Abstract
The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1–4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4, and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr, and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-chain of relaxin-3 and the A-chain of INSL5, and retains full activation potency at RXFP3 and RXFP4. All resultant R3/I5 mutants retained high activation potency towards RXFP4, but most displayed significantly decreased or even abolished activation potency towards RXFP3, confirming the role of these four INSL5 determinants in distinguishing RXFP4 from RXFP3.
Highlights
The relaxin family is a group of peptide hormones, including relaxin, relaxin-3, and insulin-like peptides 3–6 (INSL3, INSL4, insulin-like peptide 5 (INSL5), and INSL6)[1,2,3,4,5]
When the four determinants were all replaced by the corresponding residues of relaxin-3, the resultant INSL5 mutant acquired high activation potency towards both RXFP4 and RXFP3
Our present study discloses the mechanism by which INSL5 distinguishes RXFP4 from the homologous RXFP3, and sheds new light on how the relaxin family peptides interact with their receptors
Summary
The relaxin family is a group of peptide hormones, including relaxin (primates express two relaxins, namely relaxin-1 and relaxin-2), relaxin-3 ( known as INSL7), and insulin-like peptides 3–6 (INSL3, INSL4, INSL5, and INSL6)[1,2,3,4,5]. Among the four known relaxin family peptide receptors, the homologous RXFP1 and RXFP2 belong to the leucine-rich repeat (LRR)-containing G protein-coupled receptor subfamily. The LRR module forms the high affinity ligand-binding site that primarily interacts with the essential B-chain residues of their ligands[12,13,14], and the LDLa module is critical for receptor activation[15,16,17,18]. INSL5 and relaxin-3 contain unique residues in their B-chains (Fig. 1A), and some of these residues are likely responsible for their distinct selectivity against RXFP3. Our present study discloses the mechanism by which INSL5 distinguishes RXFP4 from the homologous RXFP3, and sheds new light on how the relaxin family peptides interact with their receptors
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