Reduced prostasin (CAP1/PRSS8) activity eliminates HAI-1 and HAI-2 deficiency-associated developmental defects by preventing matriptase activation.

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.

Discovery of a novel insect neuropeptide signaling system closely related to the insect adipokinetic hormone and corazonin hormonal systems.

Neuropeptides and their G protein-coupled receptors (GPCRs) play a central role in the physiology of insects. One large family of insect neuropeptides are the adipokinetic hormones (AKHs), which mobilize lipids and carbohydrates from the insect fat body. Other peptides are the corazonins that are structurally related to the AKHs but represent a different neuropeptide signaling system. We have previously cloned an orphan GPCR from the malaria mosquito Anopheles gambiae that was structurally intermediate between the A. gambiae AKH and corazonin GPCRs. Using functional expression of the receptor in cells in cell culture, we have now identified the ligand for this orphan receptor as being pQVTFSRDWNAamide, a neuropeptide that is structurally intermediate between AKH and corazonin and that we therefore named ACP (AKH/corazonin-related peptide). ACP does not activate the A. gambiae AKH and corazonin receptors and, vice versa, AKH and corazonin do not activate the ACP receptor, showing that the ACP/receptor couple is an independent and so far unknown peptidergic signaling system. Because ACP is structurally intermediate between AKH and corazonin and the ACP receptor between the AKH and corazonin receptors, this is a prominent example of receptor/ligand co-evolution, probably originating from receptor and ligand gene duplications followed by mutations and evolutionary selection, thereby yielding three independent hormonal systems. The ACP signaling system occurs in the mosquitoes A. gambiae, Aedes aegypti, and Culex pipiens (Diptera), the silkworm Bombyx mori (Lepidoptera), the red flour beetle Tribolium castaneum (Coleoptera), the parasitic wasp Nasonia vitripennis (Hymenoptera), and the bug Rhodnius prolixus (Hemiptera). However, the ACP system is not present in 12 Drosophila species (Diptera), the honeybee Apis mellifera (Hymenoptera), the pea aphid Acyrthosiphon pisum (Hemiptera), the body louse Pediculus humanus (Phthiraptera), and the crustacean Daphnia pulex, indicating that it has been lost several times during arthropod evolution. In particular, this frequent loss of hormonal systems is unique for arthropods compared with vertebrates.

The Drosophila genes CG14593 and CG30106 code for G-protein-coupled receptors specifically activated by the neuropeptides CCHamide-1 and CCHamide-2.

Recently, a novel neuropeptide, CCHamide, was discovered in the silkworm Bombyx mori (L. Roller et al., Insect Biochem. Mol. Biol. 38 (2008) 1147-1157). We have now found that all insects with a sequenced genome have two genes, each coding for a different CCHamide, CCHamide-1 and -2. We have also cloned and deorphanized two Drosophila G-protein-coupled receptors (GPCRs) coded for by genes CG14593 and CG30106 that are selectively activated by Drosophila CCH-amide-1 (EC(50), 2×10(-9) M) and CCH-amide-2 (EC(50), 5×10(-9) M), respectively. Gene CG30106 (symbol synonym CG14484) has in a previous publication (E.C. Johnson et al., J. Biol. Chem. 278 (2003) 52172-52178) been wrongly assigned to code for an allatostatin-B receptor. This conclusion is based on our findings that the allatostatins-B do not activate the CG30106 receptor and on the recent findings from other research groups that the allatostatins-B activate an unrelated GPCR coded for by gene CG16752. Comparative genomics suggests that a duplication of the CCHamide neuropeptide signalling system occurred after the split of crustaceans and insects, about 410 million years ago, because only one CCHamide neuropeptide gene is found in the water flea Daphnia pulex (Crustacea) and the tick Ixodes scapularis (Chelicerata).

Identification of the Drosophila and Tribolium receptors for the recently discovered insect RYamide neuropeptides.

One year ago, we discovered a new family of insect RYamide neuropeptides, which has the C-terminal consensus sequence FFXXXRYamide, and which is widely occurring in most insects, including the fruitfly Drosophila melanogaster and the red flour beetle Tribolium castaneum (F. Hauser et al., J. Proteome Res. 9 (2010) 5296-5310). Here, we identify a Drosophila G-protein-coupled receptor (GPCR) coded for by gene CG5811 and its Tribolium GPCR ortholog as insect RYamide receptors. The Drosophila RYamide receptor is equally well activated (EC(50), 1×10(-9)M) by the two Drosophila RYamide neuropeptides: RYamide-1 (PVFFVASRYamide) and RYamide-2 (NEHFFLGSRYamide), both contained in a preprohormone coded for by gene CG40733. The Tribolium receptor shows a somewhat higher affinity to Tribolium RYamide-2 (ADAFFLGPRYamide; EC(50), 5×10(-9)M) than to Tribolium RYamide-1 (VQNLATFKTMMRYamide; EC(50), 7×10(-8)M), which might be due to the fact that the last peptide does not completely follow the RYamide consensus sequence rule. There are other neuropeptides in insects that have similar C-terminal sequences (RWamide or RFamide), such as the FMRFamides, sulfakinins, myosuppressins, neuropeptides F, and the various short neuropeptides F. Amazingly, these neuropeptides show no cross-reactivity to the Tribolium RYamide receptor, while the Drosophila RYamide receptor is only very slightly activated by high concentrations (>10(-6)M) of neuropeptide F and short neuropeptide F-1, showing that the two RYamide receptors are quite specific for activation by insect RYamides, and that the sequence FFXXXRYamide is needed for effective insect RYamide receptor activation. Phylogenetic tree analyses and other amino acid sequence comparisons show that the insect RYamide receptors are not closely related to any other known insect or invertebrate/vertebrate receptors, including mammalian neuropeptide Y and insect neuropeptide F and short neuropeptide F receptors. Gene expression data published in Flybase (www.flybase.org) show that the Drosophila CG5811 gene is significantly expressed in the hindgut of adult flies, suggesting a role of insect RYamides in digestion or water reabsorption.

Cloning and identification of an oxytocin/vasopressin-like receptor and its ligand from insects.

More than 20 years ago, an oxytocin/vasopressin-like peptide, CLITNCPRGamide, was isolated from the locust, Locusta migratoria [Proux JP, et al. (1987) Identification of an arginine vasopressin-like diuretic hormone from Locusta migratoria. Biochem Biophys Res Commun 149:180-186]. However, no similar peptide could be identified in other insects, nor could its prohormone be cloned, or its physiological actions be established. Here, we report that the recently sequenced genome from the red flour beetle Tribolium castaneum contains a gene coding for an oxytocin/vasopressin-like peptide, identical to the locust peptide, which we named inotocin (for insect oxytocin/vasopressin-like peptide) and a gene coding for an inotocin G protein-coupled receptor (GPCR). We cloned the Tribolium inotocin preprohormone and the inotocin GPCR and expressed the GPCR in CHO cells. This GPCR is strongly activated by low concentrations of inotocin (EC(50), 5 x 10(-9) M), demonstrating that it is the inotocin receptor. Quantitative RT-PCR (qPCR) showed that in adult Tribolium, the receptor is mainly expressed in the head and much less in the hindgut and Malpighian tubules, suggesting that the inotocin/receptor couple does not play a role in water homeostasis. Surprisingly, qPCR also showed that the receptor is 30x more expressed in the first larval stages than in adult animals. The inotocin/receptor couple can also be found in the recently sequenced genome from the parasitic wasp Nasonia vitripennis but not in any other holometabolous insect with a completely sequenced genome (12 Drosophila species, the malaria mosquito Anopheles gambiae, the yellow fever mosquito Aedes aegypti, the silk worm Bombyx mori, and the honey bee Apis mellifera), suggesting that this neuropeptide system is confined to basal holometabolous insects. Furthermore, we identified an oxytocin/vasopressin-like peptide and receptor in the recently sequenced genome from the water flea Daphnia pulex (Crustacea). To our knowledge, this is the first report on the molecular cloning of an oxytocin/vasopressin-like receptor and its ligand from arthropods.

Epidermal loss of Gαq confers a migratory and differentiation defect in keratinocytes.

G-protein coupled receptors (GPCRs), which activate heterotrimeric G proteins, are an essential class of transmembrane receptors that are responsible for a myriad of signaling events in normal and pathologic conditions. Two members of the G protein family, Gαq and Gα11, activate one of the main GPCR pathways and function as oncogenes by integrating mitogen-stimulated signaling cascades that are active under malignant conditions. Recently, it has been shown that targeted deletion of Gα11 and Gαq from endothelial cells impairs the Rho-mediated formation of focal adherens junctions, suggesting that Gα11/q signaling may also play a significant role in cytoskeletal-mediated cellular responses in epithelial cells. Indeed, combined deletion of Gα11 and Gαq confers a significant migratory defect in keratinocytes that delays cutaneous wound healing in an in vivo setting. This delay can be attributed to a defect during the reepithelialization phase due to significantly attenuated migratory capacity of Gαq-null keratinocytes under combined Gα11 deficiency. In fact, cells lacking Gα11/q demonstrate a severely reduced ability to respond to mitogenic and migratory signals in the microenvironment, leading to inappropriate and premature terminal differentiation. These results suggest that Gα11/q signaling pathways may be critical for integrating mitogenic signals and cytoskeletal function to achieve normal physiological responses. Emergence of a malignant phenotype may therefore arise from both under- and overexpression of Gα11/q signaling, implicating its upstream regulation as a potential therapeutic target in a host of pathologic conditions.

Cloning and characterization of the adipokinetic hormone receptor from the cockroach Periplaneta americana.

Cockroaches have long been used as insect models to investigate the actions of biologically active neuropeptides. Here, we describe the cloning and functional expression in Chinese hamster ovary cells of an adipokinetic hormone (AKH) G protein-coupled receptor from the cockroach Periplaneta americana. This receptor is only activated by various insect AKHs (we tested eight) and not by a library of 29 other insect or invertebrate neuropeptides and nine biogenic amines. Periplaneta has two intrinsic AKHs, Pea-AKH-1, and Pea-AKH-2. The Periplaneta AKH receptor is activated by low concentrations of both Pea-AKH-1 (EC50, 5 x 10(-9)M), and Pea-AKH-2 (EC50, 2 x 10(-9)M). Insects can be subdivided into two evolutionary lineages, holometabola (insects with a complete metamorphosis during development) and hemimetabola (incomplete metamorphosis). This paper describes the first AKH receptor from a hemimetabolous insect.