Specific, High Affinity Relaxin-like Factor Receptors

Büllesbach, Erika E.; Schwabe, Christian

doi:10.1074/jbc.274.32.22354

Cited by 42 publications

(26 citation statements)

References 29 publications

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“…Membrane preparations were prepared as described previously (Büllesbach & Schwabe 1999) using germ cell fractions. Purified INSL3 was labeled with 4…”

Section: Receptor Binding Assaymentioning

confidence: 99%

Dynamics of insulin-like factor 3 and its receptor expression in boar testes

Minagawa¹,

Sagata²,

Pitia³

et al. 2014

Journal of Endocrinology

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Relaxin-like factor (RLF), now mainly known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. As a major step toward understanding the as-yet-unknown function of INSL3 in boars, this study aimed to develop a time-resolved fluoroimmunoassay for boar INSL3, characterize the dynamics of INSL3 expression during development, and demonstrate the expression of the INSL3 hormone-receptor system in the testis. All samples were collected from Duroc boars. The sensitivity of the assay system established was 8.2 pg/well (164 pg/ml), and no cross-reactivity with other hormones, such as porcine relaxin, was observed. Circulating INSL3 was shown to increase progressively during development. INSL3 secreted from the Leydig cells was released not only into the blood circulation but also into the interstitial and seminiferous compartments in sufficient concentrations. A testicular fractionation study revealed that its receptor RXFP2 transcripts were expressed mainly in testicular germ cells. In addition, INSL3 bound to the germ cell membranes in a hormone-specific and saturable manner. These results reveal that INSL3 secreted into the interstitial compartment from the Leydig cells is transported into the seminiferous compartments, where its receptor RXFP2 is expressed mainly in the germ cells to which INSL3 binds, suggesting that INSL3 functions as a paracrine factor on seminiferous germ cells.

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“…Membrane preparations were prepared as described previously (Büllesbach & Schwabe 1999) using germ cell fractions. Purified INSL3 was labeled with 4…”

Section: Receptor Binding Assaymentioning

confidence: 99%

Dynamics of insulin-like factor 3 and its receptor expression in boar testes

Minagawa¹,

Sagata²,

Pitia³

et al. 2014

Journal of Endocrinology

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“…Radioactive labeling of Tyr-B30 with 125 I Ϫ was performed by the chloramine T method, followed by the removal of the indole protecting groups (19). Phe-A3-125 I-Tyr-B30 relaxin di-sulfoxide was isolated by HPLC on an Aquapore 300 column using a 60-min linear gradient from 25 to 40% B.…”

Section: Biochemical Characterizationmentioning

confidence: 99%

The Relaxin Receptor-binding Site Geometry Suggests a Novel Gripping Mode of Interaction

Büllesbach

Schwabe

2000

Journal of Biological Chemistry

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Relaxin has a unique, clearly identifiable, mixed function receptor-binding region comprising amino acid residues that evolve sequentially from the central portion of the B chain ␣-helix. Two arginine residues in positions B13 and B17 that project like forefinger and middle finger from the helix provide the electrostatic element opposed by the hydrophobic (thumb) element isoleucine (B20), offset from the arginines by about 40°. The binding intensity of relaxin to its receptor decreases by 3 orders of magnitude if alanine is substituted for the newly discovered binding component isoleucine in position B20. The arginine residues cannot be replaced by other positive charges, nor can the guanidinium group be presented on a longer or shorter hydrocarbon chain. In contrast, the hydrophobic interaction is incremental in nature, and the contribution to the total binding energy is roughly proportional to the number of hydrocarbon units in the side chain. It appears that a hydrophobic surface exists on the receptor that offers optimal van der Waals' interaction with ␤-branched hydrophobic amino acids. The binding energy increases roughly 10-fold with each methylene group whereby ␤-branching is more effective per surface unit than chain elongation. Aromatic side chains appear to demarcate the extent of the binding region in so far as residues larger than phenylalanine decrease receptor binding. The exceptional clarity of binding site geometry in relaxin makes for an excellent opportunity to design peptido-mimetics.Relaxin, a small, two-chain protein, the physiological mediator of parturition in most mammals (1), has recently been shown to influence significantly the symptoms of scleroderma (2, 3). Since its discovery (4) relaxin has provided a share of unusual features including an insulin-like structure (5-9) and a receptor-binding site composed of two charged residues, i.e. arginine in position B13 and B17 (10, 11). The binding residues are positioned one turn apart on the major B chain helix and are projecting parallel into the surrounding water (12). This observation led to the suggestion that relaxin would bind to the receptor by a dual prong mechanism involving the interaction of the guanidinium groups with two negative charges at the bottom of a binding pocket in the receptor (11). Although both arginines are indispensable, the fact that arginine-containing peptides would not interfere with binding suggested that other binding site members might exist. In this paper, we are reporting that the receptor-binding site of relaxin includes isoleucine in position B20, which is located three-quarter of one turn further toward the C-terminal end of the same helix so that the hydrophobic side chain opposes the two arginines forming a quasi-prehensile unit that points to a novel binding mechanism. Evidence presented in this paper supports the conclusion that Ile-B20 is as important for receptor-binding as either of the critical arginines and that the relaxin/receptor interaction is trivalent. EXPERIMENTAL PROCEDURES MaterialsA...

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“…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.…”

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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

390

299

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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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Specific, High Affinity Relaxin-like Factor Receptors

Cited by 42 publications

References 29 publications

Dynamics of insulin-like factor 3 and its receptor expression in boar testes

Dynamics of insulin-like factor 3 and its receptor expression in boar testes

The Relaxin Receptor-binding Site Geometry Suggests a Novel Gripping Mode of Interaction

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

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