Lack of bombesin receptor-activated protein homologous protein impairs hippocampal synaptic plasticity and promotes chronic unpredictable mild stress induced behavioral changes in mice.

Bombesin receptor-activated protein (BRAP) and its homologous protein in mice, which is encoded by bc004004 gene, were expressed abundantly in brain tissues with unknown functions. We treated bc004004-/- mice with chronic unpredictable mild stress (CUMS) to test whether those mice were more vulnerable to stress-related disorders. The results of forced swimming test, sucrose preference test, and open field test showed that after being treated with CUMS for 28 days or 35 days both bc004004-/- and bc004004+/+ mice exhibited behavioural changes and there was no significant difference between bc004004+/+ and bc004004-/-. However, behavioural changes were observed only in bc004004-/- mice after being exposed to CUMS for 21 days, but not in bc004004+/+ after 21-day CUMS exposure, indicating that lack of BRAP homologous protein may cause vulnerability to stress-related disorders in mice. In addition, bc004004-/- mice showed a reduction in recognition memory as revealed by novel object recognition test. Since memory changes and stress related behavioural changes are all closely related to the hippocampus function we further analyzed the changes of dendrites and synapses of hippocampal neurons as well as expression levels of some proteins closely related to synaptic function. bc004004-/- mice exhibited decreased dendritic lengths and increased amount of immature spines, as well as altered expression pattern of synaptic related proteins including GluN2A, synaptophysin and BDNF in the hippocampus. Those findings suggest that BRAP homologous protein may have a protective effect on the behavioural response to stress via regulating dendritic spine formation and synaptic plasticity in the hippocampus.

Application of Alanine Scanning to Determination of Amino Acids Essential for Peptide Adsorption at the Solid/Solution Interface and Binding to the Receptor: Surface-Enhanced Raman/Infrared Spectroscopy versus Bioactivity Assays.

The article describes the application of the alanine-scanning technique used in combination with Raman, surface-enhanced Raman, attenuated total reflection Fourier transform infrared, and surface-enhanced infrared absorption (SEIRA) spectroscopies, which allowed defining the role of individual amino acid residues in the C-terminal 6-14 fragment of the bombesin chain (BN6-14) on the path of its adsorption on the surface of Ag (AgNPs) and Au nanoparticles (AuNPs). A reliable analysis of the SEIRA spectra of these peptides was possible, thanks to a curve fitting of these spectra. By combining alanine-scanning with biological activity studies using cell lines overexpressing bombesin receptors and the intracellular inositol monophosphate assay, it was possible to determine which peptide side chains play a significant role in binding a peptide to membrane-bound G protein-coupled receptors (GPCRs). Based on the analysis of spectral profiles and bioactivity results, conclusions for the specific peptide-metal and peptide-GPCR interactions were drawn and compared.

Bombesin, endothelin, neurotensin and pituitary adenylate cyclase activating polypeptide cause tyrosine phosphorylation of receptor tyrosine kinases.

Numerous peptides including bombesin (BB), endothelin (ET), neurotensin (NTS) and pituitary adenylate cyclase-activating polypeptide (PACAP) are growth factors for lung cancer cells. The peptides bind to G protein-coupled receptors (GPCRs) resulting in elevated cAMP and/or phosphatidylinositol (PI) turnover. In contrast, growth factors such as epidermal growth factor (EGF) or neuregulin (NRG)-1 bind to receptor tyrosine kinases (RTKs) such as the EGFR or HER3, increasing tyrosine kinase activity, resulting in the phosphorylation of protein substrates such as PI3K or phospholipase (PL)C. Peptide GPCRs can transactivate numerous RTKs, especially members of the EGFR/HER family resulting in increased phosphorylation of ERK, leading to cellular proliferation or increased phosphorylation of AKT, leading to cellular survival. GRCR antagonists and tyrosine kinase inhibitors are useful agents to prevent RTK transactivation and inhibit proliferation of cancer cells.

Pharmacological Effects of Two Novel Bombesin-Like Peptides from the Skin Secretions of Chinese Piebald Odorous Frog (Odorrana schmackeri) and European Edible Frog (Pelophylax kl. esculentus) on Smooth Muscle.

Bombesin-like peptides, which were identified from a diversity of amphibian skin secretions, have been demonstrated to possess several biological functions such as stimulation of smooth muscle contraction and regulation of food intake. Here, we report two novel bombesin-like peptides, bombesin-OS and bombesin-PE, which were isolated from Odorrana schmackeri and Pelophylax kl. esculentus, respectively. The mature peptides were identified and structurally confirmed by high performance Scliquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). Subsequently, the effects of these purified chemically-synthetic peptides on smooth muscle were determined in bladder, uterus, and ileum. The synthetic replications were revealed to have significant pharmacological effects on these tissues. The EC50 values of bombesin-OS for bladder, uterus and ileum, were 10.8 nM, 33.64 nM, and 12.29 nM, respectively. Furthermore, compared with bombesin-OS, bombesin-PE showed similar contractile activity on ileum smooth muscle and uterus smooth muscle, but had a higher potency on bladder smooth muscle. The EC50 value of bombesin-OS for bladder was around 1000-fold less than that of bombesin-PE. This suggests that bombesin-OS and bombesin-PE have unique binding properties to their receptors. The precursor of bombesin-OS was homologous with that of a bombesin-like peptide, odorranain-BLP-5, and bombesin-PE belongs to the ranatensin subfamily. We identified the structure of bombesin-OS and bombesin-PE, two homologues peptides whose actions may provide a further clue in the classification of ranid frogs, also in the provision of new drugs for human health.

Synthesis and exploration of novel radiolabeled bombesin peptides for targeting receptor positive tumor.

Increasing evidence of peptide receptor overexpression in various cancer cells, warrant the development of receptor specific radiolabeled peptides for molecular imaging and therapy in nuclear medicine. Gastrin-releasing-peptide (GRP) receptor, are overexpressed in a variety of human cancer cells. The present study report the synthesis and biological evaluation of new bombesin (BBN) analogs, HYNIC-Asp-[Phe13]BBN(7-13)-NH-CH2-CH2-CH3:BA1, HYNIC-Pro-[Tyr13Met14]BBN(7-14)NH2:BA2 as prospective tumor imaging agent with compare to BBN(7-14)NH2:BS as standard. The pharmacophores were radiolabeled in high yields with 99mTc, characterized for their stability in serum and saline, cysteine/histidine and were found to be substantially stable. Internalization/externalization and receptor binding studies were assessed using MDA-MB-231 cells and showed high receptor binding-affinity and favourable internalization. Fluorescence studies revealed that BA1 changed the morphology of the cells and could localize in the nucleus more effectively than BA2/BS. Cell-viability studies displayed substantial antagonistic and nuclear-internalization effect of BA1. BA1 also exhibited antiproliferative effect on MDA-MB-231 cell by inducing apoptosis. In vivo behaviour of the radiopeptides was evaluated in GRP receptor positive tumor bearing mice. The 99mTc-BA1/99mTc-BA2 demonstrated rapid blood/urinary clearance through the renal pathway and comparatively more significant tumor uptake image and favourable tumor-to-non-target ratios provided by 99mTc-BA1. The specificity of the in vivo uptake was confirmed by co-injection with BS. Moreover, 99mTc-BA1 provided a much clearer tumor image in scintigraphic studies than others. Thus the combination of favourable in vitro and in vivo properties renders BA1 as more potential antagonist bombesin-peptide for targeting GRP-receptor positive tumor. These properties are encouraging to carry out further experiments for non-invasive receptor targeting potential diagnostinc and therapeutic agent for tumors.

Identification of Miscellaneous Peptides from the Skin Secretion of the European Edible Frog, Pelophylax kl. Esculentus.

The chemical compounds synthesised and secreted from the dermal glands of amphibian have diverse bioactivities that play key roles in the hosts' innate immune system and in causing diverse pharmacological effects in predators that may ingest the defensive skin secretions. As new biotechnological methods have developed, increasing numbers of novel peptides with novel activities have been discovered from this source of natural compounds. In this study, a number of defensive skin secretion peptide sequences were obtained from the European edible frog, P. kl. esculentus, using a 'shotgun' cloning technique developed previously within our laboratory. Some of these sequences have been previously reported but had either obtained from other species or were isolated using different methods. Two new skin peptides are described here for the first time. Esculentin-2c and Brevinin-2Tbe belong to the Esculentin-2 and Brevinin-2 families, respectively, and both are very similar to their respective analogues but with a few amino acid differences. Further, [Asn-3, Lys-6, Phe-13] 3-14-bombesin isolated previously from the skin of the marsh frog, Rana ridibunda, was identified here in the skin of P. kl. esculentus. Studies such as this can provide a rapid elucidation of peptide and corresponding DNA sequences from unstudied species of frogs and can rapidly provide a basis for related scientific studies such as those involved in systematic or the evolution of a large diverse gene family and usage by biomedical researchers as a source of potential novel drug leads or pharmacological agents.

Parallel peptidome and transcriptome analyses of amphibian skin secretions using archived frozen acid-solvated samples.

Amphibian skin secretions are unique sources of bioactive peptides and their donor species are currently rapidly disappearing from the biosphere. Here, we report that both peptides and polyadenylated mRNAs from skin granular glands remain amenable to study in samples of stimulated skin secretions following their storage in 0.1 % aqueous trifluoroacetic acid at -20 °C for many years. Frozen acidified solutions of toad (Bombina variegata) skin secretions, stored for 12 years, were thawed and samples removed for direct reverse phase HPLC fractionation. Additional samples were removed, snap frozen and lyophilised for construction of cDNA libraries following polyadenylated mRNA capture using magnetic oligo-dT beads and reverse transcription. Using the bombesin and bradykinin peptides found in bombinid toad skin as models, individual variant peptides of each type were located in reverse phase HPLC fractions and their corresponding biosynthetic precursor-encoding mRNA transcripts were cloned from the cDNA library using a RACE PCR strategy. This study illustrates unequivocally that both amphibian skin secretion peptides and their biosynthetic precursor-encoding polyadenylated mRNAs are stable in frozen acid-solvated skin secretion samples for considerable periods of time-a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

Extraintestinal roles of bombesin-like peptides and their receptors: lung.

PURPOSE OF REVIEW: Description of the recent findings of the biological roles of bombesin-like peptides and their receptors in lungs. RECENT FINDINGS: Gastrin-releasing peptide (GRP) was involved in the airway inflammation in murine models of airway hyperreactivity. The circulating proGRP could serve as a valuable tumor marker for small-cell lung cancers, and the plasma level of proGRP is more stable compared with that of serum proGRP. Recent studies also shed light on the intracellular signaling pathways of bombesin receptor subtype-3 (BRS-3) activation in cultured human lung cancer cells. SUMMARY: The relevant biology of BLPs and their receptors in lung cancers and other lung diseases still remains largely unknown. With the development of several highly specific BRS-3 agonists, recent studies provided some insights into the biological effects of BRS-3 in lungs.

The structures of four bombesins and their cloned precursor-encoding cDNAs from acid-solvated skin secretion of the European yellow-bellied toad, Bombina variegata.

Four different bombesins (bombesin, His(6)-bombesin, Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin) have been identified by a systematic sequencing study of peptides in reverse phase HPLC fractions of the skin secretion of the European yellow-bellied toad, Bombina variegata, that had been solvated in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) and stored frozen at -20°C for 12 years. By using a 3'- and 5'-RACE PCR strategy, the corresponding biosynthetic precursor-encoding cDNAs of all four peptides were cloned from a cDNA library made from the same long-term frozen, acid-solvated skin secretion sample following thawing and lyophilization. Canonical bombesin and His(6)-bombesin are classical bombesin sub-family members, whereas Phe(13)-bombesin and Asp(2)-, Phe(4)-SAP-bombesin, belong to the litorin/ranatensin sub-family of bombesin-like peptides (BLPs). Assignment of these peptides to respective sub-families, was based upon both their primary structural similarities and their comparative pharmacological activities. An interesting observation in this study, was that the nucleotide sequences of the open-reading frames of cloned cDNAs encoding bombesin and its His(6)-substituted analog, were identical except for a single base that was responsible for the change observed at the position 6 residue in the mature peptide from Asn to His. In contrast, the precursor cDNA nucleotide sequences encoding the Phe(13)-bombesins, exhibited 53 base differences. The pharmacological activities of synthetic replicates of each bombesin were compared using two different mammalian smooth muscle preparations and all four peptides were found to be active. However, there were significant differences in their relative potencies.

A novel bombesin-like peptide from skin of Rana shuchinae.

Bombesin and its receptors have been demonstrated to be involved in a larger array of physiological and pathophysiological conditions including memory and fear behavior, lung development and injury, and tumor growth. A bombesin-like peptide named bombesin-RS was purified and characterized from the skin secretions of the frog, Rana shuchinae. Its amino acid sequence (pETSFMAPSWALGHLM-NH2) was determined by auto Edman degradation and mass spectrometry. The cDNA encoding bombesin-RS precursor was cloned from skin cDNA library of this frog. The precursor is composed of 67 amino acid residues including a copy of mature bombesin-RS. Two predicted enzymatic processing sites (-Lys-Lys- and -Lys-Arg-) are flanked at the mature bombesin-RS sequence. In the in vitro myotropic contraction experiment, the synthesized bombesin-RS displayed stimulating effect toward rat stomach strips, suggesting that bombesin-RS acts as agonist as most other bombesin-related peptides do.

BDNF and PDE4, but not the GRPR, regulate viability of human medulloblastoma cells.

Medulloblastoma is the most common brain tumor of childhood. Emerging molecular targets in medulloblastoma include neurotrophin and neuropeptide receptors. In the present study, we have examined the influence of brain-derived neurotrophic factor (BDNF)/TrkB receptor- and gastrin-releasing peptide receptor (GRPR)-mediated signaling on the viability of human medulloblastoma cells. The expression of TrkB and GRPR was confirmed by immunohistochemistry and mRNA for both BDNF and GRPR was detected by reverse transcriptase polymerase chain reaction in Daoy, D283, and ONS76 cells. Treatment with BDNF significantly inhibited the viability of Daoy and D283, but not ONS76 cells, measured with the MTT assay. Neither the GRPR agonists GRP and bombesin nor the GRPR antagonist RC-3095 affected cell viability. Because previous findings have indicated that the viability of glioma cells might be enhanced by GRP when combined with the cAMP phosphodiesterase-4 (PDE4) inhibitor rolipram, we also examined the effects of rolipram alone or combined with GRP on cell viability. Rolipram significantly reduced the viability of all three cell lines, and the inhibitory effect of rolipram in Daoy cells was not modified by cotreatment with GRP. The results suggest that BDNF/TrkB and PDE4, but not the GRPR, regulate the viability of medulloblastoma cells.

Observations favouring the occurrence of local production and marked effects of bombesin/gastrin-releasing peptide in the synovial tissue of the human knee joint--comparisons with substance P and the NK-1 receptor.

We have previously shown that levels of the neuropeptides substance P (SP) and bombesin/gastrin-releasing peptide (BN/GRP) in blood and synovial fluid correlate with levels of pro-inflammatory cytokines in patients with rheumatoid arthritis (RA). It is well-established that SP is present in nerve endings in the synovium whilst the source of BN/GRP in human joints is completely unknown. Nor is it known whether GRP-receptors (GRP-R) are present in human synovial tissue. This study aimed to investigate the expression pattern of SP, BN/GRP and their receptors (NK-1R and GRP-R) in synovial tissue. Synovial tissue specimens from patients with RA or osteoarthritis (OA) were processed for immunohistochemistry, in situ hybridisation and ELISA. The results show the presence of BN/GRP, but not SP, in cells in the synovial tissue at both the protein and mRNA level. We did not find immunoreactive BN/GRP in nerve structures. NK-1R and GRP-R were also expressed at both protein and mRNA levels in cells associated with blood vessels and cells in the interstitial tissue. ELISA analyses revealed both SP and BN/GRP to be present in synovial tissue extracts and that synovial levels of SP were higher in RA patients than those with OA. Our results indicate that BN/GRP is produced by non-neuronal cells in the synovial tissue. Furthermore, both BN/GRP and SP may exert their effects on the synovial tissue through the respective receptors. These results suggest that BN/GRP and SP may modulate inflammation and vascular events, and possibly healing processes in the synovium. Finally, nerves should not be considered as the source of BN/GRP in synovial tissue although this peptide is presumably intimately involved functionally in synovial tissue, a previously unrecognised fact.

Investigation of molecular structure of bombesin and its modified analogues nonadsorbed and adsorbed on electrochemically roughened silver surface.

This work describes the molecular structure of bombesin (BN) and its analogs on the basis of the absorption infrared and Raman results described below. In these analogues is replaced one ([D-Phe12]BN, [Tyr4]BN, and [Lys3]BN) or two ([Tyr4,D-Phe12]BN, [D-Phe12,Leu14]BN, and [Leu13-(R)-Leu14]BN) amino acid residues within the peptide chain with a synthetic amino acid, creating antagonists to bombesin, which are useful in the treatment of cancer. It is also used surface enhanced Raman scattering (SERS) to study the differences and changes in the vibrational spectra of BN and its analogs, which were attached to an electrochemically roughened silver surface as these peptides interacted with target proteins. This work explores the use of SERS for molecules anchored to a macroscopic silver surface to interrogate the interaction of these peptides with protein receptors. The results presented here show that all peptides coordinate to the macroscopic silver surface through an indole ring and the methylene group of Trp8, the C==O fragment, and an amide bond; however, the orientation of these fragments on the electrochemically roughened silver surface and the strength of the interactions with this surface is slightly different for each peptide. For example, the interaction of --CH2-- of [D-Phe12]BN, [Tyr4,D-Phe12]BN, [D-Phe12,Leu14]BN, [Leu13-(R)-Leu14]BN, and [Lys3]BN with the silver surface perturbed the vertical orientation of the Trp8 indole ring on this surface. Hence, the indole ring adopted a close to perpendicular orientation on the silver surface for BN and [Tyr4]BN, only.

Bombesin attenuates pre-mRNA splicing of glucocorticoid receptor by regulating the expression of serine-arginine protein p30c (SRp30c) in prostate cancer cells.

Although glucocorticoids are frequently administered to patients with hormone refractory prostate cancer, their therapeutic effectiveness is limited by the development of glucocorticoid resistance. The molecular mechanisms of glucocorticoid resistance are unknown but are believed to involve neuropeptide growth factors and cytokines. We examined the functional interaction between bombesin and dexamethasone in PC-3 cells and found that bombesin could act as a survival factor by interfering with dexamethasone-mediated growth inhibition. Because glucocorticoids exert their effects through glucocorticoid receptors (GRs), we measured the expression of GR alpha and GR beta isoforms in the presence of bombesin. Western blotting and real time PCR revealed bombesin induced expression of GR beta, but not GR alpha. Because GR isoforms are generated by alternative splicing of a common GR gene, we examined the expression of serine-arginine (SR) proteins involved in alternative splicing, and found that the expression of SRp30 was induced by bombesin in PC-3 cells. To characterize the role of SRp30 in splicing of GR isoforms, siRNAs specific to various SRp30 isoforms were transfected into PC-3 cells. We found that suppression of SRp30c expression by siRNA specifically antagonized bombesin's effect on glucocorticoid-mediated inhibition of PC cells, suggesting that bombesin-induced expression of SRp30c affects GR pre-mRNA splicing, leading to increased GR beta expression and contributing to glucocorticoid resistance in PC cells.

Cloning of novel bombesin precursor cDNAs from skin of Bombina maxima.

Amphibian skin is a rich resource of bioactive peptides like proline-rich bombesin from frog Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises proline-rich bombesin and a novel peptide, designated as bombestatin, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of the novel peptide is WEVLLNVALIRLELLSCRSSKDQDQKESCGMHSW, in which two cysteines form a disulfide bond. A BLAST search of databases did not detect sequences with significant similarity. Bombestatin possesses dose-dependent contractile activity on rat stomach strips. The differences between cDNAs encoding PR-bombesin plus bombestatin and PR-bombesin alone are due to fragment insertions located in 3'-coding region and 3'-untranslational region, respectively.

Development and function of bombesin-like peptides and their receptors.

Amphibian bombesin and its related peptides consist a family of neuropeptides in many vertebrate species. Bombesin and two major bombesin-like peptide in mammals, gastrin-releasing peptide (GRP) and neuromedin B (NMB), have been shown to elicit various physiological effects. These include inhibition of feeding, smooth muscle contraction, exocrine and endocrine secretions, thermoregulation, blood pressure and sucrose regulations and cell growth. Receptors for GRP and NMB (GRP-R and NMB-R), as well as third subtype of bombesin-like peptide receptor (BRS-3) have been cloned. These receptors are G-protein-coupled receptors and are expressed in various brain regions and in the digestive tract. In this paper, we will summarize studies on these peptides and their receptors, with special reference to research using gene-knockout mice. These studies clearly demonstrated the role of three receptors in vivo and in vitro. We will also discuss the phylogeny of these receptors.

Skin secretion of the toad Bombina variegata contains multiple insulin-releasing peptides including bombesin and entirely novel insulinotropic structures.

Skin secretions of the toad Bombina variegata were evaluated for the isolation and characterisation of insulinotropic peptides. Crude secretions obtained from young adult toads by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 44 peaks. In acute incubations with glucose-responsive BRIN-BD11 cells, peaks 21, 22, 23, 24 and 25 showed a 1.5-3.5-fold increase in insulin release compared with 5.6 mM glucose alone (p<0.001; n=3). Structural analyses of the purified insulin-releasing peaks were performed by automated Edman degradation and mass spectrometry. Peptides represented by peaks 21, 22 and 23 had molecular masses of 1641.7 Da, 1662.6 Da and 1619.8 Da respectively. These peptides were unblocked by removal of pyroglutamic acid from the N-terminus prior to Edman degradation, revealing lengths of 14 amino acids. Peak 21 yielded a primary structure of Pyr-QRLGHQWAVGHLM, which a data base search revealed as an analogue of bombesin (His6 bombesin), while peak 23 was an exact match of bombesin (Pyr-QRLGNQWAVGHLM) originally isolated from Bombina bombina. Peak 22 indicated a primary structure of Pyr-DSFGNQWARGHFM (72% homology with bombesin). Peaks 24 and 25 revealed entirely novel insulinotropic peptides with molecular masses and primary structures of 1650.5 Da and 2300.0 Da and GKPFYPPPIYPEDM (GM-14) and IYNAICPCKHCNKCKPGLLAN (IN-21) respectively. Preliminary studies on the mechanisms underlying the insulinotropic actions of peaks 21, 22, 23 and 24 suggest possible involvement of a cAMP-dependent, G protein-insensitive pathway. These data indicate that Bombina variegata skin secretions contain peptides with insulin-releasing activity, which may have mammalian counterparts and prove useful for possible exploitation as antidiabetic agents from natural resources.

Development of bombesin analogs with conformationally restricted amino acid substitutions with enhanced selectivity for the orphan receptor human bombesin receptor subtype 3.

The human bombesin receptor subtype 3 (hBRS-3) orphan receptor, which has a high homology to bombesin (Bn) receptors [gastrin-releasing peptide (GRP) and neuromedin B (NMB) receptors], is widely distributed in the rat central nervous system. Its natural ligand or role in physiology is unknown due to lack of selective ligands. Its target disruption leads to obesity, diabetes, and hypertension. A synthetic high-affinity agonist, [D-Tyr6,beta-Ala11,Phe13,Nle14]Bn(6-14), has been described, but it is nonselective for hBRS-3 over other Bn receptors; however, substitution of (R)- or (S)-amino-3-phenylpropionic acid (Apa) for beta-Ala11 resulted in a modestly selective ligand. In the present study, we have attempted to develop a more selective hBRS-3 ligand by using two strategies: substitutions on phenyl ring of Apa11 and the substitution of other conformationally restricted amino acids into position 11 of [D-Tyr6,beta-Ala11,Phe13,Nle14]Bn(6-14). Fifteen analogs were synthesized and affinities were determined for hBRS-3 and Bn receptors (hGRP-R and hNMB-R). Selective analogs were tested for their ability to activate each receptor by stimulating phospholipase C. One analog, [D-Tyr6,Apa-4Cl,Phe13,Nle14]Bn(6-14), retained high affinity for the hBRS-3 (Ki=8 nM) and had enhanced selectivity (>230-fold) for hBRS-3 over hGRP-R or hNMB-R. This analog specifically interacted with hBRS-3, fully activated hBRS-3 receptors, and was a potent agonist at the hBRS-3 receptor. This enhanced selectivity should allow this analog to be useful for investigating the possible role of hBRS-3 in physiological or pathological processes.

A novel proline rich bombesin-related peptide (PR-bombesin) from toad Bombina maxima.

A novel bombesin-related peptide was isolated from skin secretions of Chinese red belly toad Bombina maxima. Its primary structure was established as pGlu-Lys-Lys-Pro-Pro-Arg-Pro-Pro-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH(2.) The amino-terminal (N-terminal) 8-residue segment comprising four prolines and three basic residues is extensively different from bombesins from other Bombina species. The peptide was thus named proline rich bombesin (PR-bombesin). PR-bombesin was found to elicit concentration-dependent contractile effects in the rat stomach strip, with both increased potency and intrinsic activity as compared with those of [Leu(13)]bombesin. Analysis of different bombesin cDNA structures revealed that an 8 to 14- nucleotide fragment replacement in the peptide coding region (TGGGGAAT in the cDNAs of multiple bombesin forms from Bombina orientalis and CACCCCGGCCACCC in the cDNA of PR-bombesin) resulted in an unusual Pro-Pro-Arg-Pro-Pro motif in the N-terminal part of PR-bombesin.