Signal-dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate without activation of phospholipase C: implications on gating of Drosophila TRPL (transient receptor potential-like) channel

In Drosophila, a phospholipase C (PLC)-mediated signaling cascade, couples photo-excitation of rhodopsin to the opening of the transient receptor potential (TRP) and TRP-like (TRPL) channels. A lipid product of PLC, diacylglycerol (DAG), and its metabolites, polyunsaturated fatty acids (PUFAs) may function as second messengers of channel activation. However, how can one separate between the increase in putative second messengers, change in pH, and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) depletion when exploring the TRPL gating mechanism? To answer this question we co-expressed the TRPL channels together with the muscarinic (M1) receptor, enabling the openings of TRPL channels via G-protein activation of PLC. To dissect PLC activation of TRPL into its molecular components, we used a powerful method that reduced plasma membrane-associated PI(4,5)P₂ in HEK cells within seconds without activating PLC. Upon the addition of a dimerizing drug, PI(4,5)P₂ was selectively hydrolyzed in the cell membrane without producing DAG, inositol trisphosphate, or calcium signals. We show that PI(4,5)P₂ is not an inhibitor of TRPL channel activation. PI(4,5)P₂ hydrolysis combined with either acidification or application of DAG analogs failed to activate the channels, whereas PUFA did activate the channels. Moreover, a reduction in PI(4,5)P₂ levels or inhibition of DAG lipase during PLC activity suppressed the PLC-activated TRPL current. This suggests that PI(4,5)P₂ is a crucial substrate for PLC-mediated activation of the channels, whereas PUFA may function as the channel activator. Together, this study defines a narrow range of possible mechanisms for TRPL gating.     

Mutation of Light-dependent Phosphorylation Sites of the Drosophila Transient Receptor Potential-like (TRPL) Ion Channel Affects Its Subcellular Localization and Stability

The Drosophila phototransduction cascade terminates in the opening of the ion channel transient receptor potential (TRP) and TRP-like (TRPL). Contrary to TRP, TRPL undergoes light-dependent subcellular trafficking between rhabdomeric photoreceptor membranes and an intracellular storage compartment, resulting in long term light adaptation. Here, we identified in vivo phosphorylation sites of TRPL that affect TRPL stability and localization. Quantitative mass spectrometry revealed a light-dependent change in the TRPL phosphorylation pattern. Mutation of eight C-terminal phosphorylation sites neither affected multimerization of the channels nor the electrophysiological response of flies expressing the mutated channels. However, these mutations resulted in mislocalization and enhanced degradation of TRPL after prolonged dark-adaptation. Mutation of subsets of the eight C-terminal phosphorylation sites also led to a reduction of TRPL content and partial mislocalization in the dark. This suggests that a light-dependent switch in the phosphorylation pattern of the TRPL channel mediates stable expression of TRPL in the rhabdomeres upon prolonged dark-adaptation.     

Agonist- and Ca2+-dependent desensitization of TRPV1 channel targets the receptor to lysosomes for degradation

TRPV1 receptor agonists such as the vanilloid capsaicin and the potent analog resiniferatoxin are well known potent analgesics. Depending on the vanilloid, dose, and administration site, nociceptor refractoriness may last from minutes up to months, suggesting the contribution of different cellular mechanisms ranging from channel receptor desensitization to Ca(2+) cytotoxicity of TRPV1-expressing neurons. The molecular mechanisms underlying agonist-induced TRPV1 desensitization and/or tachyphylaxis are still incompletely understood. Here, we report that prolonged exposure of TRPV1 to agonists induces rapid receptor endocytosis and lysosomal degradation in both sensory neurons and recombinant systems. Agonist-induced receptor internalization followed a clathrin- and dynamin-independent endocytic route, triggered by TRPV1 channel activation and Ca(2+) influx through the receptor. This process appears strongly modulated by PKA-dependent phosphorylation. Taken together, these findings indicate that TRPV1 agonists induce long-term receptor down-regulation by modulating the expression level of the channel through a mechanism that promotes receptor endocytosis and degradation and lend support to the notion that cAMP signaling sensitizes nociceptors through several mechanisms.     

C-terminal acidic cluster is involved in Ca2+-induced regulation of human transient receptor potential ankyrin 1 channel

The transient receptor potential ankyrin 1 (TRPA1) channel is a Ca(2+)-permeable cation channel whose activation results from a complex synergy between distinct activation sites, one of which is especially important for determining its sensitivity to chemical, voltage and cold stimuli. From the cytoplasmic side, TRPA1 is critically regulated by Ca(2+) ions, and this mechanism represents a self-modulating feedback loop that first augments and then inhibits the initial activation. We investigated the contribution of the cluster of acidic residues in the distal C terminus of TRPA1 in these processes using mutagenesis, whole cell electrophysiology, and molecular dynamics simulations and found that the neutralization of four conserved residues, namely Glu(1077) and Asp(1080)-Asp(1082) in human TRPA1, had strong effects on the Ca(2+)- and voltage-dependent potentiation and/or inactivation of agonist-induced responses. The surprising finding was that truncation of the C terminus by only 20 residues selectively slowed down the Ca(2+)-dependent inactivation 2.9-fold without affecting other functional parameters. Our findings identify the conserved acidic motif in the C terminus that is actively involved in TRPA1 regulation by Ca(2+).     

Role of the transient receptor potential vanilloid 5 (TRPV5) protein N terminus in channel activity, tetramerization, and trafficking

The epithelial Ca(2+) channel transient receptor potential vanilloid 5 (TRPV5) constitutes the apical entry site for active Ca(2+) reabsorption in the kidney. The TRPV5 channel is a member of the TRP family of cation channels, which are composed of four subunits together forming a central pore. Regulation of channel activity is tightly controlled by the intracellular N and C termini. The TRPV5 C terminus regulates channel activity by various mechanisms, but knowledge regarding the role of the N terminus remains scarce. To study the role of the N terminus in TRPV5 regulation, we generated different N-terminal deletion constructs. We found that deletion of the first 32 residues did not affect TRPV5-mediated (45)Ca(2+) uptake, whereas deletion up to residue 34 and 75 abolished channel function. Immunocytochemistry demonstrated that these mutant channels were retained in the endoplasmic reticulum and in contrast to wild-type TRPV5 did not reach the Golgi apparatus, explaining the lack of complex glycosylation of the mutants. A limited amount of mutant channels escaped the endoplasmic reticulum and reached the plasma membrane, as shown by cell surface biotinylation. These channels did not internalize, explaining the reduced but significant amount of these mutant channels at the plasma membrane. Wild-type TRPV5 channels, despite significant plasma membrane internalization, showed higher plasma membrane levels compared with the mutant channels. The assembly into tetramers was not affected by the N-terminal deletions. Thus, the N-terminal residues 34-75 are critical in the formation of a functional TRPV5 channel because the deletion mutants were present at the plasma membrane as tetramers, but lacked channel activity.     

Activation of the Drosophila TRP and TRPL channels requires both Ca2+ and protein dephosphorylation

The Transient Receptor Potential (TRP) proteins constitute a large and diverse family of channel proteins, which is conserved through evolution. TRP channel proteins have critical functions in many tissues and cell types, but their gating mechanism is an enigma. In the present study patch-clamp whole-cell recordings was applied to measure the TRP- and TRP-like (TRPL)-dependent currents in isolated Drosophila ommatidia. Also, voltage responses to light and to metabolic stress were recorded from the eye in vivo. We report new insight into the gating of the Drosophila light-sensitive TRP and TRPL channels, by which both Ca2+ and protein dephosphorylation are required for channel activation. ATP depletion or inhibition of protein kinase C activated the TRP channels, while photo-release of caged ATP or application of phorbol ester antagonized channels openings in the dark. Furthermore, Mg(2+)-dependent stable phosphorylation event by ATPgammaS or protein phosphatase inhibition by calyculin A abolished activation of the TRP and TRPL channels. While a high reduction of cellular Ca2+ abolished channel activation, subsequent application of Ca2+ combined with ATP depletion induced a robust dark current that was reminiscent of light responses. The results suggest that the combined action of Ca2+ and protein dephosphorylation activate the TRP and TRPL channels, while protein phosphorylation by PKC antagonized channels openings.     

Allosteric modulation of Ca2+ flux in ligand-gated cation channel (P2X4) by actions on lateral portals

Human P2X receptors are a family of seven ATP-gated ion channels that transport Na(+), K(+), and Ca(2+) across cell surface membranes. The P2X4 receptor is unique among family members in its sensitivity to the macrocyclic lactone, ivermectin, which allosterically modulates both ion conduction and channel gating. In this paper we show that removing the fixed negative charge of a single acidic amino acid (Glu(51)) in the lateral entrance to the transmembrane pore markedly attenuates the effect of ivermectin on Ca(2+) current and channel gating. Ca(2+) entry through P2X4 receptors is known to trigger downstream signaling pathways in microglia. Our experiments show that the lateral portals could present a novel target for drugs in the treatment of microglia-associated disease including neuropathic pain.     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Elevated levels of expression associated with regions of the Drosophila genome that lack crossing over

The recombinational environment influences patterns of molecular evolution through the effects of Hill-Robertson interference. Here, we examine genome-wide patterns of gene expression with respect to recombinational environment in Drosophila melanogaster. We find that regions of the genome lacking crossing over exhibit elevated levels of expression, and this is most pronounced for genes on the entirely non-crossing over fourth chromosome. We find no evidence for differences in the patterns of gene expression between regions of high, intermediate and low crossover frequencies. These results suggest that, in the absence of crossing over, selection to maintain control of expression may be compromised, perhaps due to the accumulation of deleterious mutations in regulatory regions. Alternatively, higher gene expression may be evolving to compensate for defective protein products or reduced translational efficiency.     

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.     

Interference of human and Drosophila APP and APP-like proteins with PNS development in Drosophila

The view that only the production and deposition of Abeta plays a decisive role in Alzheimer's disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, we use transgenic Drosophila as a model. Upon overexpression of the APP-family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain-of-function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP-family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP-family members in neuronal development and a crosstalk between the APP family and Notch.     

Molecular cloning and functional characterization of Xenopus tropicalis frog transient receptor potential vanilloid 1 reveal its functional evolution for heat, acid, and capsaicin sensitivities in terrestrial vertebrates

The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca(2+)](i)). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ～60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca(2+)](i) increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function.     

Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene

MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.     

Properties of the intracellular transient receptor potential (TRP) channel in yeast, Yvc1

Transient receptor potential (TRP) channels are found among mammals, flies, worms, ciliates, Chlamydomonas, and yeast but are absent in plants. These channels are believed to be tetramers of proteins containing six transmembrane domains (TMs). Their primary structures are diverse with sequence similarities only in some short amino acid sequence motifs mainly within sequences covering TM5, TM6, and adjacent domains. In the yeast genome, there is one gene encoding a TRP-like sequence. This protein forms an ion channel in the vacuolar membrane and is therefore called Yvc1 for yeast vacuolar conductance 1. In the following we summarize its prominent features.     

Effects of intracellular Ca2+ chelation on the light response in Drosophila photoreceptors

In order to test the hypothesis that excitation in Drosophila photoreceptors is mediated by Ca²⁺ released from internal stores, the Ca²⁺ buffers EGTA, BAPTA and di-bromo-BAPTA (DBB) were introduced into dissociated photoreceptors via whole-cell recording pipettes. All buffers were preloaded with Ca²⁺ to provide the same free Ca²⁺ concentration (250 nM). EGTA (up to 18 mM free buffer) had only weak effects upon voltage-clamped flash responses in normal Ringer's solution (1.5 mM Ca ₀ ²⁺ ), and no effect in Ca²⁺-free solution. The maximum BAPTA concentration tested (14.4 mM free BAPTA) reduced the initial rate of rise by ca. 5000-fold in normal Ringer's solution; by ca. 500-fold in Ca²⁺free solution; and only ca. 60-fold in the absence of Mg²⁺, which preferentially blocks one component of the light-sensitive current. Although BAPTA delayed the time-to-peak in normal Ringer's solution, responses in Ca²⁺ free Ringer's solution were accelerated. These results support the role of Ca²⁺ influx in regulating sensitivity and response kinetics; however, in view of the high concentrations required to attenuate responses in Ca²⁺ free Ringer's solution, the role of Ca²⁺ release in excitation remains unclear. DBB was ca. 2–3 fold more potent than BAPTA, and at concentrations > 5 mM had a qualitatively different action, greatly delaying the time-to-peak. This suggests DBB may have distinct pharmacological actions or access to compartments inaccessible to BAPTA.The only current activated by introducing 5–500 M Ca²⁺ (buffered with nitrilo-triacetic acid) was electrogenic Na⁺/Ca²⁺ exchange. When this was blocked by removing Nao ₀ ⁺ , a novel cationic conductance was activated. However, its properties did not resemble those the light-activated conductance, and thus do not support the hypothesis that Ca²⁺ is sufficient for excitation.Abbreviations BAPTA bis-(o-aminophenoxy)-ethane-N,N,N-tetracetic acid - DBB Di-bromo-bapta - NTA nitrilo-triacetic acid - InsP ₃ inositol 1,4,5-trisphosphate     

The Drosophila juvenile hormone receptor candidates methoprene-tolerant (MET) and germ cell-expressed (GCE) utilize a conserved LIXXL motif to bind the FTZ-F1 nuclear receptor

Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains controversial. We previously found that in Drosophila Schneider 2 cells, the nuclear receptor FTZ-F1 is required for activation of the E75A gene by JH. Here, we utilized insect two-hybrid assays to show that FTZ-F1 interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE, in a JH-dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 activation function 2 (AF2) is removed, implicating AF2 as an interacting site. Through homology modeling, we found that MET and GCE possess a C-terminal α-helix featuring a conserved motif LIXXL that represents a novel nuclear receptor (NR) box. Docking simulations supported by two-hybrid experiments revealed that FTZ-F1·MET and FTZ-F1·GCE heterodimer formation involves a typical NR box-AF2 interaction but does not require the canonical charge clamp residues of FTZ-F1 and relies primarily on hydrophobic contacts, including a unique interaction with helix 4. Moreover, we identified paralog-specific features, including a secondary interaction site found only in MET. Our findings suggest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1.     

The ubiquitin ligase MYCBP2 regulates transient receptor potential vanilloid receptor 1 (TRPV1) internalization through inhibition of p38 MAPK signaling

The E3 ubiquitin ligase MYCBP2 negatively regulates neuronal growth, synaptogenesis, and synaptic strength. More recently it was shown that MYCBP2 is also involved in receptor and ion channel internalization. We found that mice with a MYCBP2-deficiency in peripheral sensory neurons show prolonged thermal hyperalgesia. Loss of MYCBP2 constitutively activated p38 MAPK and increased expression of several proteins involved in receptor trafficking. Surprisingly, loss of MYCBP2 inhibited internalization of transient receptor potential vanilloid receptor 1 (TRPV1) and prevented desensitization of capsaicin-induced calcium increases. Lack of desensitization, TRPV internalization and prolonged hyperalgesia were reversed by inhibition of p38 MAPK. The effects were TRPV-specific, since neither mustard oil-induced desensitization nor behavioral responses to mechanical stimuli were affected. In summary, we show here for the first time that p38 MAPK activation can inhibit activity-induced ion channel internalization and that MYCBP2 regulates internalization of TRPV1 in peripheral sensory neurons as well as duration of thermal hyperalgesia through p38 MAPK.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.     

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.