response-related phenotypes including hyporesponsiveness to bacterial lipopolysaccharide and increased levels of respiratory syncytial virus compared to controls. [provided by RefSeq, Sep 2015] PHENOTYPE: Homozygotes for spontaneous or targeted mutations are hyporesponsive to bacterial lipopolysaccharide and more susceptible to infection by gram negative bacteria. [provided by MGI curators]
Figure 1.Milquetoast mice exhibited reduced TNFα secretion in response to TLR4 ligand, LPS. TNFα levels were determined by ELISA. Normalized data are shown from gene-based superpedigree analysis. Data shown for pedigrees R1929 (green), R1888 (red), R3011 (purple), R1488 (cyan), and R0827 (gold). Abbreviations: WT, wild-type; REF, homozygous reference mice; HET, heterozygous variant mice; VAR, homozygous variant mice. Mean (μ) and standard deviation (σ) are indicated.
The milquetoast phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1929 by gene-based superpedigree analysis in which some mice from five pedigrees showed reduced TNFα secretion from macrophages in response to the Toll-like receptor 4 (TLR4) ligand, lipolysaccharide (LPS) (Figure 1).
Nature of Mutation
Figure 2.Linkage mapping of the reduced TNFα secretion after LPS stimulation using an additive model of inheritance and gene-based superpedigree analysis. Manhattan plot shows -log10 P values (Y-axis) plotted against the chromosome positions of 109 mutations identified in the G1 male of pedigree R0827, 99 mutations identified in the G1 male of pedigree R1888, 33 mutations identified in the G1 male of pedigree R3011, 55 mutations identified in the G1 male of pedigree R1488, and 87 mutations identified in the G1 male of pedigree R0827 (X-axis). Normalized phenotype data are shown with consideration of G2 dam identity. Horizontal pink and red lines represent thresholds of P = 0.05, and the threshold for P = 0.05 after applying Bonferroni correction, respectively.
Whole exome HiSeq sequencing of the R0827 G1 grandsire identified 109 mutations. The reduced TLR4-associated signaling phenotype was linked by continuous variable mapping to a mutation in Tlr4 using gene-based superpedigree analysis: an A to T transversion at base pair 66,839,444 (v38) on chromosome 4, or base pair 11,634 in the GenBank genomic region NC_000070. Linkage was found using an additive model of inheritance, wherein 12 variant homozygotes from five pedigrees exhibiting either null of missense Tlr4 alleles departed phenotypically from 45 homozygous reference mice and 67 heterozygous mice with a P value of 4.878 x 10-5 (Figure 2).
The mutation corresponds to residue 754 in the mRNA sequence NM_021297 within exon 3 of 3 total exons.
The mutated nucleotide is indicated in red. The mutation results in a histidine (H) to leucine (L) substitution at position 158 (H158L) in the TLR4 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 0.998).
Figure 3. Protein and domain structure of TLR4. A) Schematic representation of TLR9 based on crystalized structures of mouse TLR3 LRR (PBD 3CIG) and human TLR2 TIR (1FYW) domains. The residue affected by the Lps3 mutation is highlighted. 3D image was created using UCSF Chimera. B) TLR4 is an 835 amino acid protein with an extracellur domain (pink) of leucine rich repeats (LRR), a short transmembrane domain and a cytoplasmic Toll/Interleukin-1 receptor (TIR) domain. The milquetoast mutation (red asterisk) results in an histidine (H) to leucine (L) substitution at position 158 (H158L) in the TLR4 protein. This image is interactive. Click on the image to view other mutations found in TLR4 (red). Click on the mutations for more specific information.
TLR4 is a type I integral membrane glycoprotein containing 835 amino acids. TLR4 has 22 predicted leucine-rich repeats (LRRs) in its ectodomain at the N-terminal half of the protein (1-3), a transmembrane domain, and a cytoplasmic Toll/IL-1R (TIR) domain (Figure 3). The milquetoast mutation results in a histidine (H) to leucine (L) substitution at position 158 (H158L) in the TLR4 protein. H158 is within LRR5.
Please see the record for lps3 for information about Tlr4.
TLR4 is the receptor for LPS (4). Stimulation of TLR4 by LPS activates two branches of signaling, one defined by early NF-κB activation (MyD88-dependent pathway, mediated by MyD88), and another distinguished by late NF-κB activation as well as interferon responsive factor (IRF)-3 activation leading to type I IFN production and costimulatory molecule upregulation (MyD88-independent pathway, mediated by Trif) (5-7). The MyD88-dependent pathway activates expression of target genes including interleukin (IL)-6, IL-1, TNF, IL-12p40 and type I interferon (IFN), cytokines required for the inflammatory response. The MyD88-independent pathway results in the production of type I IFN. The reduction in TLR4-associated responses in Milquetoast indicates that the mutation results in loss of TLR4 function.
milquetoast(F):5'- ATGGCATGGCTTACACCACC -3'
milquetoast(R):5'- AGTTCATGGAGCTTAATTCCCTG -3'
milquetoast_seq(F):5'- CCTCTCAAACTTGATACTGACAGG -3'
milquetoast_seq(R):5'- CATGGAGCTTAATTCCCTGAAAGGC -3'