Application of the novel bioluminescent ligand-receptor binding assay to relaxin-RXFP1 system for interaction studies

Amino Acids
Qing-Ping WuZhan-Yun Guo

Abstract

Relaxin is a prototype of the relaxin family peptide hormones and plays important biological functions by binding and activating the G protein-coupled receptor RXFP1. To study their interactions, in the present work, we applied the newly developed bioluminescent ligand-receptor binding assay to the relaxin-RXFP1 system. First, a fully active easily labeled relaxin, in which three Lys residues of human relaxin-2 were replaced by Arg, was prepared through overexpression of a single-chain precursor in Pichia pastoris and in vitro enzymatic maturation. Thereafter, the B-chain N-terminus of the easily labeled relaxin was chemically cross-linked with a C-terminal cysteine residue of an engineered NanoLuc through a disulfide linkage. Receptor-binding assays demonstrated that the NanoLuc-conjugated relaxin retained high binding affinity with the receptor RXFP1 (K d = 1.11 ± 0.08 nM, n = 3) and was able to sensitively monitor binding of a variety of ligands with RXFP1. Using the novel bioluminescent binding assay, we demonstrated that three highly conserved B-chain Arg residues of relaxin-3 had distinct contributions to binding of the receptor RXFP1. In summary, our present work provides a novel bioluminescent ligand-receptor binding as...Continue Reading

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Citations

Jun 30, 2016·Archives of Biochemistry and Biophysics·Yu LiuZhan-Yun Guo
Jan 23, 2019·The FEBS Journal·Jia-Hui WangZhan-Yun Guo
Apr 6, 2016·Bioconjugate Chemistry·Christopher G EnglandWeibo Cai

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