Abstract
To study the binding mode of the adipokine chemerin as well as the short peptide agonist chemerin-9 (C9) to its two receptors chemokine-like receptor 1 (CMKLR1) and G protein-coupled receptor 1 (GPR1), we generated 5-carboxytetramethylrhodamine (TAMRA) modified variants of both ligands. In addition, we labeled GPR1 and CMKLR1 with a nanoluciferase at the N-terminus to perform NanoBRET binding assays. For GPR1, both ligands show high affinity and comparable binding. Significant differences were found for CMKLR1, whereby only full-length chemerin binds with high affinity in saturation and displacement assays. For TAMRA-C9 a biphasic binding consisting of two binding states has been found and no displacement studies could be performed. Thus, we conclude that CMKLR1 requires full-length chemerin for stable binding in contrast to GPR1. This work demonstrates the NanoBRET binding assay as a new tool for binding studies at chemerin receptors and it enables deeper insights into the ligand binding parameters.