Relaxin-3 and its endogenous receptor RXFP3 are involved in fundamental neurological signalling pathways, such as learning and memory, stress, feeding and addictive behaviour. Consequently, this signalling system has emerged as an attractive drug target. Development of leads targeting RXFP3 relies on assays for screening and ligand optimization. Here, we present the synthesis and in vitro characterization of a fluorescent europium-labelled antagonist of RXFP3. This ligand represents a cheap and safe but powerful tool for future mechanistic and cell-based receptor-ligand interaction studies of the RXFP3 receptor.
Haugaard-KedstrÖm LM, Wong LL, Bathgate RA, Amino Acids. 2015 Jun;47(6):1267-71. doi: 10.1007/s00726-015-1961-x. Epub 2015 Mar 20.
Relaxin-3 is a member of the insulin superfamily. It is expressed in the nucleus incertus of the brainstem, which has projections to the hypothalamus. Relaxin-3 binds with high affinity to RXFP1 and RXFP3. RXFP3 is expressed within the hypothalamic paraventricular nucleus (PVN), an area central to the stress response. The physiological function of relaxin-3 is unknown but previous work suggests a role in appetite control, stimulation of the hypothalamic-pituitary-gonadal axis and stress. Central administration of relaxin-3 induces c-fos expression in the PVN and increases plasma ACTH levels in rats. The aim of this study was to investigate the effect of central administration of human relaxin-3 (H3) on the hypothalamic-pituitary-adrenal (HPA) axis in male rodents in vivo and in vitro. Intracerebroventricular (i.c.v) administration of H3 (5 nmol) significantly increased plasma corticosterone at 30 min following injection compared with vehicle. Intra-PVN administration of H3 (1.8-1620 pmol) significantly increased plasma ACTH at 1620?pmol H3 and corticosterone at 180-1620?pmol H3 at 30?min following injection compared with vehicle. The stress hormone prolactin was also significantly raised at 15?min post-injection compared with vehicle. Treatment of hypothalamic explants with H3 (10-1000 nM) stimulated the release of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), but H3 had no effect on the release of ACTH from in vitro pituitary fragments. These results suggest that relaxin-3 may regulate the HPA axis, via hypothalamic CRH and AVP neurons. Relaxin-3 may act as a central signal linking nutritional status, reproductive function and stress.
McGowan BM, Minnion JS, Murphy KG et al., J Endocrinol. 2014 May 12;221(2):337-46. doi: 10.1530/JOE-13-0603. Print 2014 May.
Relaxin-like peptides (RLN/INSL) play diverse roles in reproductive and neuroendocrine processes in placental mammals and are functionally associated with two distinct types of receptors (RXFP) for each respective function. The diversification of RLN/INSL and RXFP gene families in vertebrates was predominantly driven by whole genome duplications (2R and 3R). Teleosts preferentially retained duplicates of genes putatively involved in neuroendocrine regulation, harboring a total of 10-11 receptors and 6 ligand genes, while most mammals have equal numbers of ligands and receptors. To date, the ligand-receptor relationships of teleost Rln/Insl peptides and their receptors have largely remained unexplored. Here, we use selection analyses based on sequence data from 5 teleosts and qPCR expression data from zebrafish to explore possible ligand-receptor pairings in teleosts. We find support for the hypothesis that, with the exception of RLN, which has undergone strong positive selection in mammalian lineages, the ligand and receptor genes shared between mammals and teleosts appear to have similar pairings. On the other hand, the teleost-specific receptors show evidence of subfunctionalization. Overall, this study underscores the complexity of RLN/INSL and RXFP ligand-receptor interactions in teleosts and establishes theoretical background for further experimental work in nonmammals.
Good S, Yegorov S, Martijn J et al., Int J Evol Biol. 2012;2012:310278. Epub 2012 Sep 13.
Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely hampered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncatedrelaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.
Haugaard-Kedström LM, Shabanpoor F, Hossain MA et al., J Am Chem Soc. 2011 Apr 6;133(13):4965-74. doi: 10.1021/ja110567j. Epub 2011 Mar 8.
