Gene name - 18 wheeler
Synonyms - Toll like receptor (tlr)
Cytological map position - 56F8--56F9
Function - Transmembrane receptor
Symbol - 18w
FlyBase ID: FBgn0004364
Genetic map position - 2-
Classification - Leucine rich repeat protein, IL1 like cytoplasmic domain
Cellular location - surface transmembrane protein
18 wheeler, so named because its segmental expression pattern resembles that of the segment polarity genes, and as well, the tied down, seemingly segmented tarpaulin over the rear half of an 18 wheeled semi (a large tractor-trailor truck), was characterized in two laboratories at the same time. In the Bellen lab (Eldon, 1994), 18w was characterized in a search for full length cDNAs of the couch potato gene (Bellen, 1992). In the Beachy lab, 18w was characterized because of the segmentally reiterated transverse stripes of beta-galactosidase expression in a P-element insertion line (Chiang, 1994).
18 wheeler is expressed early in head segments and in an alternating segment polarity pattern corresponding to the domains of wingless and engrailed expression. A comparison of the late expression patterns of 18w and Toll, coding for another leucine rich repeat protein, indicates that the genes are coexpressed in many tissues: the tracheal placodes, some midline cells, the developing salivary glands, the epidermis at the intersegmental furrows, the pharynx, the cells that form the dorsal vessel, the Malpighian tubules, and the hindgut. Although the expression patterns show differences, the genes are frequently coexpressed in regions undergoing extensive cell movements. Both proteins are approximately the same size and share similar motifs throughout most of the protein, including Interleukin 1 receptor homology in the cytoplasmic domain. Both proteins behave as heterophilic cell adhesion molecules when tested for adhesion in Schneider 2 cells, suggesting that they must be able to interact with one or more proteins that are constitutively expressed by these cultured cells. In spite of these similarities, there are no obvious genetic interactions between various mutant alleles of 18w and Toll (Eldon, 1994)
18-Wheeler (18W), is a critical component of the humoral immune response. 18-wheeler is expressed in the larval fat body, the primary organ of antimicrobial peptide synthesis. 18W is also detected in the lymph gland, garland cells and salivary glands. Transcript levels of 18w increase after bacterial infection. In the absence of the 18W receptor, larvae are more susceptible to bacterial infection. Nuclear translocation of the Rel protein Dorsal-like immunity factor (Dif) is inhibited, though nuclear translocation of another Rel protein, Dorsal, is unaffected. Induction of several antibacterial genes is reduced following infection, relative to wild-type: attacin is reduced by 95%, cecropin by 65% and diptericin by 12% (Williams, 1997).
Rho GTPase and its upstream activator, guanine nucleotide exchange factor 2 (RhoGEF2), have emerged as key regulators of actin rearrangements during epithelial folding and invagination (Nikolaidou, 2004). A Rho-GTPase-signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho activation. This study shows that Drosophila 18 wheeler (18W), a Toll-like receptor protein, is a novel component of the Rho-signaling pathway involved in epithelial morphogenesis. 18w mutant embryos have salivary gland invagination defects similar to embryos that lack components of the Rho pathway, and ubiquitous expression of 18W results in an upregulation of Rho signaling. Transheterozygous genetic interactions and double mutant analysis suggest that 18W affects the Rho-GTPase-signaling pathway not through Fog and RhoGEF2, but rather by inhibiting Rho GTPase activating proteins (RhoGAPs). RhoGAP5A and RhoGAP88C/Crossveinless-c (CV-C) are required for proper salivary gland morphogenesis, implicating them as potential targets of 18W (Kolesnikov, 2007).
Based on microarray experiments, 18w is downregulated in Scr mutants, implicating it in salivary gland development. To follow 18w expression in more detail, RNA in situ hybridizations were performed. The results show that in wild-type embryos 18w RNA is not maternally contributed but is expressed in salivary gland cells prior to and throughout their invagination. Within the salivary placode, 18w expression first becomes evident at stage 11 as a small spot in the dorsal posterior region. During early stage 12, following the beginning of invagination, 18w expression spreads throughout the placode. During the remainder of stage 12, 18w transcripts can be detected in both gland cells that have yet to invaginate as well as those that have already internalized, albeit at a reduced intensity. At stage 13, 18w transcripts cease to be expressed in salivary gland cells but are evident in salivary duct cells. In addition to salivary glands and ducts, 18w RNA is also detected in other tissues undergoing morphogenesis, including the tracheal placodes and the hindgut. As anticipated from microarray experiments, 18w transcripts are absent exclusively from parasegment two in Scr mutants while expression in the rest of the embryo remains unaltered. Overall, performing microarray experiments with Scr mutant embryos has proven to be an effective method for identifying salivary gland genes (Kolesnikov, 2007).
While 18w is expressed in several tissues undergoing morphogenesis, embryonic defects have yet to be identified in 18w mutant embryos. The striking 18w expression within the invaginating salivary gland prompted a more carefully investigation of the role of 18w in embryonic salivary gland development. In examining a null allele of 18w, 18wΔ21, it was discovered that initiation of invagination in the dorsal posterior region of the placode appears normal. However, during the next phase of invagination, in which the remaining cells of the salivary placode normally internalize in a strict sequential order, defects become apparent. In 18w mutants invagination is less synchronized; too many placode cells invaginate simultaneously rather than sequentially, thereby causing a wider lumen in 66% of 18w mutants when compared to wild-type embryos. Thus, it is the timely progression of invagination, not the invagination process itself, that is affected in 18w mutants. Due to the lack of proper coordination in 18w, the internalization of the gland cells appears to be delayed causing the cells that internalize last to remain near the ventral surface instead of reaching their final, more dorsal, position within the embryo. Eventually all of the salivary gland cells do internalize in 18w mutants, but the proximal part of the gland, which remains abnormally close to the ventral surface, becomes caught in the anterior movement of the ectoderm during head involution. As a result, the glands end up much closer to the anterior end of the embryo than in wild-type embryos. Similar defects are seen in mutants homozygous for the loss of function 18w allele, 18wΔ7–35, and in 18wΔ21, 18wΔ7–35 transheterozygous mutant embryos. Based on these results, 18w is an important component in coordinating salivary gland invagination (Kolesnikov, 2007).
Activation of the Rho-signaling pathway results in the phosphorylation of the myosin II regulatory light chain, encoded by the spaghetti squash (sqh) gene. The phosphorylated form of Sqh can interact with actin and cause actomyosin-based contractility at the apices of cells. To verify that 18W is part of the Rho pathway, whether overexpression of 18W results in an upregulation of Rho signaling, evident as an increase in phosphorylation of Sqh, was checked. Immunoblot analysis on embryonic extracts using anti-phospho-Sqh antibody reveals that overexpressing 18W throughout the embryo results in a two-fold increase of P-Sqh when compared to wild type. Similar results are seen when a constitutively active form of Rho is overexpressed ubiquitously in the embryo. Moreover, introducing one copy of a phosphomimetic sqh transgene, sqhE20E21, into an 18w mutant rescues the 18w invagination defects, indicating that 18W acts upstream of Sqh phosphorylation. Thus, both genetic and biochemical evidence indicate that 18W is a novel component of the Rho signaling pathway (Kolesnikov, 2007).
To determine whether 18W negatively regulates the RhoGAP branch of the Rho pathway, it was necessary to identify the RhoGAPs involved in salivary gland development. Of the 20 distinct RhoGAPs encoded by the Drosophila genome, 17 UAS-RhoGAP dsRNA lines were examined. Each of these lines was crossed to flies containing a salivary gland-expressing driver, scabrous-GAL4, and the progeny were screened for salivary gland defects. Only RhoGAP5A dsRNA expression resulted in salivary gland defects. Invagination defects are evident from more anteriorly placed glands, while migration defects resulted in wavy glands. Similar defects are seen upon expressing high levels of constitutively active Rho in the salivary gland with the scabrous-GAL4 driver. Overexpressing low levels of Rho in the gland results in mostly migratory defects, indicating that gland migration is more sensitive to the levels of Rho signaling than is the process of invagination. Invagination defects are seldom accompanied by migration defects, presumably because glands that have not properly internalized do not reach the visceral mesoderm upon which they normally migrate (Kolesnikov, 2007).
Since the GAL4/UAS dsRNA system may not result in a complete loss-of-function phenotype, strong alleles of two GAPs, RhoGAP68F and RhoGAP88C/Cv-c, which will be referred to simply as Cv-c. These have previously been shown to regulate Rho activity but lack salivary defects using the dsRNA interference technique. While neither rhoGAP68F nor cv-c mutations cause invagination defects, the cv-c mutants do display migration defects similar to those seen in embryos expressing RhoGAP5A dsRNA. To determine whether RhoGAP5A and Cv-c act redundantly within the salivary gland to regulate Rho activity, RhoGAP5A dsRNA within the gland was examined in a cv-c mutant. These embryos should have reduced activity of both RhoGAPs. They have gland invagination and migration defects that are more severe and penetrant than embryos that lack just one of them, indicating that these RhoGAPs are, in fact, partially redundant during salivary gland development (Kolesnikov, 2007).
In situ hybridization in wild-type embryos shows that cv-c RNA is not maternally contributed but is expressed in several tissues undergoing morphogenesis. In addition to its expression within the developing trachea and mesoderm, cv-c, similar to 18w, is expressed in the salivary glands prior to and during their invagination. Unlike 18w, however, cv-c expression does not appear to originate at the initial invagination site but rather initiates expression throughout most of the placode. During the onset of invagination at stage 12, cv-c expression intensifies and continues to be expressed within cells that have internalized until the conclusion of invagination at stage 13 (Kolesnikov, 2007).
To examine whether 18W regulates Rho activity by inhibiting Cv-c, several overexpression and genetic interaction experiments were performed. As might be expected if 18W negatively regulates Cv-c, overexpressing 18w within the salivary gland results in migratory defects similar to those seen in cv-c mutant embryos. In addition to migratory defects, however, some embryos overexpressing 18w also exhibit invagination defects, suggesting that Cv-c may not be the only RhoGAP negatively regulated by 18W. Moreover, lowering the dose of cv-c enhances the defects caused by 18w overexpression and suppresses the 18w mutant invagination defects, further supporting the role of 18W as a negative regulator of RhoGAP signaling. Therefore, genetic interaction experiments indicate that 18W regulates Rho signaling in the salivary gland by inhibiting at least one known RhoGAP (Kolesnikov, 2007).
Previous studies have shown that the Fog ligand activates RhoGEF2 through an as yet unidentified receptor, leading to the apical constriction of cells that form the ventral furrow and posterior midgut. Similar to salivary gland cells in 18w mutants, cells of the ventral furrow and posterior midgut in fog mutants do eventually invaginate but in an uncoordinated and delayed fashion. Since 18w and fog mutants have similar invagination defects, and 18W is a receptor protein that activates Rho signaling, whether 18W might be the FOG receptor was examined. This seems unlikely, however, because FOG overexpression within the salivary gland rescues the 18w mutant salivary gland defects. Since fog mutations do not completely eliminate apical constriction during ventral furrow and posterior midgut formation but RhoGEF2 mutations do, it has been argued that additional pathways must regulate apical constrictions via RhoGEF2. However, since 18w RhoGEF2 double mutants have more severe defects than either of the single mutants, 18W is not one of the additional upstream activators of RhoGEF2. Although neither present downstream of FOG nor upstream of RhoGEF2, 18W does appear to be positioned upstream of Sqh phosphorylation since the 18w salivary gland mutant phenotype can be rescued by introducing one copy of a phosphomimetic allele of sqh (Kolesnikov, 2007).
One possible way that 18W might activate Rho signaling is by negatively regulating RhoGAPs. Two RhoGAPs, RhoGAP5A and Cv-c, were identified that function partially redundantly during salivary gland morphogenesis. Embryos defective for both RhoGAPs exhibit invagination and migration defects similar to those observed when 18W is overexpressed within the salivary glands. Comparable defects are also seen upon expression of activated Rho, supporting the role of both RhoGAPs and 18W in Rho signaling (Kolesnikov, 2007).
Although overexpression and genetic interaction data demonstrate that 18W does indeed work in opposition to Cv-c activity, whether 18W actually negatively regulates RhoGAPs or if it controls Rho signaling through an alternate and unknown pathway has yet to to be deciphered. Another RhoGAP, RhoGAPp190, is regulated by the Src family of tyrosine kinases in both mammals and Drosophila. Depending on the site of phosphorylation, mammalian RhoGAPp190 can be either activated or inhibited by Src, while the Drosophila RhoGAPp190 appears to be only negatively regulated by the Drosophila Src homolog, Src64B. Genetic interactions and double mutant analysis with 18w and either Src64B or the other Drosophila Src gene, Src42A, however, suggest 18W does not regulate RhoGAPs via Src kinases in Drosophila (Kolesnikov, 2007).
Considering that 18W is a member of the Toll family of receptors, it might signal through the pathway used by Toll itself. Upon activation by its ligand, Spätzle, Toll signals via the cytoplasmic proteins MyD88, Tube, and Pelle to promote the degradation of the Cactus protein. This degradation releases the sequestered transcription factor Dorsal, allowing it to enter the nucleus and activate transcription. Although both 18w and Toll are expressed in the salivary gland, no evidence was found to suggest that 18W signals through the Toll-pathway or that it functions redundantly with Toll. Zygotic tube, pelle, MyD88, or Toll mutant embryos do not have salivary gland defects and MyD88 does not physically interact with any of the Toll-like receptors except for Toll itself. Similarly, there are no obvious genetic interactions between mutant alleles of 18w and Toll based both on lethality and salivary gland abnormalities (Kolesnikov, 2007).
Similar to the Toll family of receptors, many RhoGAPs and RhoGEFs are found in both mammals and flies. The Drosophila genome encodes 21 RhoGAPs and 20 RhoGEFs but only seven Rho-family GTPases. Since a specific RhoGTPase can be regulated by multiple RhoGAPs, there may be some redundancy in the function of the RhoGAPs. This appears to be the case during salivary gland development. Of the 17 RhoGAPs analyzed by RNAi, by available alleles, or by both, two resulted in distinct defects in the salivary glands. Mutant embryos that lack both of these RhoGAPs have more severe and penetrant gland defects than embryos that only lack one, indicating that the two have redundant roles during gland development (Kolesnikov, 2007).
Since 18W is expressed in several tissues undergoing morphogenesis, it will be interesting to establish whether it is important for the development of additional tissues other than the salivary gland. It will also be interesting to determine whether 18W functions in opposition to the particular RhoGAPs that are active within these other tissues. Overall, since very little is known about pathways controlling RhoGAP activity during apical constriction, identifying additional genes that interact with 18W may prove to be important not only in elucidating RhoGAP regulation but also in understanding the process of epithelial invagination (Kolesnikov, 2007).
After bacterial infection four novel forms of 18w transcripts are detected in addition to the expected 5.6 kb transcript. A 2.3 kb transcript is more rapidly induced than the others. Four novel 18W protein forms can be detected by Western analysis (Williams, 1997).
Bases in 5' UTR - 374
Bases in 3' UTR - 898
Two hydrophobic regions are apparent: one corresponding to a signal peptide and the second to a transmembrane domain. The putative extracellular region is unusally rich in leucine residues (17% of 971 amino acids) and contains 16 putative N-glycosylation sites and a single glycosaminoglycan site at amino acids 50-54 (Eldon, 1994).
There is a large domain with 22 consecutive 24-amino acid leucine-rich repeats (LRRs). In Drosophila, Toll, Slit, Chaoptin, Tartan and Connectin all contain these motifs. The 22 LRR repeats are terminated by a domain that contains cysteines. This domain is also found in a number of LRR-containing proteins: e.g. Slit, Toll, glycoprotein 1b and Glycoprotein IX. The C-terminal domain is followed by a second cysteine rich domain named the amino-flanking domain since it precedes the next run of LRRs. This domain is also found in Slit, GPIb, GPIX, Decorin, Biglycan, Fibromodulin (a collagen-binding protein) and oligodendrocyte-myelin glycoprotein. Finally, the extracellular moiety contains an additional five LRR followed by a carboxy flanking domain (Eldon, 1994 and Chiang, 1995).
The intracellular moiety of 18w is about 380 aa long, and consists of two domains. The first domain shows significant homology to the cytoplasmic domain of the interleukin-1-receptor. IL-1, Toll and 18w share six stretches of amino acids that are more conserved than the remaining amino acids. These regions probably correspond to functional domains since point mutations in some of these conserved regions lead to non-functional proteins. The second domain consists of a glutamine-rich OPA repeat (Eldon, 1994 and Chiang, 1995).
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. The molecular cloning of a class of putative human receptors is reported, with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors (TLRs 1-5) are probably the direct homologs of the fly molecule; as such, they 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, it is shown 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, MyD88 factors, and plant disease-resistance proteins recognizes a parallel/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 (Rock, 1998).
The 22- and 31-LRR ectodomains of Toll and 18 Wheeler, respectively, are most closely related to the comparable 18-, 19-, 24- and 22-LRR arrays of TLRs 1-4. However, a striking difference in the human TLR chains is the common loss of an approximately 90-residue cysteine-rich region that is variably embedded in the ectodomains of Toll and 18 Wheeler. These cysteine clusters are bipartite, with distinct top (ending in an LRR) and bottom (stacked atop an LRR) halves; the top module recurs in Drosophila and human TLRs as a conserved juxtamembrane spacer. It is suggested that the flexibly located cysteine clusters in Drosophila receptors (when mated top to bottom) form a compact module with paired termini that can be inserted between any pair of LRRs without altering the overall fold of TLR ectodomains (Rock, 1998)
date revised: 10 December 97
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