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

doi:10.1016/j.isci.2018.12.004

Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis

iScience. 2019 Jan 25:11:93-113. doi: 10.1016/j.isci.2018.12.004. Epub 2018 Dec 10.

Authors

Bradley L Hoare¹, Shoni Bruell², Ashish Sethi³, Paul R Gooley³, Michael J Lew⁴, Mohammed A Hossain⁵, Asuka Inoue⁶, Daniel J Scott⁷, Ross A D Bathgate⁸

Affiliations

¹ Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, VIC 3052, Australia.
² 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.
³ 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.
⁴ Department of Pharmacology and Therapeutics, The University of Melbourne, Parkville, VIC 3052, Australia.
⁵ 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.
⁶ Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
⁷ 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. Electronic address: daniel.scott@florey.edu.au.
⁸ 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. Electronic address: bathgate@florey.edu.au.

Abstract

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.

Keywords: Biochemical Assay; Biological Sciences; Molecular Biology.