Insulin-like protein (INSL3, INSL4, INSL5, INSL6 & INSL7/H3 Relaxin) belongs to the insulin-like hormone superfamily, which encompasses insulin, relaxin, and insulin-like growth factors I (IGF1) and II (IGF2). Insulin gene superfamily hormones regulate cell growth, metabolism, and tissue-specific functions.

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Gender Specific Application for INSL3 circulating hormone

Insulin-like protein (INSL3, INSL4, INSL5, INSL6 & INSL7/H3 Relaxin) belongs to the insulin-like hormone superfamily, which encompasses insulin, relaxin, and insulin-like growth factors I (IGF1) and II (IGF2). Insulin gene superfamily hormones regulate cell growth, metabolism, and tissue-specific functions. Members of this family are characterized by a signal peptide, a B chain, a connecting C chain, and an A chain. A preliminary in-house study performed at Phoenix Pharmaceuticals, Inc. found gender specific applications for INSL3. Results from analysis using INSL3 RIA Kit (RK-035-27) showed the levels of circulating INSL3 in male samples were approximately ten times greater than that found in female samples.

April 18, 2003 Phoenix Pharmaceuticals, Inc. Belmont, CA

 

Identification of relaxin-3/INSL7 as a ligand for GPCR142

We have recently identified the insulin-like peptide relaxin-3 (aka INSL7) as the endogenous ligand for an orphan G-protein-coupled receptor, GPCR135 (aka somatostatin- and angiotensin-like peptide receptor). Analysis of possible receptors related to GPCR135 revealed a single orphan receptor, GPCR142. Thus, we tested whether GPCR142 could also respond to relaxin-3 or related insulin-like molecules. Surprisingly, GPCR142 was activated by nanomolar concentrations of relaxin-3 but was completely unresponsive to all other known insulin-like peptides. We evaluated by reverse transcriptase-PCR the expression of GPCR142 mRNA in a variety of human tissues and found expression in brain, kidney, testis, thymus, placenta, prostate, salivary gland, thyroid, and colon. In an analysis of other species, we were able to find a full-length mouse homolog of GPCR142, but were unable to detect any complete GPCR142 transcripts in rat. With respect to intracellular signaling, GPCR142 is similar to GPCR135 in that it potently inhibits adenylate cyclase and stimulates 35S-GTPgammaS incorporation in response to relaxin-3. However, whereas GPCR135 signaling could be converted to calcium mobilization using a Gqi5 or Galpha16 G-proteins, GPCR142 was only capable of functioning in the presence of Galpha16. In the accompanying article (Liu, C., Eriste, E., Sutton, S., Chen, J., Roland, B., Kuei, C., Farmer, N., Jornvall, H., Sillard, R., and Lovenberg, T. W. (2003) J. Biol. Chem. 278, 50754-50764), we present the case that relaxin-3, which has previously been shown to bind to the relaxin receptor LGR7, is most likely the endogenous ligand for GPCR135. In this report, we show an additional receptor, GPCR142, which is also selectively activated by relaxin-3. However, the anatomical localization of GPCR142 suggests that GPCR142 may have different physiological functions.

Liu C, et al. J Biol Chem. 2003 Dec 12;278(50):50765-70. Epub 2003 Sep 30.

 

Physiological or pathological - a role for relaxin in the cardiovascular system?

The omnipresent 6kDa polypeptide relaxin (RLX) is emerging as a multi-functional endocrine and paracrine factor, with a broad range of target tissues that includes the cardiovascular system. Humans and other higher primates have three RLX genes, designated H1, H2 and H3, of which H2 RLX is the major stored and circulating form. Rodents have only two RLX genes: relaxin-1 (equivalent to H2 RLX) and relaxin-3 (equivalent to H3 RLX). The recent cloning of the human RLX receptor (LGR7), a member of the leucine-rich repeat family of G-protein-coupled orphan receptors, and detection of LGR7 gene transcripts in the heart confirm this organ as a target for RLX (H2). However, evidence for production of the ligand within the cardiovascular system is limited, and few studies have clearly identified the physiological effects of RLX on cardiac function. To add to the controversy, serum concentrations and expression of RLX in the heart are elevated in chronic heart failure patients and animal models of cardiomyopathy, implying that RLX may only be a marker for pathological cardiovascular conditions, rather than normal physiology.

Samuel CS, Parry LJ, Summers RJ. Curr Opin Pharmacol 2003 Apr;3(2):152-8

INSL-3 is expressed in human hyperplastic and neoplastic thyrocytes

The insulin-like hormone INSL-3, also named relaxin-like factor (RLF) or Leydig-derived insulin-like peptide (LEY-IL), is expressed in various reproductive tissues and is regarded a marker of differentiation in human testicular Leydig cells. Recently, we have identified differential expression of human INSL-3 in neoplastic Leydig cells and mammary epithelial cells suggesting an involvement of INSL-3 in tumor biology. Here we have investigated the expression of INSL-3 in human thyroid carcinoma cell lines and in the human thyroid gland which has been shown to express transcripts for the G protein coupled INSL-3 receptor LGR8. When we determined the expression of INSL-3 in eight human thyroid carcinoma cell lines, a novel INSL-3 splice variant containing a 95 bp out-of-frame insertion at the beginning of exon II of the INSL-3 gene was discovered. Treatment of the human anaplastic thyroid carcinoma cell line 8505C with diethylstilbestrol (DES) caused a significant dose-dependent transcriptional down-regulation of INSL-3 and a marked up-regulation of LGR8. Employing in situ hybridization to detect INSL-3 transcripts and specific rabbit antisera against the INSL-3 proteins, both INSL-3 isoforms were detected in patients with Graves' disease (n=10), follicular carcinomas (FTC; n=12), papillary carcinomas (PTC; n=9) and undifferentiated anaplastic carcinomas (UTC; n=15). By contrast, thyrocytes of all 15 benign goiter tissues studied were devoid of both INSL-3 isoforms, mRNA and protein. Our data indicate that INSL-3 hormone is up-regulated in hyperplastic and neoplastic human thyrocytes suggesting that the INSL-3 isoforms may serve as additional markers for hyperplastic and neoplastic human thyrocytes. In the anaplastic thyroid carcinoma cell line 8505C, the regulation of both INSL-3 and LGR8 by estrogen may be the first indication of a novel hormonally responsive, auto-/paracrine INSL-3 LGR8 ligand receptor system active in human thyroid carcinoma cells.

Hombach-Klonisch S, et al. Int J Oncol 2003 May;22(5):993-1001

H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2

Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrated that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for 33P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8, restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.

Sudo S. et al. J. Biol. Chem, Feb 2003; 278: 7855 - 7862.

 

Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain

Relaxin is a peptide hormone with known actions associated with female reproductive physiology, but it has also been identified in the brain. Only one relaxin gene had been characterized in rodents until recently when a novel human relaxin gene, human gene-3 (H3) and its mouse equivalent (M3) were identified. The current study reports the identification of a rat homologue, rat gene-3 (R3) relaxin that is highly expressed in a discrete region of the adult brain. The full R3 relaxin cDNA was generated using RT-PCR and 3' and 5' RACE protocols. The derived amino acid sequence of R3 relaxin retains all the characteristic features of a relaxin peptide and has a high degree of homology with H3 and M3 relaxin. The distribution of R3 relaxin mRNA in adult rat brain was determined and highly abundant expression was only detected in neurons of the ventromedial dorsal tegmental nucleus (vmDTg) in the pons, whereas all other brain areas were unlabelled or contained much lower mRNA levels. Relaxin binding sites and relaxin immunoreactivity were also detected in the vmDTg. These together with earlier findings provide strong evidence for a role(s) for multiple relaxin peptides as neurotransmitters and/or modulators in the rat CNS.

Burazin TC, Bathgate RA, Macris M, Layfield S, Gundlach AL, Tregear GW. J Neurochem 2002 Sep;82(6):1553-7

 

Relaxin-like bioactivity of ovine Insulin 3 (INSL3) analogues

Relaxin is an insulin-like peptide consisting of two separate chains (A and B) joined by two inter- and one intrachain disulfide bonds. Binding to its receptor requires an Arg-X-X-X-Arg-X-X-Ile motif in the B-chain. A related member of the insulin superfamily, INSL3, has a tertiary structure that is predicted to be similar to relaxin. It also possesses an Arg-X-X-X-Arg motif within its B-chain, although this is displaced by four amino acids towards the C-terminus from the corresponding position within relaxin. We have previously shown that synthetic INSL3 itself does not display relaxin-like activity although analogue (Analogue A) with an introduced arginine residue in the B-chain giving it an Arg cassette in the exact relaxin position does possess weak activity. In order to identify further the structural features that impart relaxin function, solid phase peptide synthesis was used to prepare three additional analogues for bioassay. Each of these contained point substitutions within the arginine cassette. Analogue D contained the full human relaxin binding cassette, Analogue G consisted of the native INSL3 sequence containing an Arg to Ala substitution, and Analogue E was a further modification of Analogue A, with the same substitution. Each analogue was fully chemically characterized by a number of criteria. Detailed circular dichrosim spectroscopy analyses showed that the changes caused little alteration of secondary structure and, hence, overall conformation. However, each analogue displayed only weak relaxin-like activity. These results indicate that while the arginine cassette is vital for relaxin-like activity, there are additional, as yet unidentified structural requirements for relaxin binding.

Claasz AA, Bond CP, Bathgate RA, Otvos L, Dawson NF, Summers RJ, Tregear GW, Wade JD. Eur J Biochem 2002 Dec;269(24):6287-93

 

INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent

Several orphan G protein-coupled receptors homologous to gonadotropin and thyrotropin receptors have recently been identified and named as LGR4-8. INSL3, also known as Leydig insulin-like peptide or relaxin-like factor, is a relaxin family member expressed in testis Leydig cells and ovarian theca and luteal cells. Male mice mutant for INSL3 exhibit cryptorchidism or defects in testis descent due to abnormal gubernaculum development whereas overexpression of INSL3 induces ovary descent in transgenic females. Because transgenic mice missing the LGR8 gene are also cryptorchid, INSL3 was tested as the ligand for LGR8. Here, we show that treatment with INSL3 stimulated cAMP production in cells expressing recombinant LGR8 but not LGR7. In addition, interactions between INSL3 and LGR8 were demonstrated following ligand receptor cross-linking. Northern blot analysis indicated that the LGR8 transcripts are expressed in gubernaculum whereas treatment of cultured gubernacular cells with INSL3 stimulated cAMP production and thymidine incorporation. The present study identified the ligand for an orphan G protein-coupled receptor based on common phenotypes of ligand and receptor null mice. Demonstration of INSL3 as the ligand for LGR8 facilitates understanding of the mechanism of testis descent and allows studies on the role of INSL3 in gonadal and other physiological processes.

Kumagai J, Hsu SY, Matsumi H, Roh JS, Fu P, Wade JD, Bathgate RA, Hsueh AJ. J Biol Chem 2002 Aug 30;277(35):31283-6

 

Reproductive Biology of the Relaxin-Like Factor (RLF/INSL3)

The relaxin-like factor (RLF), which is the product of the insulin-like factor 3 (INSL3) gene, is a new circulating peptide hormone of the relaxin-insulin family. In male mammals, it is a major secretory product of the testicular Leydig cells, where it appears to be expressed constitutively but in a differentiation-dependent manner. In the adult testis, RLF expression is a good marker for fully differentiated adult-type Leydig cells, but it is only weakly expressed in prepubertal immature Leydig cells or in Leydig cells that have become hypertrophic or transformed. It is also an important product of the fetal Leydig cell population, where it has been demonstrated using knockout mice to be responsible for the second phase of testicular descent acting on the gubernaculum. INSL3 knockout mice are cryptorchid, and in estrogen-induced cryptorchidism, RLF levels in the testis are significantly reduced. RLF is also made in female tissues, particularly in the follicular theca cells of small antral follicles and in the corpus luteum of the cycle and pregnancy. The ruminant ovary has a very high level of RLF expression, and analysis of primary cultures of ovarian theca-lutein cells indicated that, as in the testis, expression is probably constitutive but differentiation dependent. Female INSL3 knockout mice have altered estrous cycles, where RLF may be involved in follicle selection, an idea strongly supported by observations on bovine secondary follicles. Recently, a novel 7-transmembrane domain receptor (LGR8 or Great) has been tentatively identified as the RLF receptor, and its deletion in mice leads also to cryptorchidism.

Ivell R, Bathgate RA. Biol Reprod 2002 Sep;67(3):699-705

Identification of relaxin-3/INSL7 as a ligand for Orphan G-protein Coupled Receptors GPCR135 and GPCR142

ChangLu Liu, et al. 4th International Conference on Relaxin and Related peptides. Sep. 5-10, 2004, Wyoming, USA