Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

• Published in: The Journal of biological chemistry 2014-12, Vol.289 (50), p.34938-34952
• Main Authors: Diepenhorst, Natalie A., Petrie, Emma J., Chen, Catherine Z., Wang, Amy, Hossain, Mohammed Akhter, Bathgate, Ross A.D., Gooley, Paul R.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Extracellular Space - metabolism; G Protein-coupled Receptor (GPCR); HEK293 Cells; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nuclear Magnetic Resonance (NMR); Peptide Hormone; Protein Binding; Protein Engineering; Protein Structure, Tertiary; Receptor Structure-Function; Receptors, Peptide - chemistry; Receptors, Peptide - genetics; Receptors, Peptide - metabolism; Relaxin; Relaxin - metabolism; RXFP1; Serelaxin; Signal Transduction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.600882

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. Background: Extracellular loops of the transmembrane domain of the relaxin receptor RXFP1 are predicted to interact with relaxin. Results: RXFP1 extracellular loops displayed on a scaffold protein enabled investigation of ligand interactions. Conclusion: RXFP1 activation involves interactions between the extracellular loops with relaxin and the receptor LDLa module. Significance: Understanding the molecular mechanisms of RXFP1 activation will aid drug design at this receptor.