Preliminary Structure–Function Relationship Studies on Insulin-Like Peptide 5 (INSL5)

Abstract

Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide bonded member of insulin/relaxin superfamily of peptides that includes insulin, insulin-like growth factor I and II (IGFI and IGFII), insulin-like peptide 3, 4, 5 and 6 (INSL3, 4, 5 and 6), relaxin-1 (H1 relaxin), -2 (H2 relaxin) and -3 (H3 relaxin). Although it is expressed in relatively high levels in the gut, its biological function remains unclear. However, recent reports suggest a significant orexigenic action and a role in the regulation of insulin secretion and β-cell homeostasis, which implies that both agonists and antagonists of the peptide may have significant therapeutic applications. Modern solid phase synthesis techniques together with regioselective disulfide bond formation were employed for a preliminary structure–function relationship study of mouse INSL5. Two point mutated analogues, mouse INSL5 A-B(R24A, W25A) and mouse INSL5 A-B(K6A, R14A, Y18A) were chemically prepared, where the residues in the B-chain that may be involved in receptor activation and affinity binding, were respectively mutated. Synthetic mouse INSL5 A-B(R24A, W25A) analogue was inactive on RXFP4, the native receptor for INSL5, suggesting Arg^B24 and Trp^B25 are probably directly involved in INSL5 receptor activation. Mouse INSL5 A-B(K6A, R14A, Y18A) analogue had both decreased affinity and potency on RXFP4 (pIC₅₀ 7.7 ± 0.2, pEC₅₀ 7.87 ± 0.18) which indicated that one or more of these residues are critical for the binding to the receptor.