Journal article

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 AD Bathgate

ISCIENCE | CELL PRESS | Published : 2019

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 m..

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Grants

Awarded by National Health and Medical Research Council of Australia


Awarded by Japan Society for the Promotion of Science (JSPS) KAKENHI


Awarded by Japan Agency for Medical Research and Development (AMED)


Funding Acknowledgements

This research was supported by National Health and Medical Research Council of Australia project grants 1100676 and 1043750 (R.A.D.B., P.R.G., and D.J.S.) and the Victorian Government Operational Infrastructure Support Program. A.I. was funded by Japan Agency for Medical Research and Development (AMED) grant number JP17gm5910013 and Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 17K08264. R.A.D.B. is supported by an NHMRC Research Fellowship, and D.J.S. is supported by an NHMRC Dementia Fellowship. The authors thank Tania Ferraro and Sharon Layfield for technical assistance.