title: Investigation of interactions at the extracellular loops of the relaxin family peptide receptor 1 (RXFP1).
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.
authors.affiliation: From the Florey Institute of Neuroscience and Mental Health, the Department of Biochemistry and Molecular Biology, and.
authors.forename: Natalie A
authors.initials: NA
authors.lastname: Diepenhorst
authors.affiliation: the Department of Biochemistry and Molecular Biology, and.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Petrie
authors.affiliation: the National Center for Advancing Translational Sciences, Division of Preclinical Innovation, National Institutes of Health, Rockville, Maryland 20850.
authors.forename: Catherine Z
authors.initials: CZ
authors.lastname: Chen
authors.affiliation: the National Center for Advancing Translational Sciences, Division of Preclinical Innovation, National Institutes of Health, Rockville, Maryland 20850.
authors.forename: Amy
authors.initials: A
authors.lastname: Wang
authors.affiliation: From the Florey Institute of Neuroscience and Mental Health, the School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia and.
authors.forename: Mohammed Akhter
authors.initials: MA
authors.lastname: Hossain
authors.affiliation: From the Florey Institute of Neuroscience and Mental Health, the Department of Biochemistry and Molecular Biology, and bathgate@florey.edu.au.
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
authors.affiliation: the Department of Biochemistry and Molecular Biology, and prg@unimelb.edu.au.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Gooley
mesh_terms: D000595:Amino Acid Sequence; D005110:Extracellular Space / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012065:Relaxin / Q000378:metabolism
chemical_list: D018000:Receptors, Peptide; D012065:Relaxin
keywords: G Protein-coupled Receptor (GPCR); Nuclear Magnetic Resonance (NMR); Peptide Hormone; Protein Engineering; RXFP1; Receptor Structure-Function; Relaxin; Serelaxin