Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis
Bradley L. Hoare,
Shoni Bruell,
Ashish Sethi,
Paul R. Gooley,
Michael J. Lew,
Mohammed A. Hossain,
Asuka Inoue,
Daniel J. Scott,
Ross A.D. Bathgate
Affiliations
Bradley L. Hoare
Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia
Shoni Bruell
Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia; Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia
Ashish Sethi
Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia; Bio21 Molecular and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia
Paul R. Gooley
Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia; Bio21 Molecular and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia
Michael J. Lew
Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, VIC 3052, Australia
Mohammed A. Hossain
Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia; Department of Chemistry, The University of Melbourne, Parkville, VIC 3052, Australia
Asuka Inoue
Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan
Daniel J. Scott
Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia; Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia; Corresponding author
Ross A.D. Bathgate
Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia; Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, VIC, Australia; Corresponding author
Summary: The peptide hormone H2 relaxin has demonstrated promise as a therapeutic, but mimetic development has been hindered by the poorly understood relaxin receptor RXFP1 activation mechanism. H2 relaxin is hypothesized to bind to two distinct ECD sites, which reorientates the N-terminal LDLa module to activate the transmembrane domain. Here we provide evidence for this model in live cells by measuring bioluminescence resonance energy transfer (BRET) between nanoluciferase-tagged RXFP1 constructs and fluorescently labeled H2 relaxin (NanoBRET). Additionally, we validate these results using the related RXFP2 receptor and chimeras with an inserted RXFP1-binding domain utilizing NanoBRET and nuclear magnetic resonance studies on recombinant proteins. We therefore provide evidence for the multi-component molecular mechanism of H2 relaxin binding to RXFP1 on the full-length receptor in cells. Also, we show the utility of NanoBRET real-time binding kinetics to reveal subtle binding complexities, which may be overlooked in traditional equilibrium binding assays. : Biochemical Assay; Biological Sciences; Molecular Biology Subject Areas: Biochemical Assay, Biological Sciences, Molecular Biology
