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

• Published in: Biochemistry (Easton) Vol. 50; no. 39; pp. 8352 - 8361
• Main Authors: Belgi, Alessia, Hossain, Mohammed A, Shabanpoor, Fazel, Chan, Linda, Zhang, Suode, Bathgate, Ross A. D, Tregear, Geoffrey W, Wade, John D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 04.10.2011
• Subjects: Amino Acid Sequence; Animals; Humans; Insulin - biosynthesis; Insulin - chemistry; Insulin - metabolism; Mice; Molecular Sequence Data; Protein Conformation; Proteins - chemistry; Proteins - metabolism; Receptors, G-Protein-Coupled - metabolism; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi201093m

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.