Structural requirements for the interaction of sheep insulin-like factor 3 with relaxin receptors in rat atria.

Relaxin is a peptide with various reproductive and nonreproductive functions. The site for the peptide-receptor interaction contains two arginines (Arg) and an isoleucine (Ile) or valine (Val) residue in the B-chain with a configuration of -Arg-X-X-X-Arg-X-X-Ile/Val-X-. The sheep insulin-like peptide 3 (INSL3), a structural homologue of relaxin, also contains the n, n+4 arginines in the B-chain but they are displaced towards the carboxyl terminus by four residues (-X-X-X-X-Arg-X-X-Val-Arg-). Human INSL3 increases the activity of human relaxin in mouse bioassays. Here, we investigated whether sheep synthetic INSL3 affects the relaxin activity in rat atria. INSL3 lacked relaxin-like agonist activity but blocked the activity of relaxin and competed for relaxin binding sites at high concentrations. We also synthesized analogues of INSL3, with amino acid substitutions in the arginine-binding region. Analogues A, D and E, which have the arginines in positions identical to relaxin, showed weak relaxin-like agonist activity. These results suggest that other sites in the relaxin molecule are involved in high-affinity peptide-receptor interaction for the production of the relaxin biological responses.

Relaxin-like bioactivity of ovine Insulin 3 (INSL3) analogues.

Relaxin is an insulin-like peptide consisting of two separate chains (A and B) joined by two inter- and one intrachain disulfide bonds. Binding to its receptor requires an Arg-X-X-X-Arg-X-X-Ile motif in the B-chain. A related member of the insulin superfamily, INSL3, has a tertiary structure that is predicted to be similar to relaxin. It also possesses an Arg-X-X-X-Arg motif within its B-chain, although this is displaced by four amino acids towards the C-terminus from the corresponding position within relaxin. We have previously shown that synthetic INSL3 itself does not display relaxin-like activity although analogue (Analogue A) with an introduced arginine residue in the B-chain giving it an Arg cassette in the exact relaxin position does possess weak activity. In order to identify further the structural features that impart relaxin function, solid phase peptide synthesis was used to prepare three additional analogues for bioassay. Each of these contained point substitutions within the arginine cassette. Analogue D contained the full human relaxin binding cassette, Analogue G consisted of the native INSL3 sequence containing an Arg to Ala substitution, and Analogue E was a further modification of Analogue A, with the same substitution. Each analogue was fully chemically characterized by a number of criteria. Detailed circular dichroism spectroscopy analyses showed that the changes caused little alteration of secondary structure and, hence, overall conformation. However, each analogue displayed only weak relaxin-like activity. These results indicate that while the arginine cassette is vital for relaxin-like activity, there are additional, as yet unidentified structural requirements for relaxin binding.

Purification and characterization of relaxin from the tammar wallaby (Macropus eugenii): bioactivity and expression in the corpus luteum.

The objective of this study was to isolate and purify prorelaxin or mature relaxin from the tammar wallaby corpus luteum (CL), determine their structure and bioactivity, and test the hypothesis that enzymatic cleavage of prorelaxin occurs in late gestation. Tammar relaxin peptides were extracted from pooled corpora lutea of late pregnant tammars using a combination of HPLC methods, and they were identified using Western blotting with a human (H2) relaxin antisera and matrix-assisted laser desorption ionization time of flight mass spectrometry. Although no prorelaxin was identified, multiple 6-kDa peptides were detected, which corresponded to the predicted mature tammar relaxin amino acid sequence, with an A chain of 24 amino acids, and different B chain lengths of 28, 29, 30, and 32 amino acids. Tammar relaxin bound with high affinity to rat cortical relaxin receptors and stimulated cAMP production in the human monocytic cell line, THP-1, which expresses the relaxin receptor. Analysis of individual CL indicated that equivalent amounts of mature relaxin peptides were present throughout gestation and also in unmated tammars at equivalent stages of the luteal phase in the nonpregnant cycle. Immunoreactive relaxin was localized specifically to the luteal cells of the CL and the intensity of immunostaining did not vary between gestational stages. These data show that the CL of both pregnant and unmated tammar wallabies produces mature relaxin and suggests that relaxin expression in this species is not influenced by the conceptus. Moreover, the presence of mature relaxin throughout gestation implies that prohormone cleavage is not limited to the later stages of pregnancy

Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of the relaxin peptide family.

We have identified a novel human relaxin gene, designated H3 relaxin, and an equivalent relaxin gene in the mouse from the Celera Genomics data base. Both genes encode a putative prohormone sequence incorporating the classic two-chain, three cysteine-bonded structure of the relaxin/insulin family and, importantly, contain the RXXXRXX(I/V) motif in the B-chain that is essential for relaxin receptor binding. A peptide derived from the likely proteolytic processing of the H3 relaxin prohormone sequence was synthesized and found to possess relaxin activity in bioassays utilizing the human monocytic cell line, THP-1, that expresses the relaxin receptor. The expression of this novel relaxin gene was studied in mouse tissues using RT-PCR, where transcripts were identified with a pattern of expression distinct from that of the previously characterized mouse relaxin. The highest levels of expression were found in the brain, whereas significant expression was also observed in the spleen, thymus, lung, and ovary. Northern blotting demonstrated an approximately 1.2-kb transcript present in mouse brain poly(A) RNA but not in other tissues. These data, together with the localization of transcripts in the pars ventromedialis of the dorsal tegmental nucleus of C57BLK6J mouse brain by in situ hybridization histochemistry, suggest a new role for relaxin in neuropeptide signaling processes. Together, these studies describe a third member of the human relaxin family and its equivalent in the mouse.