Abstract
Relaxin, an emerging pharmaceutical treatment for acute heart failure, activates the relaxin family peptide receptor (RXFP1), which is a class A G-protein-coupled receptor. In addition to the classic transmembrane (TM) domain, RXFP1 possesses a large extracellular domain consisting of 10 leucine-rich repeats and an N-terminal low density lipoprotein class A (LDLa) module. Relaxin-mediated activation of RXFP1 requires multiple coordinated interactions between the ligand and various receptor domains including a high affinity interaction involving the leucine-rich repeats and a predicted lower affinity interaction involving the extracellular loops (ELs). The LDLa is essential for signal activation; therefore the ELs/TM may additionally present an interaction site to facilitate this LDLa-mediated signaling. To overcome the many challenges of investigating relaxin and the LDLa module interactions with the ELs, we engineered the EL1 and EL2 loops onto a soluble protein scaffold, mapping specific ligand and loop interactions using nuclear magnetic resonance spectroscopy. Key EL residues were subsequently mutated in RXFP1, and changes in function and relaxin binding were assessed alongside the RXFP1 agonist ML290 to monitor the functional integrity of the TM domain of these mutant receptors. The outcomes of this work make an important contribution to understanding the mechanism of RXFP1 activation and will aid future development of small molecule RXFP1 agonists/antagonists.
Highlights
Extracellular loops of the transmembrane domain of the relaxin receptor RXFP1 are predicted to interact with relaxin
Two thermostabilized GB1 (tGB1) loops were replaced by GPCR extracellular loops of CCR3, which were well tolerated (11)
The tGB1 loops were likewise replaced by RXFP1 EL1 and EL2 sequences (Fig. 1)
Summary
Extracellular loops of the transmembrane domain of the relaxin receptor RXFP1 are predicted to interact with relaxin. Molecular characterization of relaxin and LDLa interactions at the ELs of RXFP1 has been limited because of the inherent difficulties in studying membrane-associated GPCRs. more in depth analysis of the interactions involving the ELs of these receptors is limited by the presence of the high affinity ligand binding site within the LRRs, which masks the weak interactions at the ELs. A novel approach to investigate the interactions occurring at the ELs of the chemokine receptor utilized a protein engineering strategy to develop a small, soluble scaffold protein that had the ability to display the ELs of interest (11). The recently described RXFP1 agonist ML290 (13) binds RXFP1 at an allosteric site within EL3 of the TM domain and was used as a tool in this study to confirm the functional integrity of the mutated TM domain enabling the discrimination between residues that perturbs the structure of the receptor rather than contributing to ligand binding These findings will help to shed light on the complex molecular mechanism of the activation of RXFP1, which is emerging as an important clinical target
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