The Chemerin Receptor CMKLR1 Requires Full‐Length Chemerin for High Affinity in Contrast to GPR1 as Demonstrated by a New Nanoluciferase‐Based Binding Assay

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. The adipokine chemerin and the short peptide agonist chemerin‐9 activate two GPCRs: CMKLR1 and GPR1. Here, we established a nanoluciferase based binding assay to analyze differences in ligand binding. For GPR1, both ligands show high affinity and comparable binding. At CMKLR1, full‐length chemerin is required for stable binding, indicating a second interaction side for binding of the larger protein.