Widely distributed Drosophila G‐protein‐coupled receptor (CG7887) is activated by endogenous tachykinin‐related peptides

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G‐protein‐coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. We determined that HEK‐293 cells transfected with DTKR displayed dose‐dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR‐green fluorescent protein (GFP) fusion constructs in HEK‐293 cells. We generated specific antireceptor antisera and showed that DTKR is widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. Our findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand‐receptor system plays multiple functional roles. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

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 (EC₅₀, 2 × 10⁻⁹ M) and CCH-amide-2 (EC₅₀, 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).     

Regulation of Smoothened by Drosophila G-protein-coupled receptor kinases

The Hedgehog (Hh) signaling pathway plays a conserved and essential role in regulating development and homeostasis of numerous tissues. Cytoplasmic signaling is initiated by Smoothened (Smo), a G-protein-coupled receptor (GPCR) family member, whose levels and activity are regulated by the Hh receptor Patched (Ptc). In response to Hh binding to Ptc, Ptc-mediated repression of Smo is relieved, leading to Smo activation, surface accumulation, and downstream signaling. We find that downregulation of Drosophila Smo protein in Hh-responding imaginal disc cells is dependent on the activity of G-protein-coupled receptor kinase 2 (Gprk2). By analyzing gain- and null loss-of-function phenotypes, we provide evidence that Gprk2 promotes Smo internalization subsequent to its activation, most likely by direct phosphorylation. Ptc-dependent regulation of Smo accumulation is normal in gprk2 mutants, indicating that Gprk2 and Ptc downregulate Smo by different mechanisms. Finally, we show that both Drosophila G-protein-coupled receptor kinase orthologues, Gprk1 and Gprk2, act in a partially redundant manner to promote Hh signaling. Our results suggest that Smo is regulated by distinct Ptc-dependent and Gprk2-dependent trafficking mechanisms in vivo, analogous to constitutive and activity-dependent regulation of GPCRs. G-protein-coupled receptor kinase activity is also important for efficient downstream signaling.     

Trace amine-associated receptor 1 (TAAR1) is activated by amiodarone metabolites

Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) is a clinically available drug used to treat a wide variety of cardiac arrhythmias. We report here the synthesis and characterization of a panel of potential amiodarone metabolites that have significant structural similarity to thyroid hormone and its metabolites the iodothyronamines. Several of these amiodarone derivatives act as specific agonists of the G protein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR(1)). This result demonstrates a novel molecular target for amiodarone derivatives with potential clinical significance.     

Neuronal co-localization of different isoforms of tachykinin-related peptides (LemTRPs) in the cockroach brain

Seven isoforms of tachykinin-related peptides (TRPs) have been isolated from the brain of the cockroach Leucophaea maderae. These peptides (LemTRP-1, 2, and 5-9) share the C-terminal sequence GFX(1)GX(2)Ramide (where X(1) and X(2) are variable residues). In order to determine the neuronal distribution of several of these LemTRP isoforms, we raised antisera to their variable N-termini. Antisera to LemTRP-1, 2, 3, 7, and 8 were utilized for immunocytochemistry on cryostat sections of the L. maderae brain. As expected, the gut peptide LemTRP-3 was not detected in the brain, and the antisera to LemTRP-1, 2, and 7 labeled the same sets of neurons in different regions of the brain. These neurons could also be labeled with antisera raised to the more conserved C-termini of LemTRP-1 and the locust TRP LomTK-I. The antiserum to LemTRP-8 predominantly labeled a set of neurons distinct from that seen with any other N- or C-terminus-directed antisera, suggesting that it recognizes epitope(s) other than known insect TRPs. Our findings indicate that at least three of the LemTRPs are always co-localized in neurons of the L. maderae brain. We have also been able to show that LemTRP-2, which is an N-terminally extended form (17-mere) of LemTRP-1 with a dibasic putative cleavage site, is transported throughout the processes of the neurons in the same manner as LemTRP-1 and 7. Thus, LemTRP-2 may be released with the other shorter LemTRPs. This is the first investigation of LemTRP distribution in the cockroach central nervous system utilizing antisera to native peptides.     

G2A is a proton-sensing G-protein-coupled receptor antagonized by lysophosphatidylcholine

G2A (from G2 accumulation) is a G-protein-coupled receptor (GPCR) that regulates the cell cycle, proliferation, oncogenesis, and immunity. G2A shares significant homology with three GPCRs including ovarian cancer GPCR (OGR1/GPR68), GPR4, and T cell death-associated gene 8 (TDAG8). Lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC) were reported as ligands for G2A and GPR4 and for OGR1 (SPC only), and a glycosphingolipid psychosine was reported as ligand for TDAG8. As OGR1 and GPR4 were reported as proton-sensing GPCRs (Ludwig, M. G., Vanek, M., Guerini, D., Gasser, J. A., Jones, C. E., Junker, U., Hofstetter, H., Wolf, R. M., and Seuwen, K. (2003) Nature 425, 93-98), we evaluated the proton-sensing function of G2A. Transient expression of G2A caused significant activation of the zif 268 promoter and inositol phosphate (IP) accumulation at pH 7.6, and lowering extracellular pH augmented the activation only in G2A-expressing cells. LPC inhibited the pH-dependent activation of G2A in a dose-dependent manner in these assays. Thus, G2A is another proton-sensing GPCR, and LPC functions as an antagonist, not as an agonist, and regulates the proton-dependent activation of G2A.     

ERK activation by G-protein-coupled receptors in mouse brain is receptor identity-specific.

In transfected cells and non-neuronal tissues many G-protein-coupled receptors activate p44/42 MAP kinase (ERK), a kinase involved in both hippocampal synaptic plasticity and learning and memory. However, it is not clear to what degree these receptors couple to ERK in brain. G(s)-coupled beta-adrenergic receptor activation of ERK in neurons is critical in the regulation of synaptic plasticity in area CA1 of the hippocampus. In addition, alpha(1)- and alpha(2)-adrenergic receptors, present in CA1, could potentially activate ERK. We find that, like the beta-adrenergic receptor, the G(q)-coupled alpha(1)AR activates ERK in adult mouse CA1. However, activation of the G(i/o)-coupled alpha(2)AR does not activate ERK, nor does activation of a homologous G(i/o)-coupled receptor enriched in adult mouse CA1, the 5HT(1A) receptor. In contrast, the nonhomologous G(i/o)-coupled gamma-aminobutyric acid type B receptor does activate ERK in adult mouse CA1. Surprisingly, activation of alpha(2)ARs in CA1 from immature animals where basal phospho-ERK is low induces ERK phosphorylation. These data suggest that although most G-protein-coupled receptor subtypes activate ERK in non-neuronal cells, the coupling of G(i/o) to ERK is tightly regulated in brain.     

Transglutaminase from Streptomyces mobaraensis is activated by an endogenous metalloprotease.

Streptomyces mobaraensis secretes a Ca2+-independent transglutaminase (TGase) that is activated by removing an N-terminal peptide from a precursor protein during submerged culture in a complex medium [Pasternack, R., Dorsch, S., Otterbach, J. T., Robenek, I. R., Wolf, S. & Fuchsbauer, H.-L. (1998) Eur. J. Biochem. 257, 570-576]. However, an activating protease could not be identified, probably because of the presence of a 14-kDa protein (P14) belonging to the Streptomyces subtilisin inhibitor family. In contrast, if the microorganism was allowed to grow on a minimal medium, several soluble proteases were extracted, among them the TGase-activating protease (TAMEP). TAMEP was purified by sequential chromatography on DEAE- and Arg-Sepharose and used to determine the cleavage site of TGase. It was clearly shown that the peptide bond between Phe(-4) and Ser(-5) was hydrolyzed, indicating that at least one additional peptidase is necessary to complete TGase processing, even if TAMEP cleavage was sufficient to obtain total activity. Sequence analysis from the N-terminus of TAMEP revealed the close relationship to a zinc endo-protease from S. griseus. The S. griseus protease differs from other members of the M4 protease family, such as thermolysin, in that it may be inhibited by the Streptomyces subtilisin inhibitor. P14 likewise inhibits TAMEP in approximately equimolar concentrations, suggesting its important role in regulating TGase activity.     

Discoidin domain receptor 1 is activated independently of beta(1) integrin

Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals independent of the epidermal growth factor (EGF) receptor. In cells that endogenously express both DDR1 and the EGF receptor, stimulation with EGF does not induce DDR activation. Third, we detected full DDR1 activation after collagen stimulation in cells that have been treated with blocking antibodies for alpha(2)beta(1) integrin or in cells with a targeted deletion of the beta(1) integrin gene. Finally, we show that overexpression of dominant negative DDR1 in the myoblast cell line C2C12 blocks cellular differentiation and the formation of myofibers.     

Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll(10b)), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll(10b), and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.     

Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation

Treatment of Chinese hamster ovary (CHO) cells over-expressing the human insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadate, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyi phosphorylation of theβ-subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen-activated protein (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose- and time-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44^mapk and p42^mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44^mapk and p42^mapk in CHO-HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over-expressing a protein tyrosine kinase-deficient insulin receptor (CHO-1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediated tyrosyl phosphorylation and activation of MAP kinases.     

Identification and cardiotropic actions of brain/gut-derived tachykinin-related peptides (TRPs) from the American lobster Homarus americanus

Two tachykinin-related peptides (TRPs) are known in decapods, APSGFLGMRamide and TPSGFLGMRamide. The former peptide appears to be ubiquitously conserved in members of this taxon, while the latter has been suggested to be a genus (Cancer)- or infraorder (Brachyura)-specific isoform. Here, we characterized a cDNA from the American lobster Homarus americanus (infraorder Astacidea) that encodes both TRPs: six copies of APSGFLGMRamide and one of TPSGFLGMRamide. Mass spectral analyses of the H. americanus supraoesophageal ganglion (brain) and commissural ganglia confirmed the presence of both peptides in these neural tissues; both isoforms were also detected in the midgut. Physiological experiments showed that both APSGFLGMRamide and TPSGFLGMRamide are cardioactive in H. americanus, eliciting identical increases in both heart contraction frequency and amplitude. Collectively, our data represent the first genetic confirmation of TRPs in H. americanus and of TPSGFLGMRamide in any species, demonstrate that TPSGFLGMRamide is not restricted to brachyurans, and show that both this peptide and APSGFLGMRamide are brain-gut isoforms, the first peptides thus far confirmed to possess this dual tissue distribution in H. americanus. Our data also suggest a possible role for TRPs in modulating the output of the lobster heart.     

Glucagon like-peptide-1 receptor is covalently modified by endogenous mono-ADP-ribosyltransferase

Our previous study revealed a mono-ADP-ribosyltransferase mediated in vitro mono-ADP-ribosylation of IC₃ peptide, a peptide with sequence corresponded to third intracellular loop of glucagon like-peptide-1 (GLP-1) receptor. Furthermore, Arg³⁴⁸ was shown to be modified amino acid residue although its mutation did not eliminate mono-ADP-ribosylation completely. In order to further study the signaling mechanisms of GLP-1 receptor, we took on lease a possibility that an alternative site of enzymatic modification exist so mono-ADP-ribosylation of Cys³⁴¹ was hypothesized. The results confirmed both Arg³⁴⁸ and Cys³⁴¹ as a site of mono-ADP-ribosylation where Arg³⁴⁸ is modified predominantly. Sum of mono-ADP-ribosylation rate of both single IC₃ mutants coincided with IC₃ rate. What is in vivo role of Cys³⁴¹ mono-ADP-ribosylation is entirely speculative but our study represents an important step toward a complete understanding of signaling via GLP-1 receptor.     

The Drosophila ecdysone receptor (EcR) gene is required maternally for normal oogenesis

Oogenesis in Drosophila is regulated by the steroid hormone ecdysone and the sesquiterpenoid juvenile hormone. Response to ecdysone is mediated by a heteromeric receptor composed of the EcR and USP proteins. We have identified a temperature-sensitive EcR mutation, EcR(A483T), from a previously isolated collection of EcR mutations. EcR(A483T) is predicted to affect all EcR protein products (EcR-A, EcR-B1, and EcR-B2) since it maps to a common exon encoding the ligand-binding domain. In wild-type females, we find that both EcR-A and EcR-B1 are expressed in nurse cells and follicle cells throughout oogenesis. EcR mutant females raised at permissive temperature and then shifted to restrictive temperature exhibit severe reductions in fecundity. Oogenesis in EcR mutant females is defective, and the spectrum of oogenic defects includes the presence of abnormal egg chambers and loss of vitellogenic egg stages. Our results demonstrate a requirement for EcR during female reproduction and suggest that EcR is required for normal oogenesis.     

T-cell antigen receptor (TCR) transmembrane peptides

Cell surface membranes are generally considered as inert and hydrophobic providing a stable physical barrier that anchor proteins and maintain cellular homeostasis between the intra- and the extra-cellular environment. The integral proteins that transverse membranes do so once or multiple times and can function alone or as part of a larger complex. Far from being inert, there is a multiplicity of biophysical factors that drive protein-protein and protein-lipid interactions within membranes that are being increasingly recognised as very important for cellular function. Unravelling these “hot-spots” on the contact surface of transmembrane (TM) proteins and targeting peptides to these sites to interrupt the cohesive interaction between the proteins provides both an enormous challenge and a huge therapeutic potential that as yet remains unrecognized. Indeed, with biopharmaceutical research on the rise, TM peptides may prove a useful innovation. Using the T-cell antigen receptor (TCR) as a model system of multi-subunits interacting at the TM via electrostatic charges the potential for peptides as therapeutic agents to interfere with normal immune responses is discussed. The principles of such can be extended to other similar receptor systems including those involved in cancer or infection.