Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

Muda, Marco; He, Chaomei; Martini, Paolo; Ferraro, Tania; Layfield, Sharon; Taylor, Deanne; Chevrier, Colette; Schweickhardt, Rene; Kelton, Christie; Ryan, Peter L.; Bathgate, Ross A. D.

doi:10.1093/molehr/gah205

Cited by 61 publications

(61 citation statements)

References 32 publications

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“…It has been reported that RXFP1 and Rxfp1 are expressed in different splice variants in humans and mice, whereby often only w100 bp of regions encoding the ectodomain can be missing, thereby making the resulting products nonfunctional (Muda et al 2005, Scott et al 2006. In the present study, the RT-PCR data with primers amplifying the endodomain of the RXFP1 only was not able to resolve the issue of whether splice variants involving the ectodomaincoding region are present or not.…”

Section: Discussioncontrasting

confidence: 58%

Evidence for expression of relaxin hormone-receptor system in the boar testis

Kato¹,

Siqin²,

Minagawa³

et al. 2010

Journal of Endocrinology

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Although the physiological role of relaxin (RLN) in males remains largely unknown, there is limited evidence that the testis might be a candidate source and target of RLN in boars, as RLN transcripts are detected in the boar testis and it contains RLN-binding sites. To determine whether the boar testis acts as a source and target tissue of RLN, we characterised the expression pattern and cellular localisation of both RLN and its own receptor LGR7 (RXFP1) in boar testes during postnatal development by molecular and immunological approaches. Testes were collected from Duroc boars, and partial cDNA sequences of the boar homologue of human RXFP1 were identified. RLN expression increased through puberty onwards, while RXFP1 expression changed little during development.RLN mRNA and protein expression were restricted to the Leydig cells, whereas both Leydig cells and seminiferous epithelial cells expressed RXFP1 mRNA and protein.Interestingly, RLN was expressed in the testis as an 18 kDa form (the expected size of proRLN), but not as the 6 kDa mature form, during development because of a lack of the enzyme required for proRLN processing. In contrast, RXFP1 was detected at all stages as specific bands of 75 and 91-95 kDa (likely non-glycosylated and glycosylated RXFP1 respectively). Thus, we provide evidence for expression of RLN-RXFP1 ligand-receptor system in the boar testis, suggesting that the testis act as a source and possible target tissue of RLN.

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Section: Discussioncontrasting

confidence: 58%

Evidence for expression of relaxin hormone-receptor system in the boar testis

Kato¹,

Siqin²,

Minagawa³

et al. 2010

Journal of Endocrinology

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“…The dramatic effects of L283F and T222P mutations prove that both hydrophobic core and hydrophilic phase of LRR play an important role in this process. Interestingly, the minor splice variants of LGR7 missing some of the LRRs also failed to be expressed on cell surface membranes in vitro (17).…”

Section: Discussionmentioning

confidence: 99%

T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane

Bogatcheva

Ferlin

Feng

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

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“…Ligand Binding Assays-The method to study the binding of 125 I-INSL3 to HEK-293T cells stably transfected with human LGR8 has been described previously (15).…”

Section: Methodsmentioning

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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Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

Cited by 61 publications

References 32 publications

Evidence for expression of relaxin hormone-receptor system in the boar testis

Evidence for expression of relaxin hormone-receptor system in the boar testis

T222P mutation of the insulin-like 3 hormone receptor LGR8 is associated with testicular maldescent and hinders receptor expression on the cell surface membrane

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

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