Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation
Relaxin family peptides perform a variety of biological functions by binding and activating relaxin family peptide receptor 1–4 (RXFP1–4), four A-class G protein-coupled receptors. In the present work, we developed a novel ligand binding assay for RXFP3 and RXFP4 based on NanoLuc complementation technology (NanoBiT). A synthetic ligation version of the low-affinity small complementation tag (SmBiT) was efficiently ligated to the A-chain N terminus of recombinant chimeric agonist R3/I5 using recombinant circular sortase A. After the ligation product R3/I5-SmBiT was mixed with human RXFP3 or RXFP4 genetically fused with a secretory large NanoLuc fragment (sLgBiT) at the N terminus, NanoLuc complementation was induced by high-affinity ligand–receptor binding. Binding kinetics and affinities of R3/I5-SmBiT with sLgBiT-fused RXFP3 and RXFP4 were conveniently measured according to the complementation-induced bioluminescence. Using R3/I5-SmBiT and the sLgBiT-fused receptor as a complementation pair, binding potencies of various ligands with RXFP3 and RXFP4 were quantitatively measured without the cumbersome washing step. The novel NanoBiT-based ligand binding assay is convenient for use and suitable for automation, thus will facilitate interaction studies of RXFP3 and RXFP4 with ligands in future. This assay can also be applied to some other plasma membrane receptors for pharmacological characterization of ligands in future studies.
KeywordsRelaxin family Receptor Binding assay NanoLuc Complementation
This work was supported by grants from the National Natural Science Foundation of China (31670773, 31470767).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
- Dabo S, Maillard P, Collados Rodriguez M, Hansen MD, Mazouz S, Bigot DJ, Tible M, Janvier G, Helynck O, Cassonnet P, Jacob Y, Bellalou J, Gatignol A, Patel RC, Hugon J, Munier-Lehmann H, Meurs EF (2017) Inhibition of the inflammatory response to stress by targeting interaction between PKR and its cellular activator PACT. Sci Rep 7:16129CrossRefPubMedPubMedCentralGoogle Scholar
- Dixon AS, Schwinn MK, Hall MP, Zimmerman K, Otto P, Lubben TH, Butler BL, Binkowski BF, Machleidt T, Kirkland TA, Wood MG, Eggers CT, Encell LP, Wood KV (2016) NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells. ACS Chem Biol 11:400–408CrossRefPubMedGoogle Scholar
- Dupuis N, Laschet C, Franssen D, Szpakowska M, Gilissen J, Geubelle P, Soni A, Parent AS, Pirotte B, Chevigné A, Twizere JC, Hanson J (2017) Activation of the orphan G protein-coupled receptor GPR27 by surrogate ligands promotes β-arrestin 2 recruitment. Mol Pharmacol 91:595–608CrossRefPubMedGoogle Scholar
- Halls ML, Bathgate RA, Sutton SW, Dschietzig TB, Summers RJ (2015) International Union of Basic and Clinical Pharmacology. XCV. Recent advances in the understanding of the pharmacology and biological roles of relaxin family peptide receptors 1–4, the receptors for relaxin family peptides. Pharmacol Rev 67:389–440CrossRefPubMedPubMedCentralGoogle Scholar