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. 1998 Jan 20;95(2):588-93.
doi: 10.1073/pnas.95.2.588.

A family of human receptors structurally related to Drosophila Toll

F L Rock  1 , G Hardiman, J C Timans, R A Kastelein, J F Bazan
Affiliations

A family of human receptors structurally related to Drosophila Toll

F L Rock et al. Proc Natl Acad Sci U S A. .

Abstract

The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors--named TLRs 1-5--are probably the direct homologs of the fly molecule and, as such, could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role--akin to Toll in the dorsoventralization of the Drosophila embryo--as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, we also show that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors and MyD88 factors, and plant disease-resistance proteins recognizes a parallel beta/alpha fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.

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Figures

Figure 1
Figure 1
Schematic comparison of the protein architectures of Drosophila and human TLRs and their relationship to vertebrate IL-1Rs and plant disease-resistance proteins. Three Drosophila (Dm) TLRs (Toll, 18w, and the Mst ORF fragment) (7, 11, 13, 28) are arrayed beside the four complete (TLRs 1–4) and one partial (TLR5) human (Hu) receptors. Individual LRRs in the receptor ectodomains that are flagged by PRINTS (19) are explicitely noted by boxes; top and bottom Cys-rich clusters that flank the C- or N-terminal ends of LRR arrays are respectively drawn by apposed half circles. The loss of the internal Cys-rich region in TLRs 1–5 largely accounts for their smaller ectodomains (558, 570, 690 and 652 aa, respectively) when compared with the 784- and 977-aa extensions of Toll and 18w. The incomplete chains of DmMst and HuTLR5 (519- and 153-aa ectodomains, respectively) are represented by dashed lines. The intracellular signaling module common to TLRs, IL-1Rs, the intracellular protein Myd88, and the tobacco disease-resistance gene N product (DRgN) is indicated below the membrane. Additional domains include the trio of Ig-like modules in IL-1Rs (disulfide-linked loops); the DRgN protein features a nucleotide-binding domain (box) and Myd88 has a death domain (solid oval).
Figure 2
Figure 2
Conserved structural patterns in the signaling domains of Toll- and IL-1-like cytokine receptors and two divergent modular proteins. (A) Sequence alignment of the common TH domain. TLRs are labeled as in Fig. 1; the human (Hu) or mouse (Mo) IL-1 family receptors (IL-1R1–6) are sequentially numbered as proposed (14); Myd88 and the disease-resistance protein (DRP) sequences from tobacco (To), Arabidopsis thaliana (At), and Linum usitatissimum (Lu) represent C- and N-terminal domains, respectively, of larger multidomain molecules. Ungapped blocks of sequence (numbered 1–10) are boxed. Triangles indicate deleterious mutations, and truncations N-terminal of the arrow eliminate bioactivity in human IL-1R1 (33). phd (20) and dsc (21) secondary structure predictions of α-helix (H), β-strand (E), or coil (L) are marked. The amino acid coloring scheme depicts chemically similar residues: green (hydrophobic), red (acidic), blue (basic), yellow (Cys), orange (aromatic), black (structure breaking), and grey (tiny). Diagnostic sequence patterns for IL-1Rs, TLRs, DRPs, and full alignment (ALL) were derived by consensus at a stringency of 75%. Symbols for amino acid subsets are as follows (see consensus site for detail): o, alcohol; l, aliphatic; a dot, any amino acid; a, aromatic; c, charged; h, hydrophobic; −, negative; p, polar; +, positive; s, small; u, tiny; t, turnlike. (B) Topology diagram of the proposed CheY-like TH β/α domain fold. Parallel β-sheets (with β-strands A–E as yellow triangles) are seen at their C-terminal ends; α-helices (red circles labeled 1–5) link the β-strands; chain connections are to the front (visible) or back (hidden). Conserved charged residues at the C-terminal end of the β-sheets are noted in a shaded (Asp) or black (Arg) circle (see text).
Figure 3
Figure 3
Evolution of a signaling domain superfamily. The multiple TH module alignment of Fig. 2 was used to derive a phylogenetic tree by the neighbor-joining method (18); 10,000 bootstrapping replications were conducted to assess the reliability of the branching patterns (noted at selected nodes as percents). Proteins are labeled as in the alignment; the tree was rendered with treeview.
Figure 4
Figure 4
FISH chromosomal mapping of human TLR genes. Denatured chromosomes from synchronous cultures of human lymphocytes were hybridized to biotinylated TLR cDNA probes for localization. (A) TLR2. (B) TLR3. (C) TLR4. (D) TLR5. Assignment of the FISH mapping data (Left) with chromosomal bands was achieved by superimposing FISH signals with 4,6-diamidino-2-phenylindole-banded chromosomes (Center) (27). Analyses are summarized in the form of human chromosome ideograms (Right).
Figure 5
Figure 5
mRNA blot analyses of human TLRs. Human multiple tissue blots (He, heart; Br, brain; Pl, placenta; Lu, lung; Li, liver; Mu, muscle; Ki, kidney; Pn, Pancreas; Sp, spleen; Th, thymus; Pr, prostate; Te, testis; Ov, ovary, SI, small intestine; Co, colon; PBL, peripheral blood lymphocytes) and cancer cell line (promyelocytic leukemia, HL60; cervical cancer, HELAS3; chronic myelogenous leukemia, K562; lymphoblastic leukemia, Molt4; colorectal adenocarcinoma, SW480; melanoma, G361; Burkitt lymphoma Raji, Burkitt’s; colorectal adenocarcinoma, SW480; lung carcinoma, A549) containing approximately 2 μg of poly(A)+ RNA per lane were probed with radiolabeled cDNAs encoding TLR1 (A–C), TLR2 (D), TLR3 (E), and TLR4 (F). Blots were exposed to x-ray film for 2 days (A–C) or 1 week (D–F) at −70°C with intensifying screens. An anomalous 0.3-kb species appears in some lanes; hybridization experiments exclude a message encoding a TLR cytoplasmic fragment.
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