Gene name - Toll
Cytological map position - 97D1-2
Function - receptor
Symbol - Tl
Genetic map position - 3-91
Classification - IL-1 type receptor
Cellular location - surface
|Recent literature||Kanoh, H., Kuraishi, T., Tong, L. L., Watanabe, R., Nagata, S. and Kurata, S. (2015). Ex vivo genome-wide RNAi screening of the Drosophila Toll signaling pathway elicited by a larva-derived tissue extract. Biochem Biophys Res Commun 467: 400-406. PubMed ID: 26427875
Damage-associated molecular patterns (DAMPs), so-called "danger signals," play important roles in host defense and pathophysiology in mammals and insects. In Drosophila, the Toll pathway confers damage responses during bacterial infection and improper cell-fate control. However, the intrinsic ligands and signaling mechanisms that potentiate innate immune responses remain unknown. This study demonstrate that a Drosophila larva-derived tissue extract strongly elicits Toll pathway activation via the Toll receptor. Using this extract, an ex vivo genome-wide RNAi screening was performed in Drosophila cultured cells, and several signaling factors were identified that are required for host defense and antimicrobial-peptide expression in Drosophila adults. These results suggest that the larva-derived tissue extract contains active ingredients that mediate Toll pathway activation, and the screening data will shed light on the mechanisms of damage-related Toll pathway signaling in Drosophila.
|Liu, B., Zheng, Y., Yin, F., Yu, J., Silverman, N. and Pan, D. (2016). Toll receptor-mediated Hippo signaling controls innate immunity in Drosophila. Cell 164: 406-419. PubMed ID: 26824654
The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. This study reports an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. These results elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings.
|Capilla, A., Karachentsev, D., Patterson, R. A., Hermann, A., Juarez, M. T. and McGinnis, W. (2017). Toll pathway is required for wound-induced expression of barrier repair genes in the Drosophila epidermis. Proc Natl Acad Sci U S A. PubMed ID: 28289197
The epidermis serves as a protective barrier in animals. After epidermal injury, barrier repair requires activation of many wound response genes in epidermal cells surrounding wound sites. Two such genes in Drosophila encode the enzymes dopa decarboxylase (Ddc) and tyrosine hydroxylase (ple). This paper explores the involvement of the Toll/NF-kappaB pathway in the localized activation of wound repair genes around epidermal breaks. Robust activation of wound-induced transcription from ple and Ddc requires Toll pathway components ranging from the extracellular ligand Spatzle to the Dif transcription factor. Epistasis experiments indicate a requirement for Spatzle ligand downstream of hydrogen peroxide and protease function, both of which are known activators of wound-induced transcription. The localized activation of Toll a few cell diameters from wound edges is reminiscent of local activation of Toll in early embryonic ventral hypoderm, consistent with the hypothesis that the dorsal-ventral patterning function of Toll arose from the evolutionary cooption of a morphogen-responsive function in wound repair. Furthermore, the combinatorial activity of Toll and other signaling pathways in activating epidermal barrier repair genes can help explain why developmental activation of the Toll, ERK, or JNK pathways alone fail to activate wound repair loci.
|Coll, O., Guitart, T., Villalba, A., Papin, C., Simonelig, M. and Gebauer, F. (2018). Dicer-2 promotes mRNA activation through cytoplasmic polyadenylation. RNA 24(4):529-539. PubMed ID: 29317541
Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation. In vertebrates, this process requires two sequence elements in target 3' UTRs, the U-rich cytoplasmic polyadenylation element and the AAUAAA hexanucleotide. In Drosophila melanogaster, cytoplasmic polyadenylation of Toll mRNA occurs independently of these canonical elements and requires a machinery that remains to be characterized. This study identified Dicer-2 as a component of this machinery. Dicer-2, a factor previously involved in RNA interference (RNAi), interacts with the cytoplasmic poly(A) polymerase Wispy. Depletion of Dicer-2 from polyadenylation-competent embryo extracts and analysis of wispy mutants indicate that both factors are necessary for polyadenylation and translation of Toll mRNA. r2d2 mRNA, encoding a Dicer-2 partner in RNAi, was identified as a Dicer-2 polyadenylation target. These results uncover a novel function of Dicer-2 in activation of mRNA translation through cytoplasmic polyadenylation.
|Louradour, I., Sharma, A., Morin-Poulard, I., Letourneau, M., Vincent, A., Crozatier, M. and Vanzo, N. (2017). Reactive oxygen species-dependent Toll/NF-kappaB activation in the Drosophila hematopoietic niche confers resistance to wasp parasitism. Elife 6: e25496. PubMed ID: 29091025
Hematopoietic stem/progenitor cells in the adult mammalian bone marrow ensure blood cell renewal. Their cellular microenvironment, called 'niche', regulates hematopoiesis both under homeostatic and immune stress conditions. In the Drosophila hematopoietic organ, the lymph gland, the posterior signaling center (PSC) acts as a niche to regulate the hematopoietic response to immune stress such as wasp parasitism. This response relies on the differentiation of lamellocytes, a cryptic cell type, dedicated to pathogen encapsulation and killing. This study established that Toll/NF-kappaB pathway activation in the PSC in response to wasp parasitism non-cell autonomously induces the lymph gland immune response. The data further establish a regulatory network where co-activation of Toll/NF-kappaB and EGFR signaling by ROS levels in the PSC/niche controls lymph gland hematopoiesis under parasitism. Whether a similar regulatory network operates in mammals to control emergency hematopoiesis is an open question (Louradour, 2017).
|Papagianni, A., Fores, M., Shao, W., He, S., Koenecke, N., Andreu, M. J., Samper, N., Paroush, Z., Gonzalez-Crespo, S., Zeitlinger, J. and Jimenez, G. (2018). Capicua controls Toll/IL-1 signaling targets independently of RTK regulation. Proc Natl Acad Sci U S A 115(8): 1807-1812. PubMed ID: 29432195
The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. This study characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-kappaB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, these results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.
Toll is a transmembrane receptor and a member of the 12 gene dorsal group responsible for dorsoventral polarity in the fly. The ligand for Toll is Spätzle, and immediate targets include Pelle, Tube, Dorsal and Cactus. These proteins are held in a state of readiness while in the unfertilized egg. They are primed to carry out the transition from egg to zygote after fertilization occurs. Spätzle is available only in the ventral portion of the egg in the extracellular perivitelline space; when subjected to proteolysis, Spätzle becomes an active ligand for Toll.
Toll signals are first picked up by Tube, a protein in contact with the cell membrane. The signal is transduced to Pelle and subsequently to Cactus, which until its destruction holds Dorsal in the cytoplasm. With Cactus's bond destroyed, Dorsal enters the nucleus where it can serve as activator and repressor of genes involved in dorso-ventral polarity.
Toll is required zygotically in the development of a number of tissues, but Spätzle has not been documented as the ligand in these circumstances. Toll's mammalian homolog is Interleukin-1 (IL-1) receptor, involved in the activation of the immune response. Similarly, the Toll-Cactus-Dorsal system in the fly also activates an immune response.
The extracellular region of the Toll protein does not bear any similarity to the extracellular ligand-binding portion of the IL-1 receptor. Instead, it carries leucine rich repeats (LRR) that are found in molecules as diverse as proteoglycans, adhesion molecules, enzymes, tyrosine kinase receptors and G-protein coupled receptors. LRRs in the fly are found in Toll, Chaoptin and Connectin adhesion molecules and the Slit secreted protein. All these have roles in cell differentiation, morphogenetic events and the migration of cells and axons (Ollendorff, 1994).
The localized activation of Toll was first suggested based on the results of injection of wild-type cytoplasm into mutant flies. Wherever the Toll rescuing activity occurs defines the region from which ventral structures arise. Rescuing activity is not localized ventrally, but distributed uniformly in the wild-type embryo. This implies that the Toll gene product is normally present throughout the embryo but its activity is somehow restricted to ventral regions (Anderson, 1985 and Hashimoto, 1991). It is now understood that the ventral stimulation of Toll is caused by Spätzle, activated only on the ventral side of the egg (Morisato, 1994 and Roth, 1994).
Toll is dynamically expressed later in development by the embryonic musculature. Growth cones of RP3 and other motoneurons normally grow past muscle cells expressing Toll on their surface and innervate more distal muscle cells (muscles 6 and 7), which have down-regulated their Toll expression. The RP3 growth cone likely responds positively to Fasciclin III, an Ig-like cell adhesion molecule expressed on the target muscle cells, but still manages to avoid targeting errors in embryos lacking Fas III. Toll protein preferentially accumulates at muscle-muscle contact sites or "clefts," (the apposition between muscle cells). Later, the Toll-positive ventral muscle cells gradually lose Toll. Late-arriving growth cones innervate the clefts just as Toll expression becomes undetectable (Rose, 1997).
Reciprocal genetic manipulations of Toll proteins can produce reciprocal RP3 phenotypes. In Toll null mutants, the RP3 growth cone sometimes innervates the wrong muscle cells, including those that are normally Toll-positive. In contrast, heterochronic misexpression of Toll in the musculature leads to the same growth cone reaching its correct target region but delaying synaptic initiation. It is proposed that Toll acts locally to inhibit synaptogenesis of specific motoneuron growth cones and that both temporal and spatial control of Toll expression is crucial for its role in development (Rose, 1997).
The LRR (leucine-rich repeats) motif is shared by a number of other Drosophila cell surface molecules: Connectin, Chaoptin, 18-wheeler, Kekkon and Toll-like, as well as mammalian neural receptors, such as NLRR-1 and GARP. Structural analyses indicate that the LRR motifs could mediate protein-lipid as well as homophilic and heterophilic protein-protein interactions. The structurally related Connectin protein, when ectopically expressed in some of the ventral muscle cells, can function as a repulsive signal to motoneuron growth cone. All this evidence suggests a pivotal role for LRR proteins in axon guidance (Rose, 1997 and references).
Little is known about the genetic program that generates synaptic specificity. This study shows that a putative transcription factor, Teyrha-Meyhra (Tey), controls target specificity, in part by repressing the expression of a repulsive cue, Toll. This study focused on two neighboring muscles, M12 and M13, which are innervated by distinct motoneurons in Drosophila. It was found that Toll, which encodes a transmembrane protein with leucine-rich repeats, is preferentially expressed in M13. In Toll mutants, motoneurons that normally innervate M12 (MN12s) form smaller synapses on M12 and instead appear to form ectopic nerve endings on M13. Conversely, ectopic expression of Toll in M12 inhibited synapse formation by MN12s. These results suggest that Toll functions in M13 to prevent synapse formation by MN12s. Tey was identified as a negative regulator of Toll expression in M12. In tey mutants, Toll is strongly upregulated in M12. Accordingly, synapse formation on M12 was inhibited. Conversely, ectopic expression of tey in M13 decreased the amount of Toll expression in M13 and changed the pattern of motor innervation to the one seen in Toll mutants. These results suggest that Tey, which contains no known transcription factor motifs, determines target specificity by repressing the expression of Toll. These results reveal a mechanism for generating synaptic specificity that relies on the negative regulation of a repulsive target cue (Inaki, 2010).
A remarkable feature of the nervous system is the precision of its circuitry. A neural circuit develops through a series of neuronal recognition events. First, neurons find their path, turn at mid-way guideposts, and fasciculate or defasciculate before reaching their final target area. Then, neurons select and form synapses with specific target cells in the target region. The final matching of pre- and post-synapses is thought to be mediated by specific cues expressed on the target cells. However, the regulation and function of such cues remain poorly understood (Inaki, 2010).
The process of neuromuscular targeting in Drosophila features highly stereotypic matchings between 37 motoneurons and 30 target muscle cells, providing a unique model system for the study of neuronal target recognition. Several target cues, including Capricious, Netrin-B and Fasciclin 3, have been identified that are expressed in specific target cells and mediate attractive interactions between the synaptic partners. It has recently been shown that target specificity is also regulated by repulsion from non-target cells. Wnt4, a member of the Wnt family of secreted glycoproteins, is expressed in muscle 13 (M13) and prevents synapse formation by motoneurons targeted to a neighboring muscle, M12. In the absence of Wnt4, motoneurons targeted to M12 form ectopic nerve endings on M13, indicating that Wnt4 repulsion on M13 is required for proper targeting of the motoneurons. In addition to Wnt4, Toll and Semaphorin II (Sema-2a - FlyBase) are known to function as negative regulators of synapse formation in this system. However, whether they have a role in target selection remains unknown (Inaki, 2010).
Another unsolved issue is how the expression of such attractive or repulsive target-recognition molecules is regulated. It is amazing that the expression of these molecules is so precisely regulated that they are present at the right time and place. It is likely that the expression of these molecules is determined as part of the differentiation program of the target cells. However, little is known about the molecules and mechanisms involved. Several transcription factors, such as S59, Krüppel and Vestigial, have been identified as being expressed in subsets of muscle cells. They are expressed from the progenitor stage, and their loss-of-function (LOF) and gain-of-function (GOF) alter the specific characteristics of the individual muscles, such as their size, shape, orientation and attachment sites to the epidermis, indicating that they function as determinants of a particular muscle fate. However, whether these transcription factors regulate the expression of target-recognition molecules and thus determine the innervation pattern is unknown (Inaki, 2010).
A comparative microarray analysis has been conducted of two neighboring target muscles, M12 and M13, that are innervated by distinct motoneurons (Inaki, 2007). By comparing the expression profile of the two muscles, attempts were made to understand the molecular mechanisms that make these muscles distinct targets for the motoneurons. From this screening, ~25 potential target-recognition molecules were identified as preferentially expressed in either muscle cell. Among them was Wnt4, mentioned above. This study reports the functional analyses of two additional genes that were identified in the screening: Toll and teyrha-meyrha (tey). Toll encodes a transmembrane protein with extracellular leucine-rich repeats, and has multiple functions in development. Toll is expressed in subsets of muscles, including 6, 7 and 15-17. Previous studies have shown that Toll inhibits synapse formation by RP3, a motoneuron targeted to muscles 6 and 7. This study shows that Toll is preferentially expressed in M13 over M12 and, like Wnt4, inhibits synapse formation by motoneurons targeted to M12. It was also shown that tey, a previously uncharacterized gene, regulates the expression of Toll in specific muscles. tey is expressed specifically in M12, where it negatively regulates Toll expression. In the absence of tey, Toll is ectopically expressed in M12 and innervation of M12 is inhibited. These results suggest that Tey regulates targeting by downregulation of the repulsive cue Toll specifically in M12. Based on these results, a mechanism is proposed for the generation of synaptic specificity that relies on negative regulation of repulsive target cues (Inaki, 2010).
Toll is preferentially expressed in M13 over M12. The size of M12 terminals was decreased in Toll mutants, with concomitant expansion of M13 terminals. This phenotype is very similar to that of Wnt4 LOF and is likely to be caused by MN12s forming ectopic synapses with M13, although it remains possible that some of the ectopic nerve endings on M13 are formed by other motoneurons. Furthermore, it was observed that the size of M12 terminals is reduced when Toll is misexpressed on the muscle. The LOF and GOF analyses suggest that Toll functions as a repulsive factor in M13 that is important for target selection by MN12s. Thus, Toll provides another example of a repulsive factor that is involved in target selection. How Toll mediates the repulsive signal to motoneurons is currently unknown. A model is that Toll functions as a ligand that is expressed in muscles and signals through receptor(s) expressed in motoneurons. However, no receptor has been identified for Toll. Toll has been shown to function as a receptor, not a ligand, in other systems, such as in dorsoventral patterning and innate immunity. Another possibility is that Toll might mediate the modification or regulation of other targeting molecules, such as Wnt4 (Inaki, 2010).
M13 expresses at least two repulsive cues, Wnt4 and Toll, that are important for the targeting of M12 and M13. It seems that these two molecules contribute to target specificity in a manner that is redundant with yet other molecules because in both single and double mutants of these genes, the connectivity is only partially disrupted. Previously, other potential repulsive cues that are expressed in M13 were identified, including Beat-IIIc and Glutactin (Inaki, 2007). Ectopic expression of these molecules in M12 inhibits synapse formation by MN12s, as observed when Toll and Wnt4 are misexpressed. Although the precise roles of these molecules remain to be verified by LOF analyses, these results suggest the possibility that a single muscle, M13, expresses a number of repulsive cues that are involved in targeting of motoneurons. This is consistent with previous hypotheses that Drosophila neuromuscular connectivity is determined by highly redundant mechanisms. It will be important to determine how the signals from multiple cues are integrated to generate the precise pattern of synaptic connections. It will also be interesting to examine whether other muscles similarly express repulsive cues to prevent inappropriate innervation. The phenotypes of Wnt4 Toll double mutants were of similar severity to those of the single mutants. This might be due to the presence of other targeting molecules, as described above. Toll and Wnt4 might also function in the same signaling pathway. For example, Toll may be involved in the regulation of Wnt4 activity through influencing its secretion, localization or protein modification. Toll and Wnt4 might also act as repellents for distinct MN12s (Inaki, 2010).
This study has shown that a novel nuclear protein, Tey, regulates the expression of Toll and is important for the determination of target specificity. tey regulates the position, orientation and attachment sites of M12. Thus, Tey seems to act as a determinant of several important properties of M12, regulating both the differentiation of the muscle itself and the specificity of nerve innervation. Expression of tey is remarkably specific, being limited within the somatic musculature to a single muscle, M12. Other, known muscle-determinant genes were expressed in broader subsets of muscles (Inaki, 2010).
Tey negatively regulates the expression of Toll in M12. In tey mutants, Toll expression is strongly upregulated in M12. This indicates that tey is required in this muscle to specifically suppress Toll expression. Consistent with this, ectopic expression of tey in M13 partially suppressed Toll expression. Toll is normally expressed in most of the other ventral muscles, including muscles 6, 7, 13-17, but not in M12, suggesting that some positive transcriptional regulator(s) higher up in the hierarchy activate Toll expression in this group of muscles and that negative regulation by Tey is required to suppress Toll expression only in M12. The regulation of Toll by Tey should be at the transcriptional level because the expression of the exogenously introduced Toll enhancer-trap lacZ reporter is affected in tey mutants or when tey is misexpressed. It remains to be determined whether Tey binds directly to the regulatory region of the Toll gene or regulates Toll transcription in an indirect manner (e.g. by regulating other transcription factors). Tey contains no known transcription factor motifs. The expression of another M13-enriched gene, Wnt4, was not affected in tey mutants or when tey was misexpressed. Unlike Toll, Wnt4 is expressed in only two ventral muscles: 13 and 30. Thus, expression of Wnt4 might be regulated in a different manner to Toll, possibly by positive transcription factors that are specifically expressed in these muscles. It will be interesting to determine how the expression of target-recognition molecules is precisely regulated by the combinatorial action of positive and negative transcription factors (Inaki, 2010).
In tey mutants or when tey is misexpressed, neuromuscular connectivity was also altered in a manner consistent with the misregulation of Toll expression. The inappropriate presence of Toll repulsion in tey LOF mutants suppressed synapse formation on M12. Conversely, suppression of Toll expression in M13 in tey GOF mutants led to changes in the innervations of M12 and M13, similar to those observed in Toll mutants. Furthermore, the effects of tey GOF were dramatically reversed when Toll was co-expressed with tey, suggesting that Toll is the major target of tey in causing the GOF phenotypes. These results suggest that Tey regulates neuromuscular connectivity by specifically repressing Toll expression in M12. As noted above, Toll is normally expressed in a number of ventral muscles, but not in M12. Furthermore, Toll is expressed in M12 in the absence of Tey suppression in tey mutants. This suggests that the default state is for Toll to be expressed in all ventral muscles, possibly by the action of positive transcription factor(s) expressed in these muscles. Tey might therefore generate target specificity by suppressing the expression of Toll in one among a group of muscle cells expressing the repulsive cue. The data thus suggest a mechanism of transcriptional control of target specificity, namely, the negative regulation of repulsive cues (Inaki, 2010).
β-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. This study reports a new in vivo function of the Drosophila β-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-κB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. These findings uncover a new in vivo function of β-arrestins and present a new mechanism of ERK inhibition by the Drosophila β-arrestin Krz (Tipping, 2010).
This study demonstrate that the Krz protein is necessary for setting a precise level of activation of two maternal signalling pathways, Torso and Toll. This activity of Krz helps to establish the correct domains of expression of developmental patterning regulators that are under the control of these pathways (Tipping, 2010).
Genetic and protein interaction data suggest a new mechanism by which Krz may limit the activity of Torso. It was observed that Krz preferentially binds and sequesters an inactive form of ERK, thereby making it unavailable for activation by the upstream kinases such as MEK. Such a mechanism of direct inhibition of ERK activation by β-arrestin binding has not been previously reported. This mechanism is consistent with the observed in vivo effects of loss of krz on ERK activity. In krz maternal mutant embryos, ERK is not sequestered and therefore more ERK is available to transduce Torso signals, resulting in hyperactivation of Torso target genes, tll and hkb. Furthermore, consistent with this model is the observation that Krz and MEK apparently compete for ERK when all three proteins are co-expressed in S2 cells (Tipping, 2010).
Interaction assays using mutated forms of Krz and ERK indicate that the conformations of both proteins have an effect on their binding affinity. On binding to an activated GPCR, the arrestin molecule undergoes a dramatic conformational change that can be mimicked by specific mutations (Gurevich, 2004). In immunoprecipitation experiments it was observed that such 'pre-activated' form of Krz (R209E) has a much greater affinity for ERK, compared with the wild-type Krz protein, and that this higher affinity is also observed for the equivalent mutant of human β-arrestin2. This suggests that the ERK-binding ability of β-arrestin may be affected by its conformation, but it is unknown at present whether any upstream signals convert Krz into an activated form in the embryo. Overexpression of Krz-R209E using the da-GAL4 driver did not result in any observable phenotype and could rescue zygotic loss of krz, suggesting that it retains most of the functions of wild-type Krz (data not shown) (Tipping, 2010).
It was observed that the conformation of ERK itself has a large effect on its interactions with Krz. In the binding experiments, activated forms of ERK bind Krz (and human β-arrestin2) with lower affinity, compared with wild-type inactive ERK. Moreover, mutations in the TEY motif, which render ERK constitutively inactive, also lower its affinity for Krz, which is at a first glance a surprising result. However, previous studies have shown that both types of mutations in the TEY motif, which is a part of the activation loop, increase disorder in the lip region and cause a conformational change in the ERK molecule that makes it different from the basal state. It is therefore speculated that the activation loop may be involved in mediating an interaction of ERK with β-arrestin. Consistent with the current results, deviation of ERK structure from the basal state would decrease its association with β-arrestin (Tipping, 2010).
Other studies have reported formation of protein complexes containing β-arrestins and an activated form of ERK. It is possible that in those experimental conditions other binding partners, such as Raf or the activated receptor, assist in stabilizing the complex of MAP kinases with β-arrestin. This study has shown that although Krz can bind to the Drosophila homologues of both MEK and Raf, overexpression of Krz does not increase production of dpERK by the MAPK cascade downstream of activated RTKs, but instead appreciably inhibits it in the absence of overexpressed Raf. The data do not rule out a possibility that Krz may still promote ERK activation in other biological contexts, particularly downstream of activated GPCRs, but this question awaits further investigation (Tipping, 2010).
Interestingly, the sequestration mechanism of ERK inhibition described in this study is different from the effects of Krz on Notch. Previous studies have shown that Krz inhibits Notch activity by forming a ternary complex with Deltex and the Notch receptor. Formation of this complex increases Notch turnover and thereby downregulates Notch signalling (Mukherjee, 2005). No change was observed in ERK turnover in the presence of wild-type overexpressed Krz, suggesting that Krz is unlikely to be involved in the regulation of ERK stability. However, given the versatility of molecular functions displayed by β-arrestins, it is possible that there are other, as yet uncharacterized mechanisms by which Krz controls signalling downstream of RTKs (Tipping, 2010).
The inhibitory effects of Krz on ERK activity are not limited to the Torso pathway and early embryogenesis, but are also observed in other tissues and at later developmental stages. Thus, broadening of the dpERK patterns activated by EGFR and Btl was observed in krz maternal mutant embryos. An increase in the overall levels of dpERK during mid-to-late embryogenesis was also detected on western blots. Later in development, ERK is activated by EGFR in the wing and both EGFR and Sevenless in the eye. Genetic data suggest that Krz also inhibits ERK activity in these tissues during larval development. A broad involvement of Krz in inhibiting ERK activity suggests that Krz has a general inhibitory role to limit the activity of different RTKs in Drosophila development (Tipping, 2010).
In addition to its effects on RTK signalling, it was observed that Krz has an important role in limiting the activity of the Toll receptor, which specifies the development of the ventral structures. Other studies have reported that mammalian β-arrestins can downregulate NF-κB signalling by binding and stabilizing the NF-κB inhibitor IκBα. The inhibitory effects of Krz on Dorsal may involve a similar mechanism. It was observed that Krz can directly bind to the Drosophila orthologue of IκBα, Cactus, suggesting that the mechanism of NF-κB inhibition by β-arrestins at the level of IκBα may be conserved. Consistent with this finding, a decrease was detected in the level of the Cactus protein in krz maternal mutants at 0-4 h of development, which may explain the observed expansion of the nuclear gradient of Dorsal in these mutants. It is still unclear why expansion of Dorsal nuclear localization is more pronounced in the posterior half of the embryo (Tipping, 2010).
In the developing embryo, the Torso and Toll pathways do not work in isolation, but are involved in cross-regulatory interactions on certain common targets, such as zen. zen is repressed by nuclear Dorsal in the ventral part of the embryo, and relieved of this repression (de-repressed) by the signalling activity of Torso emanating from the embryo poles. The molecular mechanism of this de-repression is still unknown. It was observed that loss of krz shifts the balance of the effects of Torso on Toll, which results in an inappropriate expansion of zen expression at the embryo poles. It is speculated that Krz helps Torso to achieve a precise level of de-repression of zen by limiting the activity of ERK. Krz is thus able to control the separate activities of the Torso and Toll pathways (reflected in its effects on tll, hkb, twi, and rho), as well as regulate common Torso and Toll targets such as zen. For such pathways that are engaged in cross-regulatory interactions, Krz ensures that a proper level of signalling activity from one pathway reaches the other. This function adds an important new mechanism to understanding of the ways in which signalling pathways are coordinately regulated during development (Tipping, 2010).
A ubiquitous distribution of Krz in the embryo agrees with the dysregulation of multiple pathways observed in krz mutant animals. As overexpression of Krz does not cause any obvious defects, the level of Krz itself is not limiting for the regulation of signalling. Instead, Krz apparently makes other signalling co-factors limiting for their respective pathways, essentially working as a molecular 'sponge' to prevent pathway hyperactivity. Specificity of Krz function is likely to be determined by its selective interactions with specific pathway co-factors. Maternal loss of krz function thus affects multiple developmental signalling pathways, resulting in an accumulation of defects that ultimately lead to severe morphological abnormalities such as a disruption of gastrulation movements. By analysing the effects of loss of krz on individual pathways in vivo, this study has been able to show its role in the regulation of RTK and Toll signalling. Future studies will likely reveal other pathways and levels of regulation that are under the control of the Drosophila β-arrestin Krz (Tipping, 2010).
Bases in 5' UTR - 574
Bases in 3' UTR - 1257
A 5.3 kb poly(A)+ ovarian transcript of Toll was purified by hybrid selection with cloned DNA. The sequence of cDNAs suggests that the Toll protein is an integral membrane protein with a cytoplasmic domain and a large extracytoplasmic domain. The putative extracytoplasmic domain contains two blocks (for a total of 15 repeats) of a 24 amino acid, leucine-rich sequence found in both human and yeast membrane proteins. The transmembrane domain is between residues 804 and 828. There are 17 potential glycosylation sites and 17 cysteine residues in 3 clusters (Hashimoto, 1988).
The Toll protein has sequences held in common with the human membrane receptor platelet glycoprotein 1b (Gp1b). These sequences in Toll form disulphide linked extracellular domains that are important for the binding of ligands in the perivitelline space of the embryo. Expression of Toll protein induced in a non-adhesive cell line promotes cellular adhesion, a property held in common with the related Drosophila glycoprotein Chaoptin. Toll protein in such aggregates accumulates at sites of cell-cell interaction, a characteristic displayed by other cellular adhesion molecules (Keith, 1990).
Unusual properties are found for a synthetic LRR peptide derived from the sequence of the Drosophila membrane receptor Toll. In neutral solution the peptide forms a gel revealed by electron microscopy to consist of extended filaments approximately 8 nm in thickness. As the gel forms, the circular dichroism spectrum of the peptide solution changes from one characteristic of random coil to one associated with beta-sheet structures. Molecular modelling suggests that the peptide forms an amphipathic structure with a predominantly apolar and charged surface. Based on these results, models for the gross structure of the peptides filaments and a possible molecular mechanism for cellular adhesion are proposed. The finding that Toll-LRR forms intramolecular ß-sheet structures supports the view that LRRs can participate in protein-protein interactions and homotypic cellular adhesion. It could be that LRRs expressed on the cell surface are initially of disordered structure and that interactions with similarly disordered LRRs on an adjacent cell causes the formation of an extended and stable intermolecular ß structure. Such a mechansim could provide a molecular basis for cellular adhesion mediated by LRRs (Gay, 1991).
date revised: 3 July 97
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