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A group of ten hormones in humans are structurally related and known as the secretin superfamily. These hormones bind to G-protein-coupled receptors that activate the cAMP pathway and are clustered as the secretin or B family. We used an evolutionary approach with zebrafish as a model to understand why some of these hormones, such as peptide histidine-methionine (PHM) and pituitary adenylate cyclase-activating polypeptide (PACAP)-related peptide (PRP) in humans lack a receptor. We used molecular techniques to clone two full-length receptor cDNAs in zebrafish, which were analyzed for amino acid sequence and ligand-binding motifs, phylogenetic position, synteny, tissue expression, functional response, and signaling pathway. Evidence is provided that the two cDNAs encoded the peptide histidine-isoleucine (PHI) receptor and PRP receptor, which is known as GHRH-like peptide (GHRH-LP) receptor in non-mammals. Further, we cloned a zebrafish cDNA encoding the peptides PHI and vasoactive intestinal peptide (VIP). The PHIR had been previously labeled as one type of a VIP–PACAP (VPAC2R) shared receptor based only on sequence data. The PHIR cDNA, transfected into COS7 cells, responded to zebrafish PHI in a sensitive and dose-dependent manner (EC50=1.8×10−9 M) but not to PACAP and VIP. The GHRH-LP receptor responded to both zebrafish GHRH-LP1 and GHRH with a 3.5-fold greater response to the former. For comparison, two zebrafish receptors (PAC1R and VPAC1R) and two human receptors (VPAC2R and GHRHR) were tested with human and/or zebrafish peptides. Unexpectedly, zebrafish VIP activated its PAC1R suggesting that in evolution, PAC1R is not always a specific receptor for PACAP. We conclude that zebrafish, like goldfish, have a specific receptor for PHI and GHRH-LP. Our evidence that zebrafish PHI is more potent than human PHM in activating the human VPAC2R (EC50=7.4×10−9 M) supports our suggestion that the VPAC2R and PHIR shared a common ancestral receptor.

GnRH agonists or antagonists are currently utilized as therapeutic agents in a number of diseases. A side-effect of prolonged treatment with GnRH analogues is hypoestrogenism. In this study, we tested the in vitro potency of different GnRH analogues originally found to be partial agonists (i.e. analogues with decreased efficacy for activating or stimulating their cognate receptor) as well as novel analogues, to identify compounds that might potentially be useful for partial blockade of gonadotrophin release. Cultured COS-7 cells transiently expressing the rat or human GnRH receptor (GnRHR) were exposed to increasing concentrations (10−8 to 10−5 M) of GnRH analogues (c(4–10)[Asp4,DNal6,Dpr10]-GnRH; c(4–10) [Dpr4,DNal6,Asp10]-GnRH; c(4–10)[Cys4,10,DNal6]-GnRH; c[Eaca1,DNal6]-GnRH; c[Gly1,DNal6]-GnRH; c[βAla1,DTrp6]-GnRH; c[Dava1,DNal6]-GnRH; c[Gaba1, DNal6]-GnRH), and the ability of these analogues to provoke or antagonize GnRH-stimulated inositol phosphate production was assessed. With both human and rat GnRHRs, c[Eaca1,DNal6]-GnRH, c[Gly1,DNal6]-GnRH, c[βAla1,DTrp6]-GnRH and c[Dava1,DNal6]-GnRH exhibited partial agonist activity (35–87% of the maximal efficacy shown by 10−6 M GnRH), whereas c[Gaba1,DNal6]-GnRH behaved as a partial agonist with the human GnRHR and as full agonist with the rat GnRHR. c(4–10)[Asp4, DNal6,Dpr10]-GnRH and c(4–10)[Dpr4,DNal6,Asp10]-GnRH exhibited full antagonist activity with both GnRHRs, and c(4–10) [Cys4,10,DNal6]-GnRH was a weak, partial agonist with the human GnRHR and a full antagonist with the rat GnRHR. With the exception of c[Gaba1,DNal6]-GnRH stimulation of the human GnRHR, and c[Dava1,DNal6]-GnRH and c[Gaba1, DNal6]-GnRH stimulation of the rat GnRHR, all partial agonists also exhibited antagonist activity in the presence of the exogenous full agonist. The results demonstrate that structurally similar analogues display variable potencies and efficacies in vitro for a specific GnRHR as well as for the human versus the rat GnRHR. Their ultimate in vivo usefulness to treat clinical conditions in which complete suppression of gonadotroph activity is not required remains to be investigated.

The venoms of cone snails are rich in multiply disulfide-crosslinked peptides, the conotoxins. Conotoxins are grouped into families on the basis of shared cysteine patterns and homologous molecular targets. For example, both the kappaA- and alphaA-conotoxin families share the same Class IV Cys pattern (-CC-C-C-C-C-), but differ in their molecular targets. The kappaA-conotoxins are excitatory toxins that purportedly block potassium channels, while the alphaA-conotoxins are paralytic conotoxins that inhibit nicotinic acetylcholine receptors (nAChRs). In this work, we describe the isolation and characterization of a novel Conus peptide from venom milked from Hawaiian specimens of Conus obscurus. This peptide shares the Class IV Cys pattern but differs from both previously characterized alphaA- and kappaA-conotoxins in the spacing of amino acids between Cys resides. However, the peptide is similar to previously characterized alphaA-conotoxins in its paralytic effects on fish and its antagonist activity on the neuromuscular nAChR. Unexpectedly, the peptide differs in its disulfide bonding from alphaA-conotoxin PIVA. We have named this unique peptide alphaA-conotoxin OIVA, and we consider it the defining member of a subfamily of alphaA-conotoxins that we designate the alphaA(1-3)-conotoxins to identify them by their unique disulfide bonding framework. These results indicate that the alphaA-conotoxin family is both more structurally diverse and broadly distributed than previously believed.

To investigate the involvement of pituitary adenylate cyclase- activating polypeptide (PACAP) and GH-releasing factor (GRF) during early chick brain development, we established neuroblast- enriched primary cell cultures derived from embryonic day 3.5 chick brain. We measured increases in cAMP generated by several species-specific forms of the peptides. Dose-dependent increases up to 5-fold of control values were measured in response to physiological concentrations of human/salmon, chicken, and tunicate PACAP27. Responses to PACAP38 were more variable, ranging from 5-fold for human PACAP38 to 4-fold for chicken PACAP38, to no significant response for salmon PACAP38, compared with control values. The responses to PACAP38 may reflect a greater difference in peptide structure compared with PACAP27 among species. Increases in cAMP generated by human, chicken, and salmon/carp GRF were not statistically significant, whereas increases in response to lower-range doses of tunicate GRF27-like peptide were significant, but small. We also used immunocytochemistry and Western blot to show synthesis of the PACAP38 peptide. RT-PCR was used to demonstrate that messenger RNAs for PACAP and GRF and a PACAP-specific receptor were present in the cells. This is a first report suggesting an autocrine/paracrine system for PACAP in early chick brain development, based on the presence of the ligand, messages for the ligand and receptor, and activation of the receptor in neuroblast-enriched cultures.

In three earlier papers, the structures and biological potencies of numerous mono- and dicyclic antagonists of GnRH were reported. Among these, two families, each containing two to four members were identified that had very high antagonist potencies in an antiovulatory assay (within a factor of 2 of those of the most potent linear analogues) and high affinities (K(i) 20-fold) was observed with the substitution of [DAsp(3)] in 14 by [DGlu(3)] in 15 (K(i) = 23 nM). Dicyclic analogues possessing the (4-10) cycle and selected (1-6), (2-6), and (2-8) cycles led to analogues that were inactive at doses of 500 microgram/rat or larger. Two analogues with (1-8/4-10) cycles (16, K(i) = 1.1 nM) or (3-8/4-10) cycles (22, K(i) = 17 nM) showed full antiovulatory potency at 250 microgram/rat. None of these substitutions yielded analogues potent enough (>80% inhibition of ovulation at 5 microgram/rat or less and K(i) < 0.5 nM) to be candidates for structural analysis by NMR. On the other hand, four dicyclic (1, 1'-5/4-10) analogues met this criterion: dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DTrp(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (32, K(i) = 0.22 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(Gly),DCpa(2),DNal(3),Asp(4),Dbu(5 ), DNal(6), Dpr(10)]GnRH (34, K(i) = 0.38 nM), dicyclo(1, 1'-5/4-10)[Ac-Asp(1)(betaAla),DCpa(2), DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (40, K(i) = 0.15 nM), and dicyclo(1, 1'-5/4-10)[Ac-Glu(1)(Gly), DCpa(2),DTrp(3),Asp(4),Dbu(5),DNal(6), Dpr(10)]GnRH (41, K(i) = 0.24 nM). Since they differed slightly in terms of the (1,1'-5) bridge length (21 and 22 atoms) and bridgehead configuration, we may hypothesize that they assume similar bioactive conformations that satisfy a very discriminating receptor, since many other closely related analogues were significantly less potent.

We have synthesized significant amounts of the most potent member of the rat corticostatins that inhibits ACTH-induced corticosteroid and compared its structure to that of the natural hormone. The cystine bridging arrangement that corresponds to that reported for a human defensin (3–31, 5–20, 10–30) was determined. The in vitro corticostatic activity of the synthetic rat corticostatin R4 paralleled that of the natural R4. Biological studies in vivo showed that doses of 8 or 12 mg corticostatin/kg effectively interfered with corticosterone release in stressed rats. We conclude that in the assays that were used, the biological activity of the synthetic and natural molecules was identical. The availability of significant amounts of synthetic material will make possible studies investigating the physiological role played by corticostatins in modulating the activity of the hypothalamic-pituitary-adrenal axis.

The C-terminally amidated CRF antagonist astressin binds to CRF-R1 or CRF-R2 receptors with low nanomolar affinity while the corresponding astressin-acid has >100 times less affinity. To understand the role of the amide group in binding, the conformations of astressin-amide and astressin-acid were studied in DMSO using NMR techniques. The 3D NMR structures show that the backbones of both analogs prefer an α-helical conformation, with a small kink around Gln26. However, astressin-amide has a well-defined helical structure from Leu27 to Ile41 and a conformation very similar to the bioactive conformation reported by our group (Grace et al., Proc Natl Acad Sci USA 2007, 104, 4858–4863). In contrast, astressin-acid has an irregular helical conformation from Arg35 onward, including a rearrangement of the side chains in that region. This structural difference highlights the crucial role of the C-terminal amidation for stabilization of astressin's bioactive conformation. © 2007 Wiley Periodicals, Inc. Biopolymers 87: 196–205, 2007. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Complementary deoxyribonucleic acid (cDNA) encoding goldfish preproghrelin was identified using rapid amplification of the cDNA ends (RACE) and reverse transcription (RT)-polymerase chain reaction (PCR). The 490 bp cDNA encodes a 103 amino acid preproghrelin which has a 26 amino acid signal region, 19 amino acid mature peptide and a 55 amino acid C-terminal peptide region. The mature peptide region of goldfish ghrelin has two putative cleavage sites and amidation signals (GRR); one after 12 amino acids and the other after 19 amino acids. The serine (S) in the second amino acid position in the "active core" of ghrelin is substituted with threonine (T). The goldfish ghrelin gene has four exons and three short introns and resembles the human ghrelin gene. Ghrelin messenger RNA (mRNA) expression was detected in the brain, pituitary, intestine, liver, spleen and gill by RT-PCR followed by Southern blot analysis, and in the intestine by Northern blot. Intracerebroventricular (ICV) injection of n-octanoylated goldfish ghrelin (1-19) stimulates food intake in goldfish.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

Strategies to generate competitive antagonists of bioactive peptides include several possible structural modifications such as the introduction of D-residues and of reduced amide bonds, the substitution of amino acid side chains, dimerization of fragments, and deletion of part of the sequence, among others. Whereas we have identified the two most likely residues responsible for receptor activation in corticotropin releasing factor (CRF) (Ser7 and Leu8)1 and generated potent antagonists by deleting residues 1-8,2,3 the question remained as to whether we could generate CRF antagonists with enhanced affinity after reduction of amide bonds at the N-terminus of CRF or through subtle alteration of those residues' side chains. Reduced amide bond replacements (psi[CH2NH]) between residues 6-9 in oCRF(5-41) (11, 12, 15) analogues consistently yielded potencies of1% that of oCRF. Except for the 10psi11 and 12psi13 analogues 19 and 20, reduced amide bond replacements were generally well-tolerated in the longer hCRF(4-41) analogues, with the 7psi8-, 8psi9-, and 9psi10-modified peptides (13, 14, 18) yielding potencies that were 2-4 times that of hCRF. Although somewhat promising as agonists, they were, however, 3-7 times less potent than the parent [D-Pro4Nle21,38]-hCRF(4-41) (2). Since O-alkylation of Tyr3 in vasopressin yields an antagonist, and since Ser7 is one of the eight fully conserved residues in the CRF family (inclusive of sauvagine, urocortins, and urotensins) and likely to be critical for receptor binding, we synthesized cyclo(30-33)[Ser(OMe)7,D-Phe12,Nle21,Glu30,Lys33 ,Nle38]Ac-hCRF(7-41) (22), which was found to exhibit full efficacy and 40% of the potency of cyclo(30-33)[D-Phe12,Nle21,Glu30,Lys33, Nle38]Ac-hCRF(7-41) (5). Other substitutions at position 7 included aminoglycine (23, 24) and alkylated and/or acylated [alpha or alpha'-methyl (25-28), alpha'-formyl (29, 30), alpha'-formyl, alpha'-methyl (31), alpha'-acetyl (32), alpha'-acetyl, alpha'-methyl (33)], D- or L-aminoglycines. All analogues were active although less potent than the parent compound 2, and all elicited maximal ACTH response as compared to hCRF. The most potent analogue in this series (33) had the bulkiest side chain, Agl(Me, Ac), and was 60% and 80% as potent as the Ser7 analogue 5 and the Ala7 analogue 6, respectively. In conclusion, we found that neither reduction of the individual amide linkages between residues 6-11 and 12-13 nor introduction of a carbamide moiety in lieu of the side chain of Ser7 led to CRF antagonists.

Les vinyllithiums derives des triisopropyl-2,4,6 benzenesulfonylhydrazones correspondantes subissent une addition intramoleculaire pour donner des composes cycliques lithies qui peuvent etre pieges par des electrophiles tels que l'eau