Somatostatin- and urotensin II-related peptides: molecular diversity and evolutionary perspectives

Regulatory Peptides 69 (1997) 95–103

Somatostatin- and urotensin II-related peptides: molecular diversity and evolutionary perspectives a, b b J. Michael Conlon *, Herve Tostivint , Hubert Vaudry a

b

Regulatory Peptide Center, Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology INSERM U413, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France Received 18 November 1996; revised 5 February 1997; accepted 5 February 1997

Abstract Recent advances in the fields of molecular cloning and peptide purification necessitate a reappraisal of our views concerning the evolution of the genes encoding somatostatin-related peptides. The currently widely held view that the genomes of tetrapods contain only the preprosomatostatin-I (PSS-I) gene, encoding somatostatin-14, with a second preprosomatostatin gene being expressed only in teleost fish is no longer tenable. Identification of genes encoding both somatostatin-14 and the somatostatin-related peptide, cortistatin in mammals, identification of the PSS-I and PSS-II preprosomatostatin genes in amphibia, and the isolation of gene products from at least two non-allelic preprosomatostatin genes in lampreys suggests the alternative hypothesis that duplication of the PSS-I gene occurred early in evolution, predating or concomitant with the appearance of the chordates. We speculate that at least two somatostatin genes are expressed in all classes of vertebrates but these genes have evolved at very different rates. It is probable that the preprosomatostatin-II (PSS-II) gene, encoding [Tyr 7 ,Gly 10 ]somatostatin-14 or a related peptide, arose from a second independent duplication of the PSS-I gene in the ancestor of present-day teleost fish at a time after the divergence of the teleost stock from the line of evolution leading to tetrapods. The recent isolation of urotensin II, a peptide which contains a region of structural similarity but is not evolutionarily related to somatostatin-14, from the central nervous systems of lampreys, elasmobranchs and amphibia necessitates that we modify the accepted view that urotensin II is exclusively a product of the caudal neurosecretory system of teleost fish.  1997 Elsevier Science B.V. All rights reserved Keywords: Somatostatin; Cortistatin; Urotensin II; Gene duplication

1. Introduction Almost 25 years have passed since the purification and characterization of somatostatin-14 [1], one of the factors present in an extract of sheep hypothalami that was responsible for the inhibition of growth hormone release from dispersed rat pituitary cells [2]. With the development of antisera specific for somatostatin for use in immunohistochemistry and radioimmunoassay, it became apparent

*Corresponding author. Tel.: 1 1 402 2801733; fax: 1 1 402 2802690; e-mail: [email protected] 0167-0115 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0167-0115( 97 )02135-6

that somatostatin, or a structurally related antigen, occurs in the nervous systems and / or alimentary tracts of a wide range of vertebrate and invertebrate species (for review see Ref. [3]). In common with other regulatory peptides, the biosynthesis of somatostatin involves the action of processing enzymes (signal peptidase and prohormone convertases) on a precursor protein (preprosomatostatin). The amino acid sequence of PSS-I, the biosynthetic precursor of somatostatin-14 and somatostatin-28, has been deduced from the nucleotide sequence of cDNAs and / or genomic fragments from a range of tetrapod species [4–12] (see Section 2). A second preprosomatostatin gene (preprosomatostatin-II; PSS-II), encoding [Tyr 7 ,Gly 10 ]somatostatin-14 or a related peptide, is ex-

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pressed, along with the PSS-I gene, in the pancreatic islets of teleost fish [13–16] (see Section 3). The currently widely held view with respect to the evolution of somatostatin genes is based upon a statistical analysis of the nucleotide sequences of the cDNAs encoding PSS-I and PSS-II [7,17]. PSS-I is regarded as the only somatostatin gene that is present in genomes of tetrapods and its structure has been strongly conserved. The less well conserved PSS-II gene is expressed only in teleost fish and is considered to have arisen from a duplication event occurring in the ancestor of these fish at a time after the divergence of the teleost stock from the line of evolution leading to tetrapods (approximately 400 million years ago). This review will highlight several recent advances in the field that necessitate a revision of our views regarding the molecular evolution of somatostatin, leading to the alternative hypothesis that the first duplication of an ancestral somatostatin gene occurred much earlier in vertebrate evolution than the time of emergence of the teleost fish. It is more than 70 years since the first description of secretory neurons in the caudal spinal cord region of fish [18], and almost 40 years since the connection between these neurons and the urophysis, the neurohemal organ of teleost fish, led to the concept of a caudal neurosecretory system [19]. Extracts of the urophyses of teleosts contain substances with a quite remarkably wide range of pharmacological activities in both mammalian and fish test systems [20,21]. The term ‘urotensin II’ (U II) was originally used to describe the smooth muscle-stimulating activity in urophysial extracts, particularly the ability to contract the trout hind-gut [20], but it was not until 1980 that structural characterization of a U II-related peptide was accomplished [22]. U II is structurally similar to somatostatin-14 and the peptides share some biological properties (for review see Ref. [21]), but sequence analysis of the cDNA encoding preprourotensin II from the carp [23] has shown that this protein and preprosomatostatin are probably not homologous (see Section 5). In general, functional studies have focused, with limited success, upon the elucidation of the physiological role of U II in teleost fish (for review see Ref. [24]), and the peptide is often regarded as exclusively the product of the teleost urophysis. In the light of the recent isolation of biologically active U II-related peptides from tissues of the central nervous systems of amphibia [25] and Agnatha [26] and from both the caudal neurosecretory systems and central nervous systems of chondrostean [26,27] and elasmobranch fish [28,29], this article presents the alternative hypothesis that U II has a much wider distribution in vertebrate tissues where its physiological roles are beginning to be understood. For the convenience of the reader, the structural organization of some of the biosynthetic precursors referred to this article (frog PSS-I, frog PSS-II, rat preprocortistatin, anglerfish PSS-II and carp preprourotensin-II) is compared schematically in Fig. 1.

Fig. 1. A schematic representation of (a) frog preprosomatostatin-1 (PSS-1), (b) frog preprosomatostatin-2 (PSS-2), (c) rat preprocortistatin (PCS), (d) anglerfish preprosomatostatin-II (PSS-II) and (e) carp preprourotensin-II. The arrows indicate the cleavage sites that lead to generation of the mature peptides.

2. Peptides derived from preprosomatostatin-I containing the somatostatin-14 sequence The full sequence of somatostatin-14 is not required for biological activity. Structure-activity studies using synthetic peptides have demonstrated that analogs containing only the residues –Phe 7 –Trp 8 –Lys 9 –Thr 10 – in an appropriate cyclic conformation function as agonists [30]. Despite this fact, somatostatin-14 represents one of the most strongly conserved peptides in vertebrate evolution. Peptide isolation and amino acid sequence analysis studies have shown that the complete primary structure of somatostatin-14 is identical in mammals (human [31], pig [32], sheep [1], rat [33], guinea-pig [34]), birds (chicken [35], pigeon [36]), reptiles (alligator, Alligator mississipiensis [37], turtle, Psuedemys scripta [38]), amphibia (the anurans Rana ridibunda [39] and Rana pipiens [40] and the urodele Amphiuma tridactylum [41]), several species of teleost (anglerfish Lophius americanus [42], catfish, Ictalurus punctatus [43], Daddy sculpin, Cottus scorpius [44], flounder Platichythes flesus [44], Coho salmon, Oncorhynchus kisutch [45], European eel, Anguilla anguilla [46], tilapia, Oreochromis nilotica [47]), Holostei (bowfin, Amia calva [48]), Chondrostei (N. American paddlefish, Polyodon spathula (J.M. Conlon, unpublished data)) and Elasmobranchii (the ray, Torpedo marmorata [49]) and in Agnatha (sea lamprey, Petromyzon marinus [50], river lamprey, Lampetra fluviatilis [51] and the Atlantic hagfish, Myxine glutinosa [52]). The primary structure of preprosomatostatin-I (PSS-I), the biosynthetic precursor of somatostatin-14, has been deduced from the nucleotide sequence of cDNAs and / or genomic fragments from four mammalian species (human [4,5], monkey [6], ox [7], rat [8–10]), chicken [11], the frog, R. ridibunda [12], anglerfish [13] and catfish [14]. A comparison of the amino sequences of the precursors [7,17] has shown that quite strong evolutionary pressure has acted to conserve the complete sequence of PSS-I, not

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just the somatostatin-14 sequence. Frog PSS-I exhibits 75 and 85% amino acid sequence identity with human and chicken preprosomatostatins, respectively, and although the tetrapod lineage diverged from the fish lineage some 350–400 million years ago, frog PSS-I shows 59 and 48% sequence identity with the corresponding regions of PSS-I from catfish and anglerfish, respectively [12]. The Nterminal propeptide segment of PSS-I has been implicated in mediating intracellular sorting of the precursor to the trans Golgi network and targeting into the regulated secretory pathway [53,54]. In mammals, the pathway of post-translational processing of the 92-amino acid residue prosomatostatin-I is tissue-specific [55]. In the pancreas, stomach and hypothalamus, cleavage at the Arg 77 –Lys 78 dibasic residue processing site and at the site of a single arginine (Arg 64 ) residue generates somatostatin-14, somatostatin-28-(1– 12)-peptide and either prosomatostatin-(1–63)-peptide [56] or prosomatostatin-(1–64)-peptide [57] (Fig. 1). In the D-cell of the intestinal mucosa, cleavage at the single arginine residue predominates so that PSS-I is processed to somatostatin-28. The convertases PC2, PC1 and PACE4 have been implicated in the formation of somatostatin-14, whereas PACE4 and furin may be involved in cleavage at the monobasic site [58]. Somatostatin-28 is more potent than somatostatin-14 in several test systems and is the preferred agonist for the widely distributed SSTR5 somatostatin receptor [59]. It is not surprising, therefore, that the amino acid sequence of the somatostatin-28 region of PSS-I has been particularly strongly conserved. For example, the primary structures of frog and human somatostatin-28 differ by only two amino acid residues (Fig. 2). PSS-I contains a second domain in the central region of the peptide –Leu 29 –Ala 30 –Glu 31 –Leu 32 –Leu 33 – that has been fully conserved between fish and mammals, and it is noteworthy that the Leu 32 –Leu 33 bond represents a cleavage site in the pig intestine [60]. Analysis of the molecular forms of somatostatin-like immunoreactivity in extracts of the islet organ and gut of

Fig. 2. A comparison of the primary structures of peptides derived from prosomatostatin-I and containing the somatostatin-14 sequence (shown in bold type) from different vertebrate taxa. (—) Sequence identity: the frog, anglerfish and catfish sequences are deduced from the nucleotide sequence of a cDNA. Gaps denoted by (*) have been introduced into the amino acid sequences of the catfish and bowfin peptides to maximize structural similarity.

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the Agnathan, the Atlantic hagfish, has shown that prosomatostatin-I is processed primarily to a 34-residue peptide in both tissues, with somatostatin-14 present only as a minor component [52] (Fig. 2). Similarly, in the pancreas of the bowfin (Amiiformes) a 26-amino acid residue peptide derived from PSS-I is the predominant molecular form [48] (Fig. 2). In these species, cleavage at the Arg–Lys dibasic residue site in PSS-I represents only a minor pathway of processing, suggesting that structural features in the N-terminal domain of the prosomatostatins may be important in targeting the processing enzyme. In hagfish somatostatin-34, an arginine residue is present at position 6 but does not function as a monobasic processing site. In contrast, somatostatin-14 was the only immunoreactive component detected in extracts of brain, stomach, pancreas and gut of the ray, Torpedo marmorata, provoking the speculation that the tissue-specific ability to regulate the production of multiple forms of somatostatin from a single precursor may have arisen relatively late in evolution [49].

3. Peptides from teleost fish derived from preprosomatostatin-II The segregation of endocrine and exocrine tissue in the Brockmann bodies or principal islets of certain highly evolved teleost fish (particularly those belonging to the order Lophiformes and order Perciformes) has greatly facilitated the task of the peptide chemist in the purification of piscine islet hormones. As shown in Fig. 3, somatostatin-related peptides of between 25 and 28 amino acid residues containing the sequence of [Tyr 7 ,Gly 10 ]somatostatin-14 at their C-termini have been isolated from extracts of islet tissue from the anglerfish [61,62], Daddy sculpin [44,63], flounder [44], Coho salmon [45], European eel [46] and tilapia [47] and from the intestine of the goldfish, Carassus auratus [64]. The

Fig. 3. A comparison of the primary structures of peptides derived from prosomatostatin-II isolated from the Brockmann bodies of teleost fish. (—) Residue identity: (*) denotes a residue deletion that has been introduced to maximize sequence similarity. The [Tyr 7 ,Gly 10 ]somatostatin-14 sequence is shown in bold type.

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anglerfish and eel peptides were also isolated in variant forms which contained a hydroxylysine residue at position 9 of the [Tyr 7 ,Gly 10 ]somatostatin-14 sequence. This posttranslational modification is similar to the hydroxylation of Lys–Gly in collagen-like structures but its significance is unknown. As shown in Fig. 3, primary structures of the PSS-IIderived gene products have been much less well conserved than the somatostatin-related peptides derived from PSS-I. The eel and tilapia peptides contain the substitutions Phe 12 → Pro and Phe 12 → Leu within the [Tyr 7 ,Gly 10 ]somatostatin-14 region and, even among fishes of the same teleost order, the amino acid sequences in the N-terminal regions of the peptides are highly variable. The complete amino acid sequence of PSS-II has been deduced from nucleotide sequence analysis of cDNAs prepared from islet mRNAs from the anglerfish [13] and from a salmonid, the rainbow trout, Oncorhynchus mykiss [16]. Anglerfish PSS-II and trout PSS-II comprise 125- and 115-amino-acid residues, respectively. A comparison of the structures of the two precursors indicates that the overall conservation of sequence has been only moderate (46% amino acid identity between trout and anglerfish PSS-II compared with 37% between trout PSS-II and human PSS-I) [16]. At this time, there is no evidence for expression of the PSS-II gene in species other than teleosts. Cleavage of the signal peptide in anglerfish PSS-II occurs at the site of the Ser 24 –Gln 25 bond [65]. Chromatographic analysis of peptides derived from prosomatostatinII in extracts of Brockmann bodies from the anglerfish [65], sculpin [44] and flounder [44] has shown that the precursors are cleaved at a dibasic residue site in the central region of the molecule (corresponding to Lys 61 – Arg 62 in anglerfish PSS-II). The resulting fragments are further cleaved at monobasic processing sites (Arg 48 and Arg 97 in anglerfish PSS-II) to yield the mature peptides shown in Fig. 2, but processing at the Lys 110 –Arg 111 site preceding the [Tyr 7 ,Gly 10 ]somatostatin-14 sequence does not occur [66] (Fig. 1). Two somatostatin-related peptides have been isolated from extracts of the Brockmann bodies of the channel catfish . One peptide is identical to somatostatin-14 [43], but the second contains 22 amino acid residues and exists in multiple O-glycosylated forms that differ in the nature of the carbohydrate residues linked to the Thr 5 residue [67]. Catfish somatostatin-22 shows only very limited sequence similarity with other somatostatins derived from PSS-II (Fig. 3). The primary structure of the biosynthetic precursor of catfish somatostatin-22 has been deduced from the nucleotide sequence of a cloned cDNA [15] and comprises 105 amino acids. The sequence of the mature peptide is preceded by an Arg–Arg processing site. A comparison of the amino acid sequence of this precursor with the corresponding sequences of PSS-II from other teleosts [16] shows that conservation of the complete

prohormone sequence has been poor (28% identity between the catfish somatostatin-22 preprohormone and anglerfish PSS-II, compared with 50% identity between catfish PSS-I and anglerfish PSS-I). Previous commentators have regarded the second catfish gene as the homologue of the anglerfish PSS-II gene and have concluded that the two duplicate genes in catfish and anglerfish were produced by a single duplication event in their common ancestor which was followed by an accelerated rate of evolution [7]. The catfish, belonging to the specialized Ostariophysi taxon, probably diverged from the Paracanthopterygii (anglerfish) relatively early in teleost evolution [68]. However, the emergence of the Ostariophysi was coincident with, or occurred just prior to, the emergence of the Salmonidae, but trout PSS-II shows so little structural similarity to the catfish somatostatin-22 precursor outside the C-terminal region so as to preclude valid statistical analysis [16]. Thus, the question of the evolutionary relationship between the gene encoding catfish somatostatin-22 and PSS-I and PSS-II remains unresolved at this time. An antiserum raised against catfish somatostatin-22 detected low levels of immunoreactivity in an extract of anglerfish islet tissue, but not in pancreatic tissues of tetrapods, raising the possibility that a third somatostatin gene may be expressed in Brockmann bodies of teleost fish [69].

4. Molecular variants of somatostatin-14 from phylogenetically ancient fish Evidence for the expression of duplicate somatostatin genes in the Agnatha has been obtained from peptide isolation studies with tissues from lampreys (Petromyzontiformes) which, together with the hagfish (Myxineformes), are the only surviving groups from the agnathan phase of early vertebrate evolution [70]. The lampreys are believed to have diverged from the lineage leading to tetrapods at least 550 million years ago [71]. Somatostatin-14, identical in structure to mammalian somatostatin-14, has been isolated from extracts of the brains of the holarctic lampreys Petromyzon marinus [50] and Lampetra fluviatilis [51], whereas higher Mr forms, with 34 and 35 amino acid residues, terminating with the sequence of [Ser 12 ]somatostatin-14, were isolated from pancreatic tissue from the same species (Fig. 3) [51,72]. The data provide evidence that at least two non-allelic somatostatin genes are expressed in lamprey tissues and the pathways of post-translational processing of the preprosomatostatins are different. Present-day lampreys are organized into three families with the holarctic lampreys (36 species) placed in the single family Petromyzontidae, and those of the southern hemisphere (four species) placed in either the Mordaciidae or Geotriidae families [73]. As the fossil record is incomplete, the phylogenetic relationships between the different

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Fig. 4. A comparison of the primary structures of molecular variants of somatostatin-14 isolated from the tissues of phylogenetically ancient fish and from frog brain with the structure of rat corticostatin-14 deduced from the nucleotide sequence of a cDNA. (—) Residue identity; (*) residue deletion. The [Ser 12 ]somatostatin-14 sequence is shown in bold type.

families remain uncertain but it has been proposed that the southern hemisphere lampreys evolved at different times in the pre-Tertiary period from stocks similar to those represented today by the holarctic genus Ichthyomyzon [74]. A somatostatin-related peptide with 33 amino acid residues, also terminating with the sequence of [Ser 12 ]somatostatin14, has been isolated from the pancreas of the southern hemisphere lamprey Geotria australis (Fig. 4) [75]. The presence of the substitution Thr 12 → Ser in somatostatins from all three species supports the assertion that holarctic and southern hemisphere lampreys arose from a common stock [74] and suggests that the putative duplication that led to two somatostatin genes occurred before the divergence of the Petromyzontidae and Geotriidae. This divergence is believed to be of a relatively ancient (pre-Tertiary) origin [74]. A molecular variant of somatostatin-14 containing the substitution Asn 5 → Ser has been isolated from the pancreas of a holocephalan fish, the Pacific ratfish Hydrolagus colliei [76], and a variant containing the substitution Gly 2 → Pro from the pituitary of the chondrostean fish, the sturgeon Acipenser gueldenstaedti Brandt [77] (Fig. 4). It is not known whether these species express more than one somatostatin gene.

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way of post-translational processing of PCS has not yet been elucidated but cleavage at the Lys 82 –Arg 83 potential processing site would generate a 29-amino acid peptide, cortistatin-29 in addition to, or instead of, cortistatin-14 (Fig. 1). Concurrently, cDNA clones encoding two distinct somatostatin precursors have been isolated from a cDNA library prepared from the brain of the frog, Rana ridibunda [12]. One of the cDNAs encodes a 115-amino acid preprosomatostatin (PSS-I) that shows strong sequence similarity with PSS-I from mammals and teleost fish. The other cDNA encodes a 103-amino acid protein (termed PSS-2) which terminates in the sequence of [Pro 2 ,Met 13 ]somatostatin-14 but, like preprocortistatin, shows very little structural similarity with either PSS-I or PSS-II from other species outside this C-terminal region (Fig. 5). Several pieces of evidence indicate that the PSS-2 gene is not an amphibian counterpart of the teleost PSS-II genes. The PSS-II gene is expressed primarily in the pancreatic islets, whereas the mRNA directing synthesis of PSS-2, like precorticostatin mRNA, was restricted to certain regions of the brain. Frog PSS-II lacks the single arginine residue that constitutes a monobasic processing site in PSS-II, but contains a dibasic residue processing site preceding the somatostatin-14 sequence. In fact, the tetradecapeptide [Pro 2 ,Met 13 ]somatostatin-14 has previously been isolated, together with somatostatin-14, from an extract of R. ridibunda brain [39]. A synthetic replicate of the peptide, like corticostatin-14, binds to somatostatin receptors in the selected brain regions with high affinity [12]. The precise evolutionary significance of the frog PSS-2 gene is not yet known. Frog PSS-2 may be an amphibian counterpart of rat preprocortistatin but the alternative hypothesis that a homolog of the amphibian PSS-2 gene is expressed in mammals in addition to the PSS-I and the preprocortistatin genes cannot be excluded. The data suggest the hypothesis that duplication of the

5. Cortistatin from rat and frog The recent article by de Lecea et al. [78] describing the characterization of a cDNA from rat brain encoding, cortistatin-14, a peptide with sleep-modulating properties and the ability to depress neuronal electrical activity, necessitates a reappraisal of our views on the molecular evolution of the somatostatin gene. Preprocortistatin (PCS) comprises a protein of 112 amino acid residues that terminates in the sequence of a lysine-extended somatostatin-related peptide (Fig. 4). The sequence is preceded by a Lys–Lys site, and contains the substitution Ala → Pro previously identified in somatostatin-14 from the sturgeon [77] and the substitution Thr → Ser present in somatostatins from lamprey pancreas [51,72] (Fig. 3). The path-

Fig. 5. A comparison of the primary structures of (A) preprosomatostatin1 (PSS-1) from rat and frog and (B) preprocortistatin (PCS) from rat and preprosomatostatin-2 (PSS-2) from frog as predicted from the nucleotide sequences of cloned cDNAs. The somatostatin-14 sequence is shown in bold type.

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preprosomatostatin gene PSS-I occurred much earlier in evolution than previously postulated, predating or concomitant with the appearance of the Agnatha, and that the PSS-I and preprocortistatin genes may be expressed in all classes of vertebrate. The lack of sequence similarity within the N-terminal and central regions of frog PSS-2 and rat preprocortistatin compared with the corresponding regions of frog PSS-I and rat PSS-I (Fig. 5) is consistent with the view that the two somatostatin-related genes are evolving at vastly different rates. This situation has a precedent in another area of neuroendocrinology. For example, successive duplications of a putative ancestral gene have led a distinct gene encoding pancreatic polypeptide whose rate of mutation greatly exceeds that of genes encoding neuropeptide Y and peptide tyrosine– tyrosine [79]. Similarly, duplications of a glucagon-related domain within the ancestral preproglucagon gene have given rise to a precursor containing the sequences of glucagon, glucagon-like peptide-1 and glucagon-like peptide-2 that have evolved at markedly different rates [80].

6. Urotensin II-related peptides

Fig. 6. A comparison of the primary structures of urotensin II-related peptides isolated from tissues of either the caudal neurosecretory system or the central nervous system of species of different vertebrate taxa. (—) Residue identity; (*) residue deletion.

Morphological and histological studies have established that the caudal portion of the spinal cord of jawed fishes incorporates a neurosecretory system. In teleost fish, columns of linearly arranged neuronal cell bodies, located in the caudal spinal cord, project axons into a well-defined and highly vascularized organ, the urophysis whereas in elasmobranch and chondrostean fish, nerve terminals are not concentrated into a compact urophysis but large caudal neurosecretory neurons project onto diffuse neurohemal areas on the ventral surface of the posterior spinal cord [81]. U II was first isolated in pure form from an extract of the urophysis of the goby, Gillichthys mirabilis using a trout hind gut contraction assay to monitor purification [22]. Goby U II is a cyclic dodecapeptide whose amino acid sequence is shown in Fig. 6. The primary structure of U II has now been established for several teleosts (sucker, Catostomus commersoni [82], carp, Cyprinus carpio [83], flounder [84] and rainbow trout [29]). The teleost U II peptides share a common cyclic sequence (–Cys–Phe– Trp–Lys–Tyr–Cys–) at positions 6–11 in the cyclic region of the peptide, whereas the amino acid sequence at the N-terminus is highly variable. The conserved sequence is structurally similar to that in the functionally important central region of somatostatin-14 (–Phe–Trp–Lys–Thr–). However, cloning and sequence analysis of the cDNA encoding carp preprourotensin II has shown that sequence identity with PSS-I outside this region is poor indicating that U II and somatostatin are probably not derived from a common ancestral precursor [23]. The isolation of multiple forms of U II, differing slightly in amino acid sequence, from the urophyses of the sucker and carp suggests that gene encoding the peptide has undergone one or more

duplication events [83]. This multiplicity may be a consequence of the ‘tetraploidization’ events known to have occurred in these fish lineages [85]. Peptide isolation studies have established that flounder prourotensin II is cleaved at the site of two single arginine monobasic processing sites in the central region of the precursor, as well as at the tribasic residue site that generates U II [84] (Fig. 1). Although U II was originally regarded as a product exclusively of the teleost urophysis, recent immunohistochemical and chemical work has shown that this view is incorrect. Using antisera raised against goby U II, neurosecretory cells and axonal pathways containing U II-like immunoreactivity have been identified in diffuse caudal neurosecretory systems of several species of elasmobranch, chondrostean and holostean fish [86]. More recently, purification of U II-related peptides from extracts of the caudal spinal cord region of chondrostean and elasmobranch fish has shown that these peptides are structurally similar to U II peptides identified in teleost urophyses (Fig. 6). U II has been isolated from the spotted dogfish, Scyliorhinus canicula [28], sturgeon, Acipenser ruthenius [27] and N. American paddlefish, Polyodon spathula [26]. Dogfish U II is a potent vasopressor in the dogfish but appears to be devoid of metabolic or osmoregulatory properties [21]. Purification and structural characterization of U II from extracts of the whole brain of the rainbow trout [29] and the long-nosed skate Raja rhina [29] has confirmed previous immunohistochemical studies [87] that U II peptides present in central nervous systems

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of fish are structurally similar to the analogous peptides previously isolated from teleost urophyses (Fig. 6). Similarly, isolation of peptides from extracts of whole brain of the sea lamprey, P. marinus, and the river lamprey, L. fluviatilis [26], identical in structure to U II from the dogfish and skate, suggests that the primary structure of U II is much better conserved among the phylogenetically ancient fish than among teleosts. The first unequivocal demonstration of the presence of U II in the tissues of a tetrapod was provided by the isolation and structural characterization of the peptide from an extract of the whole brain of an amphibian, the European green frog, R. ridibunda [25]. Frog U II contains 13 amino acid residues rather than the 12 in all other known U II peptides but the cyclic region has been fully conserved. This region of all known U II-related peptides, is preceded by an acidic residue and all the peptides contain a hydrophobic residue at their C-terminus. Conservation of these features is consistent with the limited information regarding structure-activity relationships of the U II peptides. Goby U II-(5–12)-peptide is equipotent with goby U II in producing contraction of the isolated rat thoracic aorta, whereas U II-(6–12)-peptide has reduced potency and U II-(6–11)-peptide is almost inactive [88]. The distribution of U II-containing neurons in the frog brain and spinal cord has been mapped in detail, and it has been proposed that the peptide may play a role in the control of the striated muscles of the tongue and posterior limb as well as acting as a neurotransmitter and / or neuromodulator in the CNS [89]. Frog U II contracts frog vascular smooth muscle [90] and has a spasmogenic action on frog ileum and bladder [91]. The possibility that a U II-related peptide is present in invertebrate species is suggested by the immunohistochemical demonstration of U II-like immunoreactivity in the cerebral ganglia of the mollusk, Aplysia californica [92]. Taken together, the recent data demonstrate that U II is not exclusively a product of the teleost caudal neurosecretory system, but is widely distributed in nervous tissues of a wide range of species at vastly different evolutionary levels. Clearly, further studies are warranted to investigate the presence of U II-related peptides in the tissues of reptiles, birds and mammals.

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Acknowledgments The studies carried out in the authors’ laboratories were supported by grants from the National Science Foundation, the European Community (Human Capital and Mobility ´ Program) and the Conseil Regional de Haute-Normandie.

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