The relaxin receptor (RXFP1) utilises hydrophobic moieties on a signalling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor RXFP1, little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation and it has been hypothesised that this module is the true ligand of the receptor which directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized GPCR signalling pathway. Furthermore, we have comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signalling. Loss-of-function mutations of residues in these regions demonstrated that Leu7, Tyr9 and Lys17 all contributed to the ability of the LDLa module to drive receptor activation and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues are contributing to interactions driving the active receptor conformation, providing further evidence of a unique mode of GPCR activation.