Solid-phase synthesis of ovine Leydig cell insulin-like peptide--a putative ovine relaxin?

The primary structure of ovine Leydig cell insulin-like peptide (Ley I-L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B-chain, an Arg-X-X-X-Arg sequence exists within the Ley I-L B-chain although it is located four residues towards the C-terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I-L suggested that the peptide might possess a relaxin-like function. Ovine Ley I-L was assembled by Fmoc-solid-phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain-combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin-like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I-L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I-L and relaxin, we prepared a synthetic analogue of ovine Ley I-L containing a single replacement of B-chain residue 12, His, with Arg. This was found to possess significant relaxin-like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I-L is similar to that of relaxin and highlighting the key requirement for the five-residue sequence, Arg-X-X-X-Arg, to be present in position B12-16 for characteristic relaxin activity.     

Chemical synthesis and biological activity of rat INSL3.

The recently identified protein, insulin 3 (INSL3), has structural features that make it a bona fide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure with conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum, to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. The purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the superfamily, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high beta-turn and alpha-helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure.     

Conformationally constrained single-chain peptide mimics of relaxin B-chain secondary structure.

Relaxin is a member of the insulin superfamily and has many biological actions including angiogenesis and collagen degradation. It is a 6 kDa peptide hormone consisting of two peptide chains (A and B) tethered by two disulphide bonds. Past structure-function relationship studies have shown the key receptor binding site of relaxin to be principally situated within the B-chain alpha-helix. Molecular dynamic simulations were performed to aid the design of conformationally constrained relaxin B-chain analogues that possess alpha-helical structure and relaxin-like activity. Restraints included disulphide bonds, both single and double, and lactam bonds. Each peptide was prepared by solid phase synthesis and, following purification, subjected to detailed conformational analysis by circular dichroism spectroscopy. Of 15 prepared relaxin B-chain mimetics, one was able to mimic the secondary structure of the native ligand as indicated by biomolecular recognition/interaction analysis using surface enhanced laser desorption ionization mass spectroscopy together with a relaxin antibody. However, none of the mimetics possess characteristic relaxin-like biological activity which strongly indicates that the pharmacophore comprises additional structural elements other than the relaxin B-chain alpha-helix. These findings will assist in the design and preparation of novel relaxin agonists and antagonists.     

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.     

The relaxin peptide family and their novel G-protein coupled receptors

Relaxin-1 is a heterodimeric peptide hormone primarily produced by the pregnant corpus luteum and/or placenta and is involved in many essential physiological processes centered on its action as a potent extracellular matrix (ECM) remodeling agent. Insulin-like peptide 3 (INSL3), also known as relaxin-like factor, is predominantly expressed in the Leydig cells of the testes and is an important mediator of testicular descent. The relaxin-1 equivalent peptide in humans is actually the product of the human RLN2 gene, human 2 (H2) relaxin. Recently identified and thought to be the ancestral relaxin, relaxin-3 is specifically expressed in the nucleus incertus of the mouse and rat brain and is most likely an important neuropeptide. Each of the hormones above act on cell membrane G-protein coupled receptors (GPCRs). The relaxin-1 receptor is leucine-rich repeat-containing GPCR 7 (LGR7) whereas INSL3 acts on the closely related LGR8. These receptors have large extra-cellular domains containing multiple leucine-rich repeats (LRRs) and a unique LDL receptor-like cysteine-rich motif (LDLR-domain). Relaxin-3 will bind and activate LGR7 with 50-fold lower activity than H2 relaxin. Two relaxin-3 selective GPCRs; somatostatin and angiotensin like peptide receptor (SALPR) and GPCR 142 were recently identified, these type I GPCRs are unrelated to LGR7 and LGR8. The discovery and characterisation of these receptors is greatly aiding the quest to unravel the mechanics of these important hormones, however with three other family members, insulin-like peptides 4–6 (INSL4, INSL5 and INSL6) with unknown functions and unidentified receptors, there is still much to be learnt about this hormone family.     

Design, synthesis and pharmacological evaluation of cyclic mimetics of the insulin-like peptide 3 (INSL3) B-chain.

Insulin-like peptide 3 (INSL3) is a peptide hormone belonging to the relaxin-insulin superfamily of peptides that plays important roles in testes descent, oocyte maturation and the control of male germ cell apoptosis. These actions are mediated via a specific G-protein coupled receptor, LGR8. Previous structure-activity studies have shown that the key binding site of INSL3 is situated within its B-chain. Recent studies in our laboratory have led to the identification of a cyclic peptide mimetic 2 of the INSL3 B-chain, which we have shown to compete with the binding of [33P]-relaxin to LGR8 expressed in HEK293T cells, and to inhibit cAMP-mediated signaling in these cells, i.e. it is an antagonist of INSL3. In order to further define the structure-activity relationships of cyclic analogues of the INSL3 B-chain, we used a structure-based approach to design a series of cyclic, disulfide-constrained INSL3 B-chain mimetics. To do this, we first created a model of the 3D structure of INSL3 using the crystal structure of human relaxin as a template. This model of INSL3 was then used as a template to design a series of disulfide-constrained mimetics of the INSL3 B-chain. The peptides were synthesized by solid-phase peptide synthesis using pseudoproline dipeptides to improve the synthesis outcome. Of the seven prepared INSL3 B-chain mimetics, three compounds were found to have partial displacement activity, while four were able to completely displace [33P]-relaxin from LGR8, including compounds that were markedly shorter than compound 2. The best of these, mimetic 6, showed significantly greater affinity for LGR8 than compound 2, but still displayed around 1000-fold less affinity for LGR8 than native INSL3. Analysis of selected mimetics for their alpha-helical content using circular dichroism (CD) spectroscopy revealed that, generally, the mimetics showed less than expected helicity. The inability of the compounds to display true native INSL3 structure is likely contributing to their reduced receptor binding affinity. We are currently examining alternative INSL3 B-chain mimetics that might better present key receptor binding residues in the native INSL3-like conformation.     

The Importance of Tryptophan B28 in H2 Relaxin for RXFP2 Binding and Activation

H2 relaxin (relaxin) is a member of the insulin–relaxin superfamily and exhibits several non-reproductive functions in addition to its well-known properties as a pregnancy hormone. Over the years, the therapeutic potential of relaxin has been examined for a number of conditions. It is currently in phase III clinical trials for the treatment of acute heart failure. The 53 amino acid peptide hormone consists of two polypeptide chains (A and B) which are cross-linked by two inter-chains and one intra-A chain disulfide bridge. Although its cognate receptor is relaxin family peptide receptor (RXFP) 1, relaxin is also able to cross-react with RXFP2, for which the native ligand is INSL3. The “RXXXRXXI” motif in the B-chain of H2 relaxin is responsible for primary binding to LRR of the RXFP1 receptor (Büllesbach and Schwabe, J Biol Chem 280:14051–14056, 2005). Previous RXFP2 receptor mutation and molecular modelling studies strongly suggest that, in addition to this motif, the Trp-B28 residue in the B-chain is responsible for H2–RXFP2 interaction. To confirm this finding, here we have mutated H2 relaxin in which Trp-B28 was replaced with alanine. The synthetic relaxin analogue was then tested on cells expressing either RXFP1 or 2 to determine the affinity and potency for the respective receptors. Our results confirm that Trp-B28 in the B-chain is crucial for binding and activating RXFP2, but not for RXFP1.     

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.     

Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B-C-A with full biological activity in boars

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B-C-A monomeric structure with full biological activity.     

Preliminary Structure–Function Relationship Studies on Insulin-Like Peptide 5 (INSL5)

Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide bonded member of insulin/relaxin superfamily of peptides that includes insulin, insulin-like growth factor I and II (IGFI and IGFII), insulin-like peptide 3, 4, 5 and 6 (INSL3, 4, 5 and 6), relaxin-1 (H1 relaxin), -2 (H2 relaxin) and -3 (H3 relaxin). Although it is expressed in relatively high levels in the gut, its biological function remains unclear. However, recent reports suggest a significant orexigenic action and a role in the regulation of insulin secretion and β-cell homeostasis, which implies that both agonists and antagonists of the peptide may have significant therapeutic applications. Modern solid phase synthesis techniques together with regioselective disulfide bond formation were employed for a preliminary structure–function relationship study of mouse INSL5. Two point mutated analogues, mouse INSL5 A-B(R24A, W25A) and mouse INSL5 A-B(K6A, R14A, Y18A) were chemically prepared, where the residues in the B-chain that may be involved in receptor activation and affinity binding, were respectively mutated. Synthetic mouse INSL5 A-B(R24A, W25A) analogue was inactive on RXFP4, the native receptor for INSL5, suggesting Arg^B24 and Trp^B25 are probably directly involved in INSL5 receptor activation. Mouse INSL5 A-B(K6A, R14A, Y18A) analogue had both decreased affinity and potency on RXFP4 (pIC₅₀ 7.7 ± 0.2, pEC₅₀ 7.87 ± 0.18) which indicated that one or more of these residues are critical for the binding to the receptor.     

Structure of the R3/I5 chimeric relaxin peptide, a selective GPCR135 and GPCR142 agonist

The human relaxin family comprises seven peptide hormones with various biological functions mediated through interactions with G-protein-coupled receptors. Interestingly, among the hitherto characterized receptors there is no absolute selectivity toward their primary ligand. The most striking example of this is the relaxin family ancestor, relaxin-3, which is an agonist for three of the four currently known relaxin receptors: GPCR135, GPCR142, and LGR7. Relaxin-3 and its endogenous receptor GPCR135 are both expressed predominantly in the brain and have been linked to regulation of stress and feeding. However, to fully understand the role of relaxin-3 in neurological signaling, the development of selective GPCR135 agonists and antagonists for in vivo studies is crucial. Recent reports have demonstrated that such selective ligands can be achieved by making chimeric peptides comprising the relaxin-3 B-chain combined with the INSL5 A-chain. To obtain structural insights into the consequences of combining A- and B-chains from different relaxins we have determined the NMR solution structure of a human relaxin-3/INSL5 chimeric peptide. The structure reveals that the INSL5 A-chain adopts a conformation similar to the relaxin-3 A-chain, and thus has the ability to structurally support a native-like conformation of the relaxin-3 B-chain. These findings suggest that the decrease in activity at the LGR7 receptor seen for this peptide is a result of the removal of a secondary LGR7 binding site present in the relaxin-3 A-chain, rather than conformational changes in the primary B-chain receptor binding site.     

Canine relaxin-like factor: unique molecular structure and differential expression within reproductive tissues of the dog

Employing postpubertal testicular tissue, we determined the cDNA coding sequence of a truncated canine relaxin-like factor (RLF) consisting of a signal peptide of 28 amino acids (aa), a B-domain of 23 aa, a truncated C-domain of 34 aa, and an A domain of 26 aa, respectively. Within the B-domain of canine RLF, the putative relaxin receptor binding motif contained a single substitution with the C-terminal arginine replaced by a serine residue, and the putative RLF receptor binding motif was truncated. Leydig cells specifically expressed RLF in the normal postpubertal and cryptochid testis as well as in testicular Leydig cell adenoma. The epididymis was an additional source of RLF in the dog. In the female reproductive tract, expression of immunoreactive RLF and relaxin were compared. Within the ovary, RLF, but not relaxin, was detected in follicular theca interna and granulosa cells and the corpus luteum. In the nonpregnant uterus, luminal and glandular epithelium coexpressed RLF and relaxin. Uteroplacental tissue at early stages of gestation revealed RLF expression in the proliferative fetal villous cytotrophoblast and in maternal uterine cells. In the mature canine placenta, the trophoblast surrounding the maternal blood vessels and the hemophagous cytotrophoblast of the paraplacental zone expressed RLF. Canine relaxin was absent in the paraplacental areas. Western analysis of placental tissue extracts revealed the presence of specific immunoreactive bands likely resembling unprocessed and enzymatically cleaved RLF. Differential expression of RLF and relaxin appears to reflect distinct autocrine and paracrine functions of RLF in canine reproductive tissues.     

Evolution of the relaxin-like peptide family

Background  

The relaxin-like peptide family belongs in the insulin superfamily and consists of 7 peptides of high structural but low sequence similarity; relaxin-1, 2 and 3, and the insulin-like (INSL) peptides, INSL3, INSL4, INSL5 and INSL6. The functions of relaxin-3, INSL4, INSL5, INSL6 remain uncharacterised. The evolution of this family has been contentious; high sequence variability is seen between closely related species, while distantly related species show high similarity; an invertebrate relaxin sequence has been reported, while a relaxin gene has not been found in the avian and ruminant lineages.     

Adjustment of RNA content during temperature upshift in Escherichia coli.

The sequence of the B-chain of relaxin, and ovarian peptide hormone isolated from ovaries of pregnant sows, has been shown to have the following primary structure: PCA-Ser-Thr-Asn-Asp-Phe-Ile-Lys-Ala-Cys-Gly-Arg-Glu-Leu-Val-Arg-Leu-Trp-Val-Glu-Ile-Cys-Gly-Val-Trp-Ser (2820 daltons). The heterogeneity of relaxin observed during purification procedures is likely to be due to variations in the C-terminal region of the B-chain, in particular the substitution of Gln for Glu₂₀, and the possible addition of arginine or serylarginine at the C terminus. The B-chain exhibited a distribution of sulfhydryl residues relative to one another that is identical to that found in the B-chain of insulin. A similar analogy has already been demonstrated for the A-chains of relaxin and insulin.     

Synthesis, conformational studies and biological activity of N(alpha)-mono-biotinylated rat relaxin.

Biotin-avidin immobilization can be a useful tool in structure-function studies of hormone receptors. A crucial step is the preparation of a specifically biotinylated hormone that is able to bind to its receptor while leaving the biotin group free for interaction with avidin. The receptor for relaxin, an ovarian peptidic hormone produced during pregnancy, has not yet been isolated. We therefore undertook to prepare a specifically monobiotinylated rat relaxin for use in ligand-searching strategies. Rat relaxin is a convenient analogue because reliable bioassays exist, thus allowing assessment of the effect of N-biotinylation on bioactivity. To help improve the yield of the two-chain, three-disulfide bond rat relaxin, 2-hydroxy-4-methoxybenzyl (Hmb) backbone protection was used during the solid-phase assembly of the B-chain to help prevent any possible chain aggregation. As a final step, while the protected peptide was still on the resin, the biotin label was introduced at the N-terminus of the B-chain using standard coupling protocols. The chain combination with the A-chain was accomplished in reasonable yield. Secondary structural measurements demonstrated that the biotin caused the starting B-chain to adopt a more ordered conformation. The labelled synthetic relaxin exhibited similar circular dichroism spectra to native and synthetic single B-chain peptides. In addition, the biotinylated relaxin showed no significant difference in its chronotropic activity in the rat isolated heart assay compared with the native peptide. Biosensor studies showed that antibody recognition was retained upon attachment of the synthetic relaxin to the streptavidin-derivatized surface.     

The A-chain of human relaxin family peptides has distinct roles in the binding and activation of the different relaxin family peptide receptors

The relaxin peptides are a family of hormones that share a structural fold characterized by two chains, A and B, that are cross-braced by three disulfide bonds. Relaxins signal through two different classes of G-protein-coupled receptors (GPCRs), leucine-rich repeat-containing GPCRs LGR7 and LGR8 together with GPCR135 and GPCR142, now referred to as the relaxin family peptide (RXFP) receptors 1-4, respectively. Although key binding residues have been identified in the B-chain of the relaxin peptides, the role of the A-chain in their activity is currently unknown. A recent study showed that INSL3 can be truncated at the N terminus of its A-chain by up to 9 residues without affecting the binding affinity to its receptor RXFP2 while becoming a high affinity antagonist. This suggests that the N terminus of the INSL3 A-chain contains residues essential for RXFP2 activation. In this study, we have synthesized A-chain truncated human relaxin-2 and -3 (H2 and H3) relaxin peptides, characterized their structure by both CD and NMR spectroscopy, and tested their binding and cAMP activities on RXFP1, RXFP2, and RXFP3. In stark contrast to INSL3, A-chain-truncated H2 relaxin peptides lost RXFP1 and RXFP2 binding affinity and concurrently cAMP-stimulatory activity. H3 relaxin A-chain-truncated peptides displayed similar properties on RXFP1, highlighting a similar binding mechanism for H2 and H3 relaxin. In contrast, A-chain-truncated H3 relaxin peptides showed identical activity on RXFP3, highlighting that the B-chain is the sole determinant of the H3 relaxin-RXFP3 interaction. Our results provide new insights into the action of relaxins and demonstrate that the role of the A-chain for relaxin activity is both peptide- and receptor-dependent.     

Identification of INSL5, a new member of the insulin superfamily.

A new member of the insulin gene superfamily (INSL5) was identified by searching EST databases for the presence of the conserved insulin B-chain cysteine motif. Human and murine INSL5 are both polypeptides of 135 amino acids, matching the classical signature of the insulin superfamily. Through the B- and A-chain regions, human INSL5 has 48% identity to shark relaxin, 40% identity to human relaxin, and 34% identity to human Leydig insulin-like factor. Northern blot analysis detected expression of human INSL5 in rectal, colon, and uterine tissue and of murine INSL5 only in thymic tissue. Using quantitative RT-PCR, expression of murine INSL5 was detected in the highest quantity in colon followed by thymus, and minimal expression was seen in testis. By radiation hybrid mapping and the use of surrounding markers, human INSL5 maps to chromosome 1 in the 1p31.1 to 1p22.3 region.     

Analogs of insulin-like peptide 3 (INSL3) B-chain are LGR8 antagonists in vitro and in vivo

Insulin-like peptide 3 (INSL3) is a member of the insulin superfamily that plays an important role in mediating testes descent during fetal development. More recently, it has also been demonstrated to initiate oocyte maturation and suppress male germ cell apoptosis. These actions are mediated via a specific G-protein-coupled receptor, LGR8. Little is known regarding the structure and function relationship of INSL3, although it is believed that the principal receptor binding site resides within its B-chain. We subsequently observed that the linear B-chain alone (INSL3B-(1-31)) bound to LGR8 and was able to antagonise INSL3 stimulated cAMP accumulation in HEK-293T cells expressing LGR8. Sequentially N- and C-terminally shortened linear analogs were prepared by solid phase synthesis and subsequent assay showed that the minimum length required for binding was residues 11-27. It was also observed that increased binding affinity correlated with a corresponding increase in alpha-helical content as measured by circular dichroism spectroscopy. Molecular modeling studies suggested that judicious placement of a conformational constraint within this peptide would increase its alpha-helix content and result in increased structural similarity to the B-chain within native INSL3. Consequently, intramolecularly disulfide-linked analogs of the B-chain showed a potentiation of INSL3 antagonistic activity, as well as exhibiting increased proteolytic stability, as assessed in rat serum in vitro. Administration of one of these peptides into the testes of rats resulted in a substantial decrease in testis weight probably due to the inhibition of germ cell survival, suggesting that INSL3 antagonists may have potential as novel contraceptive agents.     

Structure and function relationship of murine insulin-like peptide 5 (INSL5): free C-terminus is essential for RXFP4 receptor binding and activation

Insulin-like peptide 5 (INSL5) is a member of insulin/relaxin superfamily of peptides. It has recently been identified as the cognate ligand for the G-protein-coupled receptor, RXFP4. Although the complete physiological role of this naturally occurring peptide is still under investigation, there is evidence that it acts to both stimulate appetite and activate colon motility. This suggests that both agonists and antagonists of the peptide may have potential therapeutic applications. To further investigate the physiological role of this peptide and because of the ready availability of the mouse as an experimental animal, the preparation of mouse INSL5 was undertaken. Because of its complex structure and the intractable nature of the two constituent chains, different solid phase synthesis strategies were investigated, including the use of a temporary B-chain solubilizing tag. Unfortunately, none provided significantly improved yield of purified mouse INSL5 which reflects the complexity of this peptide. In addition to the native peptide, two mouse INSL5 analogues were also prepared. One had its two chains as C-terminal amides, and the other contained a europium chelate monolabel for use in RXFP4 receptor assays. It was found that the INSL5 amide was substantially less potent than the native acid form. A similar observation was made for the human peptide acid and amide, highlighting the necessity for free C-terminal carboxylates for function. Two additional human INSL5 analogues were prepared to further investigate the necessity of a free C-terminal. The results together provide a first insight into the mechanism whereby INSL5 binds to and activates RXFP4.