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The relaxin-like factor (RLF) is thought to be responsible for the intra-abdominal migration of the testis during mammalian development. Our latest studies of RLF and LGR8 have revealed that the N-terminal region of the A chain is not required for receptor binding but is indispensable for cyclic AMP generation. RLF derivatives with six residues deleted from the N terminus of the A chain are active, whereas further truncation, up to the first A chain cysteine (A-10), yields tightly binding ligands devoid of signaling activity. These derivatives are specific competitive inhibitors (RLFi) of RLF. Although receptor binding is dependent upon B chain residues, the N-terminal region of the A chain is a generic trigger of the trans-membrane signaling activity.

The relaxin-like factor (RLF) is a circulating hormone that binds to specific membrane-bound uterine receptors in the mouse. Mono-iodinated RLF tracers were produced and characterized specifically to study the properties of the RLF receptor. The tracers bound to the RLF receptor in uterine crude membrane preparations with high affinity (73 nm for 125I-Tyr(A9) RLF and 90 nm for 125I-Tyr(A26) RLF) as determined by Scatchard analysis. The specificity of binding was confirmed by chemical cross-linking experiments. Binding of 125I-Tyr(A9) RLF to the putative receptor was inhibited in the presence of a 640-fold excess of unlabeled human RLF but not by the same excess of human relaxin. SDS-gel electrophoresis of the RLF-receptor complex revealed a molecular mass of >200 kDa, which remained unchanged upon reduction. The size and the lack of subunit structure of the receptor is similar to the features reported for the relaxin receptor. In this regard both, the RLF and the relaxin receptor are different from the insulin- and the insulin-like growth factor-type 1 receptors. This observation supports the relaxin-likeness of this new factor not only toward potential target tissues but also as regards receptor features.

Isolation and sequencing of a genomic clone encoding the mouse gene for the relaxin-like factor (RLF), which is endogenously expressed to a high level exclusively in Leydig cells, indicated that similar sequences were also present at the 3′ end of the mouse JAK3 gene, a gene expressed predominantly in lymphoid tissues. More extensive Southern blot, polymerase chain reaction and sequencing analyses showed that the published mouse sequence for exon 23 of the JAK3 gene in fact comprises two exons, 23A and 23B, separated by an additional novel intron of 2.2 kb, and that within this intron the promoter and exon 1 of the mouse RLF gene are encoded. The two overlapping transcripts appear to use different polyadenylation signals in the common 3′ untranslated region of exon 23B. Transient transfection of different RLF promoter reporter constructs into Leydig, Sertoli, granulosa and kidney cell lines indicate that as little as 0.7 kb of the region upstream of exon 1 of the RLF gene, and within the novel intron 22 of the JAK3 gene, is sufficient to account for cell-specific expression of the RLF gene. This promoter region is specifically hypomethylated in Leydig cells compared to non-expressing tissues.

The relaxin-like factor (RLF) also named insulin-like 3 (INSL3) consists of two polypeptide chains linked by two interchain and one intrachain disulfide bond. RLF binds to its receptor (LGR8 also named RXFP2) through the B chain and initiates transmembrane communication by activating the adenylate cyclase through the N-terminal region of both chains. Cystine A11-B10 occupies a unique position on the molecular surface just outside the binding region and between the two signaling ports. We have synthesized an RLF analogue in which the disulfide A11-B10 was replaced by a peptide bond and found that cAMP production ceased while receptor binding was not affected. In contrast, replacing the disulfide A24-B22 by a peptide bond reduced potency proportional to the binding affinity and lowered efficacy to 65%, while replacing disulfide A10-A15 by a peptide bond reduced binding affinity to 32% and lowered potency to 7% but maintained 100% efficacy. The exceptional properties of the derivative bearing an A11-B10 isopeptide cross-link suggests that the disulfide has a special role in signal transduction. We propose that disulfide A11-B10 serves as an insulator between the two ports, whereas the amide functionality disturbs the signal transmission complex likely due to changes in polarity. The clear separation between receptor binding and signal activation sites within this small protein permits one to study how the relaxin-like factor initiates the signal on the receptor that induces intracellular cAMP production.

Cryptorchidism is a serious problem, which affects 2–5% of the male population. Failure of the testes to descend into the scrotal region impairs germ cell development and is associated with a greater incidence of testicular cancer. The relaxin-like factor (RLF or insulin-like-3) has been shown to be critically important for the timely descent of the testicles in mice. We have discovered that the signal initiation site of the RLF can be eliminated without measurable effects on hormone binding to its receptor and that the resulting RLF derivative is a competitive inhibitor of RLF called RLFi. RLFi administered to pregnant rats causes dose-dependent gonadal retention in the offspring. The ability to control the severity of the syndrome by altering the concentration of RLFi and the timing of administration enables us to study in detail the structural changes that are associated with the action of RLF during critical stages of development. Targeted inhibition of the physiological migration pattern of testicles by RLFi lets one dissect the physiological process such as to find a window for clinical application of RLF and to search for ancillary factors that might play a role during normal development.