Structure and Function Relationship of Murine Insulin-like Peptide 5 (INSL5): Free C-Terminus Is Essential for RXFP4 Receptor Binding and Activation

Abstract

Insulin-like peptide 5 (INSL5) is a member of insulin/relaxin superfamily of peptides. It has recently been identified as the cognate ligand for the G-protein-coupled receptor, RXFP4. Although the complete physiological role of this naturally occurring peptide is still under investigation, there is evidence that it acts to both stimulate appetite and activate colon motility. This suggests that both agonists and antagonists of the peptide may have potential therapeutic applications. To further investigate the physiological role of this peptide and because of the ready availability of the mouse as an experimental animal, the preparation of mouse INSL5 was undertaken. Because of its complex structure and the intractable nature of the two constituent chains, different solid phase synthesis strategies were investigated, including the use of a temporary B-chain solubilizing tag. Unfortunately, none provided significantly improved yield of purified mouse INSL5 which reflects the complexity of this peptide. In addition to the native peptide, two mouse INSL5 analogues were also prepared. One had its two chains as C-terminal amides, and the other contained a europium chelate monolabel for use in RXFP4 receptor assays. It was found that the INSL5 amide was substantially less potent than the native acid form. A similar observation was made for the human peptide acid and amide, highlighting the necessity for free C-terminal carboxylates for function. Two additional human INSL5 analogues were prepared to further investigate the necessity of a free C-terminal. The results together provide a first insight into the mechanism whereby INSL5 binds to and activates RXFP4.

International Union of Basic and Clinical Pharmacology. XCV. Recent Advances in the Understanding of the Pharmacology and Biological Roles of Relaxin Family Peptide Receptors 1–4, the Receptors for Relaxin Family Peptides

“…INSL5 has many of the characteristics of an incretin being secreted from enteroendocrine L cells and regulating insulin secretion and glucose homeostasis (Burnicka-Turek et al, 2012). Synthesis of human INSL5 represents considerable challenges so that some of the characterization of the peptide has been performed with mouse INSL5 that has 71% homology (Belgi et al, 2011). Compared with human INSL5, mouse INSL5 displays an 8-fold increase in affinity in binding assays and a 5-fold greater potency in Liu et al (2005a) cAMP inhibition assays, possibly related to the overall greater positive charge associated with the nonconserved residues (Belgi et al, 2011) (Table 4).…”

“…Synthesis of human INSL5 represents considerable challenges so that some of the characterization of the peptide has been performed with mouse INSL5 that has 71% homology (Belgi et al, 2011). Compared with human INSL5, mouse INSL5 displays an 8-fold increase in affinity in binding assays and a 5-fold greater potency in Liu et al (2005a) cAMP inhibition assays, possibly related to the overall greater positive charge associated with the nonconserved residues (Belgi et al, 2011) (Table 4). In addition, the amidated peptides show a considerable reduction in potency compared with the free acids (Belgi et al, 2011).…”

“…Compared with human INSL5, mouse INSL5 displays an 8-fold increase in affinity in binding assays and a 5-fold greater potency in Liu et al (2005a) cAMP inhibition assays, possibly related to the overall greater positive charge associated with the nonconserved residues (Belgi et al, 2011) (Table 4). In addition, the amidated peptides show a considerable reduction in potency compared with the free acids (Belgi et al, 2011). Although relaxin-3 binds both RXFP3 and RXFP4 with high affinity, INSL5 is actually a weak antagonist at RXFP3 (Zhu et al, 2008) (Table 4).…”

Liquid chromatography/mass spectrometry based detection and semi‐quantitative analysis of INSL5 in human and murine tissues

“…Mass spectrometric techniques have been applied to human INSL5 protein previously to characterise the prohormone cleavage sites after heterologous expression in a mammalian cell line (Cos7 co‐overexpressing furin), identifying an identical mature form to the one reported here . Whilst MS has been used to verify the sequence of recombinant and synthetically synthesised INSL5 variants, to our knowledge, no endogenously expressed INSL5 has previously been analysed using LC/MS.…”

Synthesis and pharmacological characterization of a europium-labelled single-chain antagonist for binding studies of the relaxin-3 receptor RXFP3

“…Importantly, this K i matches well with that obtained from competition binding studies with Eu-H3/I5 (Haugaard-Kedstrom et al 2011) indicating that the DTPA-cage does not interfere with the ligand binding to RXFP3. Importantly, several other relaxin ligands have been fluorescent labelled using the Eu-DTPA strategy without altered receptor selectivity profile (Belgi et al 2011;Shabanpoor et al 2008Shabanpoor et al , 2012. Competition binding experiments using cold R3B1-22R and R3 ligands demonstrate that this peptide is an excellent probe for screening of both antagonist and agonist compounds.…”