Insight into tachykinin-related peptides,their receptors,and invertebrate tachykinins: a review

Tachykinins (TKs) constitute the largest vertebrate neuropeptide family with multifunctions in central and peripheral tissues. In several invertebrate species, two types of structurally related peptides, 'tachykinin-related peptides (TKRPs)' and 'invertebrate tachykinins (inv-TKs)' have been identified. TKRPs, isolated from the nerve and/or gut tissues, contain the common C-terminal sequence -Phe-X-Gly-Y-Arg-NH(2) (X and Y are variable) analogous to the vertebrate TK consensus -Phe-X-Gly-Leu-Met-NH(2), and exhibit vertebrate TK-like contractile activity on invertebrate gut tissues. Inv-TKs have been shown to be present exclusively in the salivary gland of several species, to share vertebrate TK consensus motif, and to possess TK-like potencies on vertebrate, not invertebrate tissues. However, the functional and evolutionary relevance of TKRPs and inv-TKs to vertebrate TKs remains to be understood. Recent studies have revealed that TKRP precursors dramatically differ from vertebrate preprotachykinins in structural organization and that TKRP receptors share structural and functional properties with vertebrate TK receptors. Moreover, the C-terminal arginine in TKRPs has been shown to play an essential role in discriminating their receptors from vertebrate TK receptors. Such recent marked progress is expected to enhance further investigation of biological roles of TKRPs. This review provides an overview of the basic findings obtained previously and a buildup of new knowledge regarding TKRPs and inv-TKs. We also compare TKRPs and inv-TKs to vertebrate TKs with regard to evolutionary relationships in structure and function among these structurally related peptides.     

Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis

We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes. Double-immunostaining demonstrated that the immunoreactivity of Ci-cathepsin D was largely colocalized with that of the receptor of Ci-TK, Ci-TK-R, in test cells of the stage II oocytes. Ci-cathepsin D gene expression was detected at 2h after treatment with Ci-TK, and elevated for up to 5h, and then slightly decreased. Gene expression of Ci-carboxypeptidase B1 and Ci-chymotrypsin was observed at 5h after treatment with Ci-TK, and then decreased. The enzymatic activities of Ci-cathepsin D, Ci-carboxypeptidase B1, and Ci-chymotrypsin showed similar alterations with 1-h lags. These gene expression and protease activity profiles verified that Ci-cathepsin D is initially activated, which is followed by the activation of Ci-carboxypeptidase B1 and Ci-chymotrypsin. Collectively, the present data suggest that Ci-TK directly induces Ci-cahtepsin D in test cells expressing Ci-TK receptor, leading to the secondary activation of Ci-chymotrypsin and Ci-carboxypeptidase B1 in the follicle in the tachykininergic oocyte growth pathway.     

Characterization of a novel vasopressin/oxytocin superfamily peptide and its receptor from an ascidian, Ciona intestinalis

The vasopressin (VP)/oxytocin (OT) superfamily peptides are one of the most widely distributed neuropeptides and/or neurohypophysial hormones, but have ever not been characterized from any deuterostome invertebrates including protochordates, ascidians. In the present study, we show the identification of a novel VP/OT superfamily peptide and its receptor in the ascidian, Ciona intestinalis. Intriguingly, the Ciona VP/OT-related peptide (Ci-VP), unlike other 9-amino acid and C-terminally amidated VP/OT superfamily peptides, consists of 13 amino acids and lacks a C-terminal amidation. Mass spectrometry confirmed the presence of the 13-residue Ci-VP in the neural complex. Furthermore, 10 of 14 cysteines are conserved in the neurophysin domain, compared with other VP/OT counterparts. These results revealed that the VP/OT superfamily is conserved in ascidians, but the Ci-VP gene encodes an unprecedented VP/OT-related peptide and neurophysin protein. Ci-VP was also shown to activate its endogenous receptor, Ci-VP-R, at physiological concentrations, confirming the functionality of Ci-VP as an endogenous ligand. The Ci-VP gene was expressed exclusively in neurons of the brain, whereas the Ci-TK-R mRNA was distributed in various tissues including the neural complex, alimentary tract, gonad, and heart. These expression profiles suggest that Ci-VP, like other VP/OT superfamily peptides, serves as a multifunctional neuropeptides. Altogether, our data revealed both evolutionary conservation and specific divergence of the VP/OT superfamily in protochordates. This is the first molecular characterization of a VP/OT superfamily peptide and its cognate receptor from not only ascidians but also deuterostome invertebrates.     

Tachykinins and tachykinin receptors: effects in the genitourinary tract

Tachykinins (TKs) are a family of peptides involved in the central and peripheral regulation of urogenital functions through the stimulation of TK NK1, NK2 and NK3 receptors. At the urinary system level, TKs locally stimulate smooth muscle tone, ureteric peristalsis and bladder contractions, initiate neurogenic inflammation and trigger local and spinal reflexes aimed to maintain organ functions in emergency conditions. At the genital level, TKs are involved in smooth muscle contraction, in inflammation and in the modulation of steroid secretion by the testes and ovaries. TKs produce vasodilatation of maternal and fetal placental vascular beds and appear to be involved in reproductive function, stress-induced abortion, and pre-eclampsia. The current data suggest that the genitourinary tract is a primary site of action of the tachykininergic system.     

A novel tachykinin-related peptide receptor of Octopus vulgaris--evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide

The tachykinin (TK) and tachykinin-related peptide (TKRP) family represent one of the largest peptide families in the animal kingdom and exert their actions via a subfamily of structurally related G-protein-coupled receptors. In this study, we have identified a novel TKRP receptor from the Octopus heart, oct-TKRPR. oct-TKRPR includes domains and motifs typical of G-protein-coupled receptors. Xenopus oocytes that expressed oct-TKRPR, like TK and TKRP receptors, elicited an induction of membrane chloride currents coupled to the inositol phosphate/calcium pathway in response to Octopus TKRPs (oct-TKRP I-VII) with moderate ligand selectivity. Substance P and Octopus salivary gland-specific TK, oct-TK-I, completely failed to activate oct-TKRPR, whereas a Substance P analog containing a C-terminal Arg-NH2 exhibited equipotent activation of oct-TKRPs. These functional analyses prove that oct-TKRPs, but not oct-TK-I, serve as endogenous functional ligands through oct-TKRPR, although both of the family peptides were identified in a single species, and the importance of C-terminal Arg-NH2 in the specific recognition of TKRPs by TKRPR is conserved through evolutionary lineages of Octopus. Southern blotting of RT-PCR products revealed that the oct-TKRPR mRNA was widely distributed in the central and peripheral nervous systems plus several peripheral tissues. These results suggest multiple physiologic functions of oct-TKRPs as neuropeptides both in the Octopus central nervous system and in peripheral tissues. This is the first report on functional discrimination between invertebrate TKRPs and salivary gland-specific TKs.     

Neurokinin B- and substance P-like immunoreactivity are co-localized in enteric nerves of rat ileum

The tachykinins (TKs) substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) have conserved C-terminal sequences and mediate similar physiological responses by activating neurokinin receptors found on neural and smooth muscle cells. Many enteric nerves express preprotachykinin A (PPT A) mRNA and synthesize SP and NKA. However, it is unclear if NKB is synthesized in enteric neurons as many antibodies developed against NKB also recognize other TKs. Therefore, the cellular distribution of NKB-like-immunoreactivity (NKB-ir) in rat ileum was examined using selective antisera raised against either synthetic Cys10-NKB or peptide 2 (P2), a non-tachykinergic peptide sequence in NKB precursor protein. NKB-ir and P2-ir had a similar distribution in varicose nerve fibers in submucosal and myenteric ganglia and almost all ganglia contained immunoreactive nerves. Few submucosal or myenteric neuronal somata contained strong immunoreactivity. Preabsorption of NKB or P2 antisera with their respective cognate peptides, but not with other TK peptides, abolished specific immunostaining. Finally, co-localization of NKB-/P2-ir with SP-ir suggested that most NKB-/P2-ir nerve fibers contain SP-ir, but some SP-ir nerves do not contain detectable NKB-/P2-ir. These results indicate that PPT B products P2 and NKB are localized in a subpopulation of enteric nerves containing TKs encoded by PPT A. Stimulation of these nerves may release NKB to activate local neurokinin receptors.     

Immunohistochemical Localization of Cardio-Active Neuropeptides in the Heart of a Living Fossil, Nautilus pompilius L. (Cephalopoda, Tetrabranchiata)

Neuropeptides play an important role in modulating the effects of neurotransmitters such as acetylcholine and noradrenaline in the heart and the vascular system of vertebrates and invertebrates. Various neuropeptides, including substance P (SP), vasoactive intestinal polypeptide (VIP) and FMRFamide, have been localized in the brain in cephalopods and the neurosecretory system of the vena cava. Previous studies involving cephalopods have mainly focussed on the modern, coleoid cephalopods, whereas little attention was paid to the living fossil Nautilus. In this study, the distributions of the peptides related to tachykinins (TKs) and the high affinity receptor for the best characterized TK substance P (tachykinin NK-1), VIP, as well as FMRFamide were investigated in the heart of Nautilus pompilius L. by immunohistochemistry. TK-like immunoreactivity (TK-LI) was seen associated to a sub-population of hemocytes, VIP-LI glial cells in larger nerves entering the heart, whereas FMRFamide immunoreactivity was distributed throughout the entire heart, including the semilunar atrioventricular valves. The pattern of FMRFamide immunoreactivity matched that of Bodian silver staining for nervous tissue. The NK-1-LI receptor was located on endothelial cells, which were also positive for endothelial nitric oxide synthase-LI (eNOS). The results indicate that neuropeptides may be involved in the regulation of the Nautilus heart via different mechanisms, (1) by direct interaction with myocardial receptors (FMRFamide), (2) by interacting with the nervus cardiacus (VIP-related peptides) and (3) indirectly by stimulating eNOS in the endothelium throughout the heart (TK-related peptides).     

The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis

The G-protein coupled cannabinoid receptors CB(1) and CB(2) are activated by Delta(9)-tetrahydrocannabinol, the psychoactive ingredient of cannabis, and mediate physiological effects of endogenous cannabinoids ('endocannabinoids'). CB(1) genes have been identified in mammals, birds, amphibians and fish, whilst CB(2) genes have been identified in mammals and in the puffer fish Fugu rubripes. Therefore, both CB(1) and CB(2) receptors probably occur throughout the vertebrates. However, cannabinoid receptor genes have yet to be identified in any invertebrate species and the evolutionary origin of cannabinoid receptors is unknown. Here we report the identification of CiCBR, a G-protein coupled receptor in a deuterostomian invertebrate - the urochordate Ciona intestinalis - that is orthologous to vertebrate cannabinoid receptors. The CiCBR cDNA encodes a protein with a predicted length (423 amino-acids) that is the intermediate of human CB(1) (472 amino-acids) and human CB(2) (360-amino-acid) receptors. Interestingly, the protein-coding region of the CiCBR gene is interrupted by seven introns, unlike in vertebrate cannabinoid receptor genes where the protein-coding region is typically intronless. Phylogenetic analysis revealed that CiCBR forms a clade with vertebrate cannabinoid receptors but is positioned outside the CB(1) and CB(2) clades of a phylogenetic tree, indicating that the common ancestor of CiCBR and vertebrate cannabinoid receptors predates a gene (genome) duplication event that gave rise to CB(1)- and CB(2)-type receptors in vertebrates. Importantly, the discovery of CiCBR and the absence of orthologues of CiCBR in protostomian invertebrates such as Drosophila melanogaster and Caenorhabditis elegans indicate that the ancestor of vertebrate CB(1) and CB(2) cannabinoid receptors originated in a deuterostomian invertebrate.     

Immunohistochemical and chromatographic studies of peptides with tachykinin-like immunoreactivity in the central nervous system of the lamprey

The distribution and chemical properties of compounds with tachykinin-like immunoreactivity (TK-LI) in the spinal cord and brain of lampreys (Lampetra fluviatilis and Ichthyomyzon unicuspis) were investigated by means of immunohistochemistry and various chromatographic methods combined with radioimmunoassay. The distribution of TK immunoreactive fibers in the lamprey spinal cord was investigated with 13 different TK antisera which gave positive staining in pilot experiments. The antisera were raised against substance P (SP) (n = 6), physalaemin (PHY) (n = 1), neurokinin A (NKA) (n = 2), kassinin (KAS) (n = 2) or eledoisin (ELE) (n = 2). Pre-incubation of these antisera with their corresponding TKs abolished or reduced the immunostaining. Four different patterns of distribution were found with the 13 antisera, and they did not seem to be related to the TKs against which the antisera were raised. The different patterns could be explained by assuming the presence of the three different TKs. Six different antisera, raised against SP (n = 2), KAS (n = 2) or ELE (n = 2), were used for radioimmunoassay. The TK-LI material eluted as several separate components in various chromatographic systems. The central nervous system (CNS) of the lamprey did not contain measurable amounts of SP, NKA, neurokinin B (NKB), KAS or ELE. The present data imply that the lamprey CNS contains at least three different TKs probably different from SP, PHY, NKA, NKB, KAS or ELE; these are possibly new, not earlier described TKs. The three hypothetical TKs differ in their distribution.     

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES*

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH₂. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.     

Characterization of novel GPCR gene coding locus in amphioxus genome: gene structure, expression, and phylogenetic analysis with implications for its involvement in chemoreception

Chemosensation is the primary sensory modality in almost all metazoans. The vertebrate olfactory receptor genes exist as tandem clusters in the genome, so that identifying their evolutionary origin would be useful for understanding the expansion of the sensory world in relation to a large-scale genomic duplication event in a lineage leading to the vertebrates. In this study, I characterized a novel GPCR (G-protein-coupled receptor) gene-coding locus from the amphioxus genome. The genomic DNA contains an intronless ORF whose deduced amino acid sequence encodes a seven-transmembrane protein with some amino acid residues characteristic of vertebrate olfactory receptors (ORs). Surveying counterparts in the Ciona intestinalis (Asidiacea, Urochordata) genome by querying BLAST programs against the Ciona genomic DNA sequence database resulted in the identification of a remotely related gene. In situ hybridization analysis labeled primary sensory neurons in the rostral epithelium of amphioxus adults. Based on these findings, together with comparison of the developmental gene expression between amphioxus and vertebrates, I postulate that chemoreceptive primary sensory neurons in the rostrum are an ancient cell population traceable at least as far back in phylogeny as the common ancestor of amphioxus and vertebrates.     

Identification and characterization of a novel RF-amide peptide ligand for orphan G-protein-coupled receptor SP9155

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.     

Gene structure and function of tyrosine kinases in the marine sponge Geodia cydonium: autapomorphic characters in Metazoa

Porifera (sponges) represent the most ancient, extant metazoan phylum. They existed already prior to the 'Cambrian Explosion'. Based on the analysis of aa sequences of informative proteins, it is highly likely that all metazoan phyla evolved from only one common ancestor (monophyletic origin). As 'autapomorphic' proteins which are restricted to Metazoa only, integrin receptors, receptors with scavenger receptor cysteine-rich repeats, neuronal-like receptors and protein-tyrosine kinases (PTKs) have been identified in Porifera. From the marine sponge Geodia cydonium, a receptor tyrosine kinase (RTK) has been cloned that comprises the characteristic structural topology known from other metazoan RTKs; an extracellular domain, the transmembrane region, the juxtamembrane region and the TK domain. Only two introns, within the coding region of the RTK gene, could be found, which separate the two highly polymorphic immunoglobulin-like domains, found in the extracellular region of the enzyme. The functional role of this sponge RTK could be demonstrated both in situ (grafting experiments) and in vitro (increase of intracellular Ca2+ level). Upstream of this RTK gene, two further genes coding for tyrosine kinases (TK) have been identified. Both are intron-free. The deduced aa sequence of the first gene shows no transmembrane segment; from the second gene--so far--only half of its catalytic domain is known. A phylogenetic analysis with the TK domains from these sequences and a fourth, from a novel scavenger RTK (all domains comprise the signature for the TK class II receptors), showed that they are distantly related to the insulin and insulin-like receptors. The presented findings support the 'introns-late' hypothesis for such genes that encode 'metazoan' proteins. It is proposed that the TKs evolved from protein-serine/threonine kinases through modularization and subsequent exon shuffling. After formation of the ancestral TKs, the modules lost the framing introns to protect the evolutionary novelty. Since cell culture systems of sponges are now available, it can be expected that soon also those mechanisms that control the developmental programs will be unravelled.     

Biologic activity of the tachykinins on lamb and sheep gallbladder

In this study, we examined the activity of the tachykinins (TKs) on lamb and sheep isolated gallbladder and whether the TKs are involved in the capsaicin-induced activity in these tissues. Substance P (SP) and physalaemin (PHYS) contracted lamb gallbladder, PHYS-induced striking tachyphylaxis. This tissue was nearly insensitive to neurokinin A (NKA), neurokinin B (NKB), septide, and capsaicin. As in lamb tissues, SP and PHYS both contracted sheep gallbladder although PHYS induced no tachyphylaxis. At doses that had no effect on lamb tissue, NKA, NKB, septide, and capsaicin contracted sheep gallbladder. Our findings indicate that TK receptors differ in adult and young ovine gallbladder. The activity of PHYS on lamb gallbladder could depend on the existence of an unusual binding site, carrying one or more residues critical for the N-terminal sequence present in PHYS but not in SP.     

BfCBR: a cannabinoid receptor ortholog in the cephalochordate Branchiostoma floridae (Amphioxus)

A gene encoding an ortholog of vertebrate CB(1)/CB(2) cannabinoid receptors was recently identified in the urochordate Ciona intestinalis (CiCBR; [Elphick, M.R., Satou, Y., Satoh, N., 2003. The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis. Gene 302, 95-101.]). Here a cannabinoid receptor ortholog (BfCBR) has been identified in the cephalochordate Branchiostoma floridae. BfCBR is encoded by a single exon and is 410 amino acid residue protein that shares 28% sequence identity with CiCBR and 23% sequence identity with human CB(1) and human CB(2). The discovery of BfCBR and CiCBR and the absence of cannabinoid receptor orthologs in non-chordate invertebrates indicate that CB(1)/CB(2)-like cannabinoid receptors originated in an invertebrate chordate ancestor of urochordates, cephalochordates and vertebrates. Furthermore, analysis of the relationship of BfCBR and CiCBR with vertebrate CB(1) and CB(2) receptors indicates that the gene/genome duplication that gave rise to CB(1) and CB(2) receptors occurred in the vertebrate lineage. Identification of BfCBR, in addition to CiCBR, paves the way for comparative analysis of the expression and functions of these proteins in Branchiostoma and Ciona, respectively, providing an insight into the ancestral functions of cannabinoid receptors in invertebrate chordates prior to the emergence of CB(1) and CB(2) receptors in vertebrates.     

Characterization of the short neuropeptide F receptor from Drosophila melanogaster

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y(2) receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs.     

A new tachykinin receptor revealed by substance P analogues in the guinea pig ileum]

[Pro9]SP and septide have been described as selective agonists for the SP receptor (NK-1 type). These two peptides contract with a great efficacy the guinea-pig ileum, but unexpectedly septide was practically devoid of affinity for the NK-1 site labelled by 3H-[Pro9]SP. Like septide, SP analogues like SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP share the same peculiar properties. In addition, the contracting activity of these peptides is not explained by an interaction with NK-2 or NK-3 sites. GR 71,251, a compound which has been described as NK-1 antagonist, was more potent in inhibiting the septide- and the [Apa9-10]SP- than the [Pro9]SP-evoked contracting responses. Altogether, these results suggest that septide, SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP exert their high contracting activity in the guinea-pig ileum by acting on a new type of tachykinin receptor.     

Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase

Taurocyamine kinase (TK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase. TK is found only in certain marine annelids. In this study we used PCR to amplify two cDNAs coding for TKs from the polychaete Arenicola brasiliensis, cloned these cDNAs into the pMAL plasmid and expressed the TKs as fusion proteins with the maltose-binding protein. These are the first TK cDNA and deduced amino acid sequences to be reported. One of the two cDNA-derived amino acid sequences of TKs shows a high amino acid identity to lombricine kinase, another phosphagen kinase unique to annelids, and appears to be a cytoplasmic isoform. The other sequence appears to be a mitochondrial isoform; it has a long N-terminal extension that was judged to be a mitochondrial targeting peptide by several on-line programs and shows a higher similarity in amino acid sequence to mitochondrial creatine kinases from both vertebrates and invertebrates. The recombinant cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% of that of taurocyamine). However, the mitochondrial TK showed activity for taurocyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Neither TK catalyzed the phosphorylation of creatine. Comparison of the deduced amino acid sequences of mitochondrial CK and TK indicated that several key residues required for CK activity are lacking in the mitochondrial TK sequence. Homology models for both cytoplasmic and mitochondrial TK, constructed using CK templates, provided some insight into the structural correlation of differences in substrate specificity between the two TKs. A phylogenetic analysis using amino acid sequences from a broad spectrum of phosphagen kinases showed that annelid-specific phosphagen kinases (lombricine kinase, glycocyamine kinase and cytoplasmic and mitochondrial TKs) are grouped in one cluster, and form a sister-group with CK sequences from vertebrate and invertebrate groups. It appears that the annelid-specific phosphagen kinases, including cytoplasmic and mitochondrial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplasmic and mitochondrial isoforms of TK evolved independently.     

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.