Abstract
Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. Although its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide, insulin-like peptide 3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats of RXFP1 and RXFP2 using B-chain-specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr-3, Leu-20, and Phe-23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys-9, His-12, Lys-17, Arg-18, and Arg-22. Our results provide new insights into the mechanism of secondary activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analog, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin.
Highlights
The pleiotropic hormone Human gene-2 (H2) relaxin can bind to both RXFP1 and RXFP2 receptors
We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, residues Tyr-3, Leu-20, and Phe-23 appear to contribute
When the chimeric peptide H2A-INSL3B was tested against an RXFP2 chimeric receptor comprising of the TM domain from RXFP1, full activation of the receptor was observed. These results highlight that the A-chains of H2 relaxin and insulin-like peptide 3 (INSL3) play a key role in interacting with the TM exoloops of RXFP1 or RXFP2, and the mode by which H2 relaxin or INSL3 binds to the leucine-rich repeat (LRR) of RXFP2 is not compatible with the interaction between the A-chain of these chimeric peptides and TM exoloops of RXFP2 [21]
Summary
The pleiotropic hormone H2 relaxin can bind to both RXFP1 and RXFP2 receptors. Results: A-chain-modified H2 relaxin analogues retain RXFP1 activity and anti-fibrotic properties while ablating RXFP2 responses. Human gene-2 (H2) relaxin is currently in Phase III clinical trials for the treatment of acute heart failure It is a 53-amino acid insulin-like peptide comprising two chains and three disulfide bonds. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, residues Tyr-3, Leu-20, and Phe-23 appear to contribute These same three residues are important drivers of the. Relaxin is a pleiotropic hormone with additional roles in several non-reproductive processes that are centered around its anti-fibrotic [3], vasodilatory, and cardioprotective effects [4] Based on the latter two actions, human H2 relaxin is currently in Phase III clinical trials for the treatment of patients with acute heart failure [5]. The large extracellular domain of these receptors consists of series of LRR strands
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