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

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Author
Hoare, BL; Bruell, S; Sethi, A; Gooley, PR; Lew, MJ; Hossain, MA; Inoue, A; Scott, DJ; Bathgate, RADDate
2019-01-25Source Title
iSciencePublisher
CELL PRESSUniversity of Melbourne Author/s
Sethi, Ashish; Bathgate, Ross; Scott, Daniel; Hossain, Mohammed; Lew, Michael; Gooley, Paul; Bruell, Shoni; Bruell, ShoniAffiliation
Florey Department of Neuroscience and Mental HealthBiochemistry and Molecular Biology
Pharmacology and Therapeutics
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Journal ArticleCitations
Hoare, B. L., Bruell, S., Sethi, A., Gooley, P. R., Lew, M. J., Hossain, M. A., Inoue, A., Scott, D. J. & Bathgate, R. A. D. (2019). Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis. ISCIENCE, 11, pp.93-+. https://doi.org/10.1016/j.isci.2018.12.004.Access Status
Open AccessAbstract
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.
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