The relaxin-2 receptor, RXFP1, is amongst the most complex G-protein coupled receptors (GPCRs) identified to date. It consists of a 7 transmembrane helix domain (7TM), common to all GPCRs, and a large ectodomain of leucine-rich repeats (LRRs), unstructured linkers, and an N-terminal low density lipoprotein class A (LDLa) module. RXFP1 is activated by a multi-step mechanism involving relaxin-2 binding to both the LRRs and exoloops in the 7TM. For most GPCRs, ligand binding to an exoloop is sufficient to activate signaling, but for RXFP1 a secondary event involving the LDLa module is required. This LDLa-dependent activation is not well understood and presents not only a unique paradigm in GPCR signaling, but the potential to be exploited to develop specific therapeutics. Relaxin has successfully completed Phase III clinical trials for the treatment of acute heart failure, but has poor bioavailability as it is not orally active and has a short half-life.
We have extensively characterized the surface of the RXFP1 LDLa module via mutagenesis. Using Nuclear Magnetic Resonance (NMR) and cell-based signaling assays, we have found certain residues that are essential for activity. Whilst this approach has been successful, it is limited by the sensitivity of the overall structure of the LDLa module to mutations. Concurrently, we have identified that the LDLa module interacts with mM affinity to an engineered protein presenting exoloops 1 and 2 of the 7TM. Therefore, in order to extend our findings, we will confirm this interaction and comprehensively map residues on the activation surface of the LDLa module using saturation transfer difference (STD) NMR experiments with membrane preparations of HEK293T cells expressing RXFP1 and labeled recombinant LDLa.