Chemical synthesis and biological activity of rat INSL3

Smith, Kathryn J.; Wade, John D.; Claasz, Antonia A.; Ötvös, László; Temelcos, Catherine; Kubota, Yoshihiro; Hutson, John M.; Tregear, Geoffrey W.; Bathgate, Ross A. D.

doi:10.1002/psc.344

Cited by 31 publications

(25 citation statements)

References 16 publications

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“…Ovine and rat INSL3 were chemically synthesized and characterized as described (7,9). Human INSL3 and biotinylated ovine INSL3 were prepared similarly with the ovine INSL3 containing a single biotin molecule on the N terminus of the A chain.…”

Section: Methodsmentioning

confidence: 99%

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INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent

Kumagai

Hsu

Matsumi

et al. 2002

Journal of Biological Chemistry

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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 testisDuring fetal development, the sexual dimorphic position of the gonads in mammals is dependent on the differential development of two ligaments. In males, growth of the gubernaculum and regression of the cranial suspensory ligament results in transabdominal descent of the testes. Impaired testicular descent (cryptorchidism) is a prevalent congenital abnormality in humans, found in 2% of male births. INSL3, 1 also known as Leydig insulin-like peptide or relaxin-like factor, is one of the seven relaxin-like genes in humans known to be expressed in Leydig cells of fetal and adult testes as well as in theca and luteal cells of the postnatal ovary (1). Male mice mutant for INSL3 exhibit bilateral abdominal cryptorchidism (2, 3) whereas female mice overexpressing INSL3 showed ovary descent and displayed bilateral inguinal hernia (4). Although INSL3 binds to gubernacular homogenates (5, 6) and induces growth of rat gubernaculum in whole organ cultures (7), the exact nature of the INSL3 receptor is unknown.A recent study indicated that transgene integration in crsp mice resulted in a 550-kb deletion located upstream of the Brca2 gene, leading to defective testis descent. Because a candidate gene encoding a G protein-coupled receptor homologous to human LGR8 was deleted in these mice (8), we tested whether INSL3 is the cognate ligand for LGR8 based on the observed common phenotypes of potential ligand-receptor pairs in null mice. Here, we report that INSL3 is capable of binding LGR8, leading to the stimulation of cAMP production and thymidine incorporation in the gubernaculum. EXPERIMENTAL PROCEDURESOvine and rat INSL3 were chemically synthesized and characterized as described (7, I-Streptavidin and streptavidin conjugated to horseradish peroxidase (HRP) were purchased from Amersham Biosciences, whereas foskolin, glucagon, collagenase, and trypsin were from Sigma. Sprague-Dawley rats were obtained from Simonsen Laboratories (Gilroy, CA). Animals were anesthetized and killed using CO 2 . Animal care was consistent with institutional and NIH guidelines.Human 293T cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS), 100 g/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. When 70 -80% confluent, cells were transfected with 10 g of plasmid using the calcium phosphate precipitation method (10). After 18 -24 h of incubation, media were replaced with DMEM/F12 containing 10% FBS. Forty-eight hours after transfection, cells (10 5 /ml) were preincubated at 37°C for 30 min in the presence of 0.25 mM 3-isobutyl-1-methylxanthine (IBMX) before treatment with or without hormones for 12 h. Total cAMP content was measured in triplicate by a specific radioimmunoassay (11). All experiments we...

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Section: Methodsmentioning

confidence: 99%

“…Male mice mutant for INSL3 exhibit bilateral abdominal cryptorchidism (2,3) whereas female mice overexpressing INSL3 showed ovary descent and displayed bilateral inguinal hernia (4). Although INSL3 binds to gubernacular homogenates (5,6) and induces growth of rat gubernaculum in whole organ cultures (7), the exact nature of the INSL3 receptor is unknown.…”

mentioning

confidence: 99%

INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent

Kumagai

Hsu

Matsumi

et al. 2002

Journal of Biological Chemistry

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390

299

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“…The native peptides of INSL4 through 6 have not been isolated. There is currently no other structural information on any other relaxin peptide, although circular dichroism analysis of INSL3 generally indicates that it has a tertiary structure similar to that of relaxin (Bü llesbach and Schwabe, 1995;Dawson et al, 1999;Smith et al, 2001). …”

Section: B Structural Features Of Other Relaxin Family Peptidesmentioning

confidence: 99%

International Union of Pharmacology LVII: Recommendations for the Nomenclature of Receptors for Relaxin Family Peptides

Bathgate

Ivell²,

Sanborn³

et al. 2006

Pharmacol Rev

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“…Overexpression of the INSL3 protein in female mice causes the ovaries to descend into the inguinal region due to an overdeveloped gubernaculum (9). Synthetic INSL3 will induce the growth of the gubernaculum in whole organ cultures (10). Hence the peptide is essential for the transabdominal phase of testicular descent where the gubernaculum proliferates and results in the migration of the testis to the inguinal region.…”

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Solution Structure and Characterization of the LGR8 Receptor Binding Surface of Insulin-like Peptide 3

Rosengren

Zhang

Lin

et al. 2006

Journal of Biological Chemistry

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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. Insulin-like peptide 3 (INSL3 4; also known as relaxin-like factor and Leydig cell insulin-like peptide) was originally discovered and isolated as a cDNA clone from a boar testis cDNA library (1). It is primarily produced by prenatal and postnatal mature Leydig cells of the testis and the thecal cells of the ovary. Its primary structure showed it to be a bona fide member of the insulin-insulin-like growth factor-relaxin superfamily, which is now known to have a total of ten members in the human. Like insulin, INSL3 is firstly synthesized as a pre-prohormone precursor containing a signal peptide linked to B-C-A domains. It undergoes processing by hitherto unidentified proprotein convertases, resulting in the production of mature INSL3 consisting of a A-B heterodimer that is covalently linked by two interchain disulfide bonds and one intrachain disulfide bond (Fig. 1) (2). These disulfide bonds are essential for maintaining its expected characteristic insulin-like conformation and its unique biological activities (3). The receptor for INSL3, LGR8, is a member of the leucine-rich repeat-containing G-proteincoupled receptor family (4). It is closely related to the relaxin receptor, LGR7, and has been recently classified as relaxin family peptide (RXFP) receptor, RXFP2 (5). Importantly, relaxin peptides from some species can also bind to and activate LGR8, albeit with a lower affinity than INSL3 (5).Male mice homozygous with targeted disrup...

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Chemical synthesis and biological activity of rat INSL3

Cited by 31 publications

References 16 publications

INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent

INSL3/Leydig Insulin-like Peptide Activates the LGR8 Receptor Important in Testis Descent

International Union of Pharmacology LVII: Recommendations for the Nomenclature of Receptors for Relaxin Family Peptides

Solution Structure and Characterization of the LGR8 Receptor Binding Surface of Insulin-like Peptide 3

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