Multi-Component Mechanism of H2 Relaxin Binding to RXFP1 through NanoBRET Kinetic Analysis
Web of Science
AuthorHoare, BL; Bruell, S; Sethi, A; Gooley, PR; Lew, MJ; Hossain, MA; Inoue, A; Scott, DJ; Bathgate, RAD
University of Melbourne Author/sSethi, Ashish; Bathgate, Ross; Scott, Daniel; Hossain, Mohammed; Lew, Michael; Gooley, Paul; Bruell, Shoni; Bruell, Shoni
AffiliationFlorey Department of Neuroscience and Mental Health
Biochemistry and Molecular Biology
Pharmacology and Therapeutics
Document TypeJournal Article
CitationsHoare, 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 StatusOpen Access
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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