Across multiple organ systems, relaxin family peptide receptor 1 (RXFP1) is activated by relaxin-2, a peptide hormone that regulates cardiovascular and anti-fibrotic functions. These biological roles position RXFP1 as a compelling therapeutic target in related pathologies and in relaxin-mediated oncogenesis. However, therapeutic development remains obstructed by an incomplete understanding of the activation mechanism, which stems from an inability to resolve the large, conformationally dynamic extracellular (ecto-) domain at high resolution. As this region mediates ligand binding and receptor activation, structural characterisation would contribute meaningfully to a fuller view of RXFP1 activation.
In other GPCRs, extracellular-targeted nanobodies (single-domain heavy-chain only antibodies derived primarily from camelids) have proven effective in both assisting structural imaging and enabling functional modulation. Here, nanobodies targeting RXFP1 were generated by immunising alpacas with purified RXFP1 protein. To isolate high-affinity RXFP1 binding nanobodies, nanobody sequences from two immunised alpacas were cloned into a phage display library for magnetic-based selection against a truncated RXFP1 ectodomain protein containing only the N-terminal low-density lipoprotein A (LDLa) module and linker. This method yielded a single, highly conserved group of nanobodies with low-nanomolar affinity for RXFP1, as characterised by direct protein-protein interaction and cell-based binding assays. Importantly, the lead nanobody demonstrated functional antagonism against relaxin-induced cAMP signalling, marking the identification of the first true antagonist for RXFP1. This antagonist nanobody holds significant promise as a therapeutic candidate for targeting or localising RXFP1-implicated cancers. Interestingly, the nanobody was observed to compete with ligand binding in the presence of the TMD, but not when the TMD was absent, suggesting an unexpected dependence on the TMD for ligand binding. These findings highlight the potential for nanobodies to pharmacologically target RXFP1 and to advance our understanding of RXFP1 activation.