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

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.

Stable Binding of Full‐Length Chemerin Is Driven by Negative Charges in the CMKLR1 N Terminus

“…Here, C9 is sufficient for high affinity and full activation as confirmed by binding experiments using a bioluminescence resonance energy transfer (BRET) based assay set-up with nano-luciferase (Nluc) modified receptors. [21] Thus, in addition to the already known interaction of the C-terminal segment of ChemS157 with the transmembrane orthosteric binding pocket we hypothesize an additional interaction of ChemS157 at the CMKLR1.…”

“…Here, the activity of ChemS157 drops to values comparable to C9 at the CMKLR1 wt (227 nM and 1042 nM; Table 1). N-terminal receptor deletions have little impact on the interaction of C9 with a maximal 3-fold loss of receptor activation for the CMKLR1[ΔNterm [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] ] variant (Table 1).…”

“…All assays were performed at least three times in duplicate using transiently transfected COS-7 cells. [13][14][15][16][17] , YPDYL [18][19][20][21][22] and the full N terminus (E 4 À L 22 ), respectively. Aspartic acid and glutamic acid residues are highlighted in red.…”

“…To get detailed kinetic information about the negatively charged patches of the CMKLR1 N terminus, Nluc-based bioluminescence resonance energy transfer (NanoBRET) binding assays were performed as previously described. [21] First, the CMKLR1 alanine variants (CMKLR1[EDED 4-7 AAAA] and CMKLR1 [DED 16,17,20 AAA]) were N-terminally fused to Nluc (Figure 5A). All Nluc-labeled receptor constructs showed wt-like membrane localization (Figure S4).…”

“…[17a,20] The C-terminal nonapeptide chemerin-9 (C9) is able to display full activity at the CMKLR1 receptor in non-equilibrium Ca 2 + flux assays, [8a] whereas full-length ChemS157 is required for strong interactions as determined by equilibrium assays such as IP accumulation and competition binding studies. [21] Interestingly, this is different for GPR1. Here, C9 is sufficient for high affinity and full activation as confirmed by binding experiments using a bioluminescence resonance energy transfer (BRET) based assay set-up with nano-luciferase (Nluc) modified receptors.…”