NORMAL GENOMIC

• de Bree FM
• Trafficking of the vasopressin and oxytocin prohormone through the regulated secretory pathway.
• J Neuroendocrinol. 2000; 12: 589-94
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• The trafficking of prohormones and of regulated secretory proteins in general has been studied extensively in the last decades of the last century. Prohormone trafficking starts with correct folding and subsequently efficient sorting into the secretory granule of the regulated secretory pathway. The vasopressin/oxytocin prohormone is particularly interesting for studying protein trafficking, because the physicochemical properties and three-dimensional structure have been largely elucidated. In the case of pro-vasopressin and pro-oxytocin, folding and sorting depend completely on both intramolecular and intermolecular interactions. Proper folding is guided by the hormone-neurophysin association and the sorting event relies on the aggregative properties of the neurophysin domain in the prohormone, as well as a specific sorting signal, which is revealed when the aggregative property of the neurophysin domain is deleted. A comprehensive mechanism for trafficking of the vasopressin/oxytocin prohormone from the endoplasmic reticulum to the secretory granule is proposed.

• Satake H, Takuwa K, Minakata H, Matsushima O
• Evidence for conservation of the vasopressin/oxytocin superfamily in Annelida.
• J Biol Chem. 1999; 274: 5605-11
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• Annetocin is a structurally and functionally oxytocin-related peptide isolated from the earthworm Eisenia foetida. We present the characterization of the annetocin cDNA. Sequence analyses of the deduced precursor polypeptide revealed that the annetocin precursor is composed of three segments: a signal peptide, an annetocin sequence flanked by a Gly C-terminal amidation signal and a Lys-Arg dibasic processing site, and a neurophysin domain, similar to other oxytocin family precursors. The proannetocin showed 37.4-45.8% amino acid homology to other prohormones. In the neurophysin domain, 14 cysteines and amino acid residues essential for association of a neurophysin with a vasopressin/oxytocin superfamily peptide were conserved, suggesting that the Eisenia neurophysin can bind to annetocin. Furthermore, in situ hybridization experiments demonstrated that the annetocin gene is expressed exclusively in neurons of the central nervous system predicted to be involved in regulation of reproductive behavior. These findings confirm that annetocin is a member of the vasopressin/oxytocin superfamily. This is the first identification of the cDNA encoding the precursor of an invertebrate oxytocin-related peptide and also the first report of the identification of an annelid vasopressin/oxytocin-related precursor.

• van Kesteren RE, Geraerts WP
• Molecular evolution of ligand-binding specificity in the vasopressin/oxytocin receptor family.
• Ann N Y Acad Sci. 1998; 839: 25-34

• Barberis C, Mouillac B, Durroux T
• Structural bases of vasopressin/oxytocin receptor function.
• J Endocrinol. 1998; 156: 223-9

• Evans DA, Burbach JP, Swaab DF, van Leeuwen FW
• Mutant vasopressin precursors in the human hypothalamus: evidence for neuronal somatic mutations in man.
• Neuroscience. 1996; 71: 1025-30
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• We report here the expression of mutant proteins displaying the +1 reading frame of the vasopressin and oxytocin precursors in magnocellular neurons of the human hypothalamus. Our data demonstrate a high frequency of frameshift mutations in these neurons and thus provide the first evidence of somatic mutations in neurons of the human brain. The results imply that other neuronal populations and specific genes may also undergo similar mutational events with possible consequences for neuronal functioning and pathology.

• van Kesteren RE et al.
• Co-evolution of ligand-receptor pairs in the vasopressin/oxytocin superfamily of bioactive peptides.
• J Biol Chem. 1996; 271: 3619-26
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• In order to understand the molecular mechanisms that underlie the co-evolution of related yet functionally distinct peptide-receptor pairs, we study receptors for the vasopressin-related peptide Lys-conopressin in the mollusc Lymnaea stagnalis. In addition to a previously cloned Lys-conopressin receptor (LSCPR1), we have now identified a novel Lys-conopressin receptor subtype, named LSCPR2. The two receptors have a differential distribution in the reproductive organs and the brain, which suggests that they are involved in the control of distinct aspects of reproduction and mediate transmitter-like and/or modulatory effects of Lys-conopressin on different types of central neurons. In contrast to LSCPR1, LSCPR2 is maximally activated by both Lys-conopressin and Ile-conopressin, an oxytocin-like synthetic analog of Lys-conopressin. Together with a study of the phylogenetic relationships of Lys-conopressin receptors and their vertebrate counterparts, these data suggest that LSCPR2 represents an ancestral receptor to the vasopressin/oxytocin receptor family in the vertebrates. Based on our findings, we provide a theory of the molecular co-evolution of the functionally distinct ligand-receptor pairs of the vasopressin/oxytocin superfamily of bioactive peptides.

• van Kesteren RE et al.
• A novel G protein-coupled receptor mediating both vasopressin- and oxytocin-like functions of Lys-conopressin in Lymnaea stagnalis.
• Neuron. 1995; 15: 897-908
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• We have cloned a receptor, named LSCPR, for vasopressin-related Lys-conopressin in Lymnaea stagnalis. Lys-conopressin evokes Ca(2+)-dependent Cl- currents in Xenopus oocytes injected with LSCPR cRNA. Expression of LSCPR mRNA was detected in central neurons and peripheral muscles associated with reproduction. Upon application of Lys-conopressin, both neurons and muscle cells depolarize owing to an enhancement of voltage-dependent Ca2+ currents and start firing action potentials. Some neurons coexpress LSCPR and Lys-conopressin, suggesting an autotransmitter-like function for this peptide. Lys-conopressin also induces a depolarizing response in LSCPR-expressing neuroendocrine cells that control carbohydrate metabolism. Thus, in addition to oxytocin-like reproductive functions, LSCPR mediates vasopressin-like metabolic functions of Lys-conopressin as well.

• Lopes da Silva S, Van Helvoort A, Burbach JP
• The human vasopressin-oxytocin gene family: no evidence for additional neurophysin-related genes.
• Mol Cell Endocrinol. 1993; 98: 61-6
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• Over the last 20 years several observations at the peptide level have indicated the possible existence of additional members of the vasopressin (VP)-oxytocin (OT) gene family in mammals. In this study, the human genome was analyzed for the existence of genes structurally related to the VP and OT genes. Human genomic blots probed under low stringency conditions with exon B of the human OT gene, that codes for the conserved constant region of neurophysin, revealed the presence of two distinct bands in addition to the known VP and OT gene fragments. Five clones were obtained from a library of genomic EcoRI fragments ranging from 4-8 kb, that comprised both low stringency signals, by low stringency hybridization with the OT exon B probe. One clone of 7 kb hybridized at high stringency conditions to bands of the same size as previously detected with OT exon B on a human genomic blot. However, no similarity was observed between the open reading frames of this clone and the neurophysin portion of the OT gene. Another clone of 4.8 kb was identical to a fragment of the gene for the human bone morphogenetic factor hBMP-6, a member of the TGF-beta family. The hBMP-6 gene was not detected by low stringency hybridization of the human genomic blot with the OT exon B probe. No significant similarity was found between the amino acid sequences of human OT neurophysin and hBMP-6. Therefore, no evidence can be provided that the human genome contains additional neurophysin-related genes.(ABSTRACT TRUNCATED AT 250 WORDS)

• van Kesteren RE et al.
• A vasopressin-related peptide in the mollusc Lymnaea stagnalis: peptide structure, prohormone organization, evolutionary and functional aspects of Lymnaea conopressin.
• Prog Brain Res. 1992; 92: 47-57

• McMaster D, Kobayashi Y, Lederis K
• A vasotocin-like peptide in Aplysia kurodai ganglia: HPLC and RIA evidence for its identity with Lys-conopressin G.
• Peptides. 1992; 13: 413-21
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• The presence of a vasopressin (VP)- or vasotocin (VT)-like peptide in the central nervous system of the gastropod mollusc Aplysia has been indicated previously. In the case of Aplysia californica, HPLC and RIA evidence suggested the peptide was VT-like but not identical with the nonmammalian vertebrate peptide [Arg8]VT (AVT). In the present study, anterior ganglia extracts from the related species Aplysia kurodai were analyzed by HPLC followed by RIA. Further analysis of the major AVT-IR peak showed it to be indistinguishable, in three distinct solvent systems, from the sea snail venom peptide Lys-conopressin G, but to be different from the vertebrate peptides [Arg8]VP (AVP), [Lys8]VP (LVP), AVT, oxytocin (OT), mesotocin, isotocin, aspargtocin, glumitocin, and valitocin, from the sea snail venom peptide Arg-conopressin S, and from the peptides [Lys8]VT and [Gln8]OT. In addition, the carboxymethylated (CM) A. kurodai peptide had the same HPLC retention time as CM-Lys-conopressin G. The HPLC/RIA results suggest that (i) based on the properties of the solvent systems used, the A. kurodai peptide has two basic amino acids (like the conopressins but unlike the vertebrate peptides), and (ii) there is a high probability that the A. kurodai peptide is identical with Lys-conopressin G.

• Richter D
• Molecular events in expression of vasopressin and oxytocin and their cognate receptors.
• Am J Physiol. 1988; 255: 20719-20719
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• This article focuses on some aspects of neuropeptide gene expression using as examples the hypothalamic hormones vasopressin and oxytocin. Their transcriptional and translational expressions as polyproteins have been outlined, including the analysis of the genetic defect in the expression of vasopressin by the Brattleboro gene. The interaction of neuropeptide hormones with their target organs is discussed. A molecular approach toward the identification of the respective neuropeptide hormone receptors is described.

• Cruz LJ et al.
• Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms.
• J Biol Chem. 1987; 262: 15821-4
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• The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.

• Rehbein M et al.
• The neurohypophyseal hormones vasopressin and oxytocin. Precursor structure, synthesis and regulation.
• Biol Chem Hoppe Seyler. 1986; 367: 695-704
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• Complete cDNA sequences for the vasopressin and oxytocin precursor polyproteins have been determined for the rat, calf, human and pig (vasopressin only), indicating the essential conservation of the precursor structures throughout mammals. DNA probes specific for vasopressin or oxytocin mRNAs have been used to identify both classic (hypothalamic) and novel (thymus, corpus luteum, phaeochromocytoma) sites of hormone expression. Semiquantitative DNA/RNA hybridization suggests that in rats expression of the vasopressin and oxytocin genes is positively effected by osmotic stress, negatively by a systemically applied excess of vasopressin; in the latter experiment a reduction in the hypothalamic levels of vasopressin and oxytocin mRNAs in normal and Brattleboro rats have been observed. This suggests a feedback regulation by the hormone as a possible element in controlling the transcription of the vasopressin gene.

• Ivell R, Schmale H, Richter D
• Vasopressin and oxytocin precursors as model preprohormones.
• Neuroendocrinology. 1983; 37: 235-40
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• Using a combination of in vitro methodology, including cell-free translation, two-dimensional peptide mapping and recombinant DNA techniques, the structure of the precursors of the hypothalamic nonapeptide hormones vasopressin and oxytocin has been elucidated. Both hormone precursors are model cellular polyproteins in that they comprise several different entities within the same polypeptide molecule. In each precursor, the nonapeptide hormone follows immediately the signal peptide and is, in turn, attached to its respective carrier neurophysin. The vasopressin precursor also includes a pituitary glycoprotein at its C-terminus. The posttranslational processing of the precursors to set free the nonapeptide hormones is thus a critical regulatory step, which can in part be simulated in the quasi in vivo system of the Xenopus laevis oocyte. The preprohormones to vasopressin and oxytocin illustrate well the convenience of the in vitro experimental approach in understanding the function of the peptidergic neuron.

• Majzoub JA, Rich A, van Boom J, Habener JF
• Vasopressin and oxytocin mRNA regulation in the rat assessed by hybridization with synthetic oligonucleotides.
• J Biol Chem. 1983; 258: 14061-4
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• Vasopressin and oxytocin are nonapeptide hormones that regulate water metabolism and lactation, respectively. To study the regulation of the vasopressin and oxytocin genes at the mRNA level, we constructed a series of synthetic oligonucleotides, from 8 to 15 bases in length, for use in filter-blot hybridization assays (Northern blots) of hypothalamic mRNA levels and for primed synthesis of cDNAs from which we determined the nucleotide sequences of the 5' regions of the vasopressin and oxytocin mRNAs. A 20-fold increase occurred in the amounts of the two mRNAs present in the hypothalami of rats drinking 2% saline for three weeks. In addition, the sequence analyses of the cDNAs provided the complete amino acid sequences of the NH2-terminal signal peptides of the rat vasopressin and oxytocin precursors. Thus, synthetic oligonucleotides consisting of as few as eight nucleotides can be used to prime reverse transcription of specific cDNAs from hypothalamic RNA, and pentadecanucleotide hybridization probes readily detect changes in levels of vasopressin and oxytocin mRNAs in response to osmotic stress.

• Brownstein MJ
• Biosynthesis of vasopressin and oxytocin.
• Annu Rev Physiol. 1983; 45: 129-35