A Novel Leydig Cell cDNA-derived Protein Is a Relaxin-like Factor

Bombyxin 11, a product of the brain of the adult silkmoth, Bombyx mori, binds to ovarian cells of three different species of lepidoptera, i.e. B. mori (silkmoth), Samia Cynthia ricini (ailanthus moth), and an ovarian cell line of Spodopteru frugiperda (Sf9) (fall armyworm). Crude Sf9 cell membrane preparations were used to show that the purported bombyxin receptor binds its ligand in a specific, saturable, and reversible manner. The dissociation constant of the bombyxin-receptor complex is 260 -C 90 pM. Quantitative binding studies and Scatchard analysis suggest that every Sf9 cell displays 20000 receptors on the surface. The cross-linked bombyxin-receptor ligand complex has an apparent molecular mass of about 300 kDa as determined by SDSIPAGE. Reduction causes the bombyxin receptor to dissociate into two subunits with molecular masses of 90 kDa and 116 kDa. The size and subunit structure of the putative bombyxin receptor on Sf9 cells show some similarities to the mammalian insulin receptor.Keywords: bombyxin ; insulin ; bombyxin receptor; structure/function relationship ; photoactivatible crosslink Bombyxin, an invertebrate developmental factor similar in structure to insulin, was isolated from the brain of the silkmoth, Bombyx mori [I, 21. Bombyxin elicits metamorphosis in brain extirpated dormant pupae of the saturniid moth, Sumia Cynthia ricini [3], by acting on the prothoracic gland to stimulate the synthesis and release of ecdysone, the steroid hormone that causes molting and metamorphosis. This effect is ascribed to two structurally unrelated prothoracicotropic hormones, the smaller of which was later named bombyxin, while the larger one (30 kDa) retained the name prothoracicotropic hormone [4, 51. The structural dissimilarity of the two hormones means that there are different receptors that mediate a similar cell response. Indeed, the prothoracicotropic glands of Manduca sexta were sensitive to the small and large prothoracicotropic hormones, as demonstrated by Bollenbacher et al. [6].Although initial observations pointed to bombyxin as a prothoracicotropic hormone 171, its biological function in B. mori remained obscure [8, 91. Bombyxin titers in the hemolymph of postembryonic B. mori showed no correlation with ecdysone during molting [ 101. Furthermore, applications of physiological concentrations of bombyxin to brain extirpated dormant B. mori were ineffective in inducing metamorphosis [3].Based on the hypothesis that a structural relationship may indicate functional similarity, the insulin-like bombyxin has been suspected to have an effect on the blood sugar of insects, for instance in the metabolism of trehalose [Ill. Bombyxin was detected in the ovaries of the silkmoth [I21 and significantly higher levels of bombyxin were found in the hemolymph of the female [lo]. This observation suggests a role for bombyxin similar to that of the mammalian ovarian hormone relaxin, another member of the insulin family. It seems possible that bombyxin is involved in the ovarian development [13] of several specie...

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“…Neither insulin nor relaxin nor relaxin-like factor [21] competed for bombyxin receptor binding (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

The Prothoracicotropic Hormone Bombyxin has Specific Receptors on Insect Ovarian Cells

Fullbright

Lacy

Büllesbach

1997

European Journal of Biochemistry

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“…The other conserved region in the B chain contains the R-A-L-V-R sequence that seems to be important for the binding of INSL3 to the relaxin receptor (21). However, it is the arginine (R) at either end of this sequence that is crucial and therefore the substitution of the valine for leucine (V19L), another neutral charged hydrophobic amino acid, is probably conservative.…”

Section: Comparison Of Amino Acid Changes With a Multi-species Proteimentioning

confidence: 99%

Genetic analysis of the INSL3 gene in patients with maldescent of the testis

et al. 2001

European Journal of Endocrinology

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Objective: Testicular maldescent is important because it is a common congenital disorder that is associated with an increased risk of infertility and testicular cancer. Murine studies indicate that testicular maldescent can result from disruption of insulin-like factor 3 (INSL3) activity and that it may be more severe when there is concurrent undermasculinisation. Therefore, the INSL3 gene was screened for mutations and polymorphisms that may contribute to testicular maldescent in patients with undermasculinisation as well as those with isolated testicular maldescent. Methods and Results: The patient groups consisted of individuals with isolated testicular maldescent n 28 and patients with undermasculinised genitalia and intra-abdominal n 24 or inguinal gonads n 33X The three control groups were: normal males n 15Y males with undermasculinised genitalia and scrotal gonads n 29 and females n 82X SSCP/HA mutation screening detected eight variants, five of which were predicted to alter the protein sequence (A-1G, V19L, P25S, A36T, R78H). Three of the amino acid changes (A-1G, V19L, R78H) each occurred in a single control sample and one was identified in a male with undermasculinised genitalia and intraabdominal testes (P25S). The A36T amino acid polymorphism was found in both patient and control groups at a similar frequency. Conclusions: The evidence suggests that INSL3 mutations and polymorphisms are not a major cause of testicular maldescent with or without associated undermasculinisation.

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“…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).…”

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confidence: 99%

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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A Novel Leydig Cell cDNA-derived Protein Is a Relaxin-like Factor

Cited by 106 publications

References 18 publications

The Prothoracicotropic Hormone Bombyxin has Specific Receptors on Insect Ovarian Cells

The Prothoracicotropic Hormone Bombyxin has Specific Receptors on Insect Ovarian Cells

Genetic analysis of the INSL3 gene in patients with maldescent of the testis

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

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