Ligand-Induced and -Independent Internalization of the Chemerin Receptor GPR1

1. Tight regulation of cytokines is essential for the initiation and resolution of inammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the rst GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization and recycling, which allows GPR1 to internalize inactive peptides eciently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: As a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specicity on the other. based on arrestin recruitment as a readout were not in agreement with results obtained in uorescence microscopy, we further investigated the internalization of GPR1 and its ligands, revealing an activation-independent pathway of constitutive internalizion. at least two independent experiments performed in quadruplicates.


Introduction
G protein-coupled receptor 1 (GPR1) is a G protein-coupled receptor (GPCR) that was rst identi ed as an orphan receptor in the human hippocampus in 1994 [1]. It was not until 14 years later that Barnea et al. reported chemerin as a natural ligand for GPR1 in 2008 [2]. Chemerin is a small protein that is expressed in adipose tissue, liver, and skin [3,4]. After cleavage of an N-terminal signal peptide, it is secreted as 143 amino acid prochemerin, [5] followed by activation through C-terminal processing by proteases of the coagulation and in ammatory cascades [6,7]. The resulting chemerin biologically active isoforms are named according to their last C-terminal amino acid, ChemS157 (consisting of 137 residues) and ChemF156 (consisting of 136 residues) [8]. Chemerin is predicted to form a cystatin-like fold, thus sharing structural homology with human cathelidin [9,10]. Levels of chemerin strongly correlate with the body mass index (BMI) and further obesity-associated parameters like fasting serum insulin or hypertension [11,12]. Moreover, chemerin is linked to many co-morbidities of obesity like metabolic syndrome, psoriasis, or diabetes [13]. The role of chemerin in auto-in ammatory diseases has gained increasing interest: After it was initially isolated from the synovial joint uid from patients suffering from rheumatoid arthritis, there is emerging evidence that chemerin is a driver of in ammation in the joints of these individuals [5,14]. Chemerin levels are also elevated in early psoriatic skin lesions where it correlates with in ltration by dendritic cells [15]. More recently, several studies have demonstrated that chemerin plays essential roles in different cancer types: Either indirectly by promoting angiogenesis in, e.g., colorectal cancer, [16] or directly by stimulating the invasion of oesophageal squamous cancer cells [17].
Chemerin binds to three GPCRs with different a nities: GPR1, chemokine-like receptor 1 (CMKLR1), and C-C motif chemokine receptor-like 2 (CCRL2). While the highest a nity was found for GPR1, [18] most known functions are mediated by CMKLR1, e.g., the chemotaxis of leukocytes towards sites of in ammation, the differentiation of adipocytes, and vasoconstrictive effects of chemerin [5,19,20]. In contrast, the biological role of GPR1 is understudied. GPR1 knock-out mice displayed exacerbated glucose intolerance after being fed with a high-fat diet [21]. Other studies suggest a regulatory role of GPR1 in follicle development and hormone secretion [22]. No GPR1-selective probe molecules are known so far, which impedes the characterization of this receptor [23]. GPR1 shares high sequence identity (35.1 %) with CMKLR1, its closest homolog, which is expressed by adipocytes and cells of the innate immune system [5,24,4]. However, while CMKLR1 acts as a classical GPCR with functional homology to the chemokine receptors, no ligand-induced G protein activation has been described for GPR1. [18] The functional response induced by GPR1 upon stimulation with chemerin is limited to arrestin-recruitment and RhoA/ROCKmediated signaling [25,2]. For the third chemerin receptor, CCRL2, no detectable signaling events have been observed [26,18].
The identi cation and characterization of atypical chemokine receptors was a signi cant step in the characterizion of the chemokine system. The chemerin system displays some parallels to the chemokines, and insights into the mechanisms that control and mediate chemerin activity will be essential to understand the chemerin system. We, therefore, chose to analyze the function of GPR1. The determination of the ligand-binding mode of chemerin at GPR1 enabled us to develop the rst selective ligand for this receptor. Moreover, we demonstrate that GPR1 can scavenge and internalize peptides that fail to induce receptor activation.

Results
We devised an experimental/computational protocol outlined in Fig. 1 to study the structure-function relation of chemerin with its receptor GPR1. First, we determined the minimal portion of the chemerin protein needed for full arrestin recruitment, our functional readout, in order to identify the portion of chemerin that engages the receptor. In parallel, we constructed a comparative model of GPR1 to identify candidate residues in the extracellular loops and the upper portions of the transmembrane (TM) helices that can form the putative binding site for chemerin; this was done bearing in mind the previously identi ed binding pocket of chemerin at the CMKLR1 [27]. Consequently, we determined an interaction point by complementary mutagenesis, and probed the internal conformation of the minimal activation sequence employing cyclized peptides. Using our experimental data as restraints, we docked the minimal peptide into the GPR1 binding pocket. As our results from the studies of the binding mode based on arrestin recruitment as a readout were not in agreement with results obtained in uorescence microscopy, we further investigated the internalization of GPR1 and its ligands, revealing an activation-independent pathway of constitutive internalizion.

3.1
A C-terminal Chemerin Peptide is Su cient to Induce Rapid Recruitment of Arrestin to GPR1 Stimulation with ChemS157 led to rapid recruitment of arrestin3 to the receptor, reaching its maximum within two minutes as monitored by a bioluminescence resonance energy transfer (BRET) assay (Fig. 2a). Fluorescence microscopy showed that GPR1 recruits mCherry-labeled arrestin3 to the membrane upon stimulation with 1 µM ChemS157. However, GPR1 internalizes without arrestin3, leaving Arr3-mCherry at the membrane (Fig. 2b).
To further characterize the activation of GPR1 by chemerin, we tested the effect of truncations of the ligand on arrestin3-recruitment in a BRET assay.
Recombinantly expressed ChemS157 displayed a low nanomolar activity (EC 50 = 2.1 nM). The same activity was observed for recombinantly expressed ChemF156 (EC 50 = 2.6 nM). Next, we synthesized two peptides derived from the C-termini of ChemS157 and ChemF156, Chem 139-157 and Chem 139-156 . These peptides displayed the same activities as the full-length proteins with EC 50 values of 3.1 nM and 2.9 nM, respectively. Further truncating the peptide to yield Chem 149-157 (chemerin-9) and Chem 149-156 had no negative impact on activity. However, further N-terminal truncations resulted in a loss of activity: Chem 150-157 was tenfold less active than the full-length protein, while Chem 151-157 was completely devoid of activity at concentrations up to 1 µM. Similarly, removal of the C-terminal Phe 156 resulted in a complete loss of activity; Chem 149-155 (chemerin-7) did not reach full receptor activation at concentrations of up to 1 µM.
Two scrambled chemerin-9 peptides (scrC9 and scr2C9) failed to induce arrestin3-recruitment at concentrations of up to 10 µM. Table 1 displays an overview of all EC 50 values. For an improved understanding of the function of GPR1, knowledge of the peptide binding mode is essential. To gain insight into the three-dimensional structure of the receptor, we constructed homology models of GPR1 employing RosettaCM. We chose crystal structures of ve related receptors as templates for homology modeling: Complement 5 a receptor 1 (PDB: 6c1r), type 1 angiotensin II receptor (AT1R, PDB: 4zud), apelin receptor (APJ, PDB: 5vbl), CXC chemokine receptor 4 (CXCR4, PDB: 3odu) and CC chemokine receptor 9 (CCR9, PDB: 5lwe), which display sequence identities of 27%-35% to GPR1. In a previous benchmark of our method, these premises proved suitable for constructing highly accurate homology models from multiple templates [28]. Details on the construction of homology models are given in the methods section and SI. As the receptor N-and C-termini are expected to be highly exible, they were truncated to limit the conformational sampling space. A total of 1500 models were produced and clustered by Cα RMSD (Fig. S1), the binding pocket of the best scoring model is displayed in Fig. 3b. We selected conserved residues in the extracellular loops of GPR1 that pointed to the putative binding pocket for investigation in a nanoBRET-based ligand binding assay: Residues Y 2.63 , F 2.68 , Y 4.76 , and F 4.79 (numbering of receptor residues follows Ballesteros and Weinstein [29]) are highly conserved across species and potentially available for interaction with the aromatic residues in the peptide. Residue E 6.58 is positioned as in CMKLR1, where it is critical for binding to chemerin-9, suggesting a similar role in GPR1 [27].

3.3
Residues in the Extracellular Loops of GPR1 Interact with Chemerin-9 Mutations were introduced into a GPR1 construct N-terminally fused to NanoLuc ® . The receptor was stimulated with chemerin-9 N-terminally connected to a 6carboxy tetramethylrhodamine (Tam) uorophore by an ethylene glycol linker (Tam-EG(4)-chemerin-9). Stimulating Nluc-GPR1 WT with Tam-EG(4)-chemerin-9 yielded a nanomolar a nity (EC 50 = 6 nM). Exchanging residues Y 2.63 and F 2.68 in TM2 and ECL1 to alanine led to a pronounced loss of a nity with EC 50 values of 114 nM and 460 nM, respectively. Two residues in the ECL2, Y 4.76 , and F 4.79 , also displayed a dramatic loss of a nity when exchanged to alanine (EC 50 = 224 nM and EC 50 >1000 nM, respectively). E 6.58 was the only non-aromatic residue we found to be essential for peptide binding; the E 6.58 A mutant displayed an EC 50 > 1000 nM.

A Hydrophobic Pocket Formed by the ECL2 of GPR1 Contributes to Ligand Binding
Chemerin-9 residue F 8 binds to a hydrophobic pocket in the ECL2 of CMKLR1 [27]. The ligand-binding residues are highly conserved between CMKLR1 and GPR1. We, therefore, investigated whether an interaction between chemerin-9 residue F 8 and the ECL2 of GPR1 is involved in ligand binding as well.
Introducing the F 8 L mutation in the ligand ([L 8 ]-chemerin-9) resulted in a very slight increase of a nity at the wild-type receptor (EC 50

Chemerin-9 Activates GPR1 in a Loop-Conformation
We suspected that chemerin-9 adopts a turn-conformation for activating GPR1, which should allow for cyclization of the ligand while retaining activity. To test this hypothesis, we synthesized a highly constrained chemerin-9 derivate, connecting N-and C-terminus by a lactam bond ([N-C]-c(chemerin-9). This peptide displayed a signi cantly reduced potency but was still able to activate GPR1 (EC 50 = 132 nM). To improve the potency at the receptor for potential therapeutic applications, we synthesized a second cyclic peptide with increased exibility by exchanging positions 4 and 9 for D-homocysteine and cysteine. Oxidizing the peptide promoted the cyclization, forming a disul de between positions 4 and 9 ([4-9]-c(chemerin-9). This peptide showed high activity at GPR1 and was as active as the linear peptide (EC 50 = 3.3 nM), as displayed in Fig. 6c.

Restrained Docking Simulations Uncover the Binding Mode of Chemerin-9 at GPR1
With this data on the GPR1 ligand-binding mode at hand, we chose to construct a model of this interaction. Chemerin-9 was docked to the ten best scoring receptor models from each of the three largest clusters (i.e. a total of 30 models) using Rosetta FlexPepDock with our experimental data as restraints: All identi ed residues of the binding pocket ( Fig. 3a) were required to interact with the ligand, residues V 4.67 and F 4.69 were restrained to interact with chemerin-9 residue F 8 ( Fig. 4). Additionally, a loop conformation was enforced by a distance restraint between the ligand termini. The resulting models were clustered based on Cα RMSD, and the best scoring models from cluster 2 ( Fig. S2, selected based on their agreement with the experimental data) were subjected to Rosetta FastRelax without restraints. Finally, the 20 best scoring models by interface score ΔG separated were selected for analysis ( Fig. 6b). In these models, chemerin-9 residue F 8 interacts with a hydrophobic domain in the ECL2 consisting of F 4.69 , L 4.74 , Y 4.76, and F 4.79 . Residue F 6 displays a very pronounced interaction with F 2.68 and, to a lesser extent, with Y 2.63 . Residue S 9 is barely involved in binding to the receptor. The N-terminal residues Y 1 and F 2 interact with a wide range of residues in the receptor and highly contribute to the predicted binding energy. Chemerin-9 G 4 and Q 5 extend towards the bottom of the binding pocket and interact with T 7.39 . On the receptor side, I 7.35 interacts with a range of ligand residues and displays the highest energy contribution of all receptor residues. Taken together, the interface between chemerin-9 and GPR1 is dominated by hydrophobic interactions. The predicted binding energies of different receptor residues show a high correlation with the logarithmic EC 50 shifts of their respective alanine mutants (r 2 = 0.8), demonstrating that the models are in good agreement with the available experimental data (Fig. 6d).

GPR1 Undergoes Constitutive Internalization
In order to characterize the function of GPR1 in the cell, HEK293 cells stably expressing GPR1-eYFP were treated with 1 µM of Tam-labeled peptides, and intracellular uorescence was measured at distinct time points using an ImageExpress high content imaging system (Fig. 7). Stimulation with Tam-EG(4)chemerin-9 led to a rapid, exponential accumulation of uorescence in the cell within 15 min. This rapid accumulation was followed by a linear increase starting at around 20 min, which continues until the end of the experiment at 120 min. The truncated Tam-EG(4)-chemerin-7 peptide does not evoke any intracellular increase of uorescence over time. In contrast, both scrambled peptides displayed substantial internalization. In the rst 20 min, the growth of intracellular uorescence for Tam-EG(4)-scrC9 is delayed compared to Tam-EG(4)-chemerin-9, but the linear increase between 20 min and 120 min displays a comparable slope for both peptides (Fig. 7). A second scrambled peptide, Tam-EG(4)-scr2C9, did not display rapid intracellular accumulation in the rst 15 minutes but showed a similar, linear increase of intracellular uorescence as Tam-EG(4)-chemerin-9 and Tam-EG(4)-scrC9 (Fig. 7b). In contrast, neither of the scrambled peptides was internalized in CMKLR1-expressing HEK293 cells (Fig. S3). Comparing the slopes of the linear regression lines of all peptides revealed a signi cant deviation from zero for Tam-(EG4)-chemerin-9, Tam-EG(4)-scrC9, and Tam-EG(4)-scr2C9, but not for Tam-EG(4)-chemerin-7 (p<0.05). Moreover, the slope of the linear t for Tam-EG(4)-chemerin-7 was signi cantly lower than for all other peptides.
The scrambled scrC9 peptide is not able to induce arrestin-recruitment to GPR1, but still accumulates in cells expressing GPR1. A fraction of GPR1 always resides in intracellular vesicles (Fig. 2d), which prompted us to investigate whether they co-localized with endosomal markers. We co-transfected GPR1-eYFP and rab4-CFP or rab11-CFP in HEK293 cells and examined this by live-cell uorescence microscopy. Indeed, rab4-CFP forms distinct intracellular vesicles that co-localize with GPR1-eYFP (Fig. 8a). A one-dimensional intensity scan of CFP and eYFP uorescence in imageJ con rmed that the intracellular GPR1-eYFP was predominantly found in rab4-CFP vesicles (Fig. 8b). In contrast, no apparent co-localization of GPR1-eYFP with rab11-CFP occurs, and no distinct vesicles containing rab11-CFP are formed (Fig. 8c, d).
We hypothesized that GPR1 undergoes constitutive internalization and recycling, independent of the presence of ligand. To test this hypothesis, we selectively labeled cell surface receptors by peptide-templated transfer of a uorescent dye [30]. We genetically introduced an N-terminal Cys-E3 tag into GPR1, which speci cally interacts with a synthetic peptide probe (termed K3) by coiled-coil interactions. This high-a nity interaction brings the free N-terminal cysteine of the Cys-E3 tag in proximity to a Tam uorophore, which is bound to the K3 peptide probe by a thioester. A transthioesteri cation followed by an S-N acyl shift transfer the Tam dye to the N-terminus, forming a stable amide bond between receptor N-terminus and dye [31]. This approach allows to discriminate receptors embedded in the cell membrane from intracellular subpopulations at the time of reaction. Comparing eYFP uorescence revealed that unlabeled, Cys-E3-tagged GPR1-eYFP resides in intracellular vesicles and the cell membrane, similar to untagged GPR1-eYFP (Fig. 9a). Performing the peptide-templated labeling reaction on ice to prevent internalization visualizes only receptors in the membrane (Fig. 9b, lower panel). When the labeling reaction was performed at 37°C, signi cant amounts of labeled receptor had already internalized 15 min after starting the reaction (Fig. 9c). We quanti ed the relative distribution of Tam uorescence in the membrane and intracellular vesicles over time (Fig. 9d): After 15 min, 65 ± 18% (mean ± SD) of Tam uorescence is still in the membrane, and after 30 min, 60 ± 14% are left. The distribution of labeled GPR1 reaches an equilibrium at around 60 min when 45 ± 16% of Tam uorescence is in the membrane. This distribution of Tam uorescence after 60 min matches the distribution of eYFP uorescence for untagged GPR1-eYFP (50 ± 14%).

Discussion
Chemerin is a signaling protein involved in several physiological processes, including adipogenesis, host defense, and reproductive functions [13]. An imbalance in the regulation of these processes induces severe effects, and consequently, chemerin itself underlies tight control. This control is most striking on the level of proteolytic activation and inactivation, but also on the receptor level. Three chemerin receptors GPR1, CMKLR1, and CCRL2 display distinct expression patterns and pharmacology, indicating a complex interplay between these receptors and their shared ligand.
GPR1 lacks functional coupling to G proteins but rapidly recruits arrestin3 in response to stimulation with chemerin. The C-terminus of chemerin is responsible for activating the receptor, as described for CMKLR1. Our BRET ratios of GPR1 correspond to the values obtained by de Henau et al. in a similar set-up [18]. Interestingly, GPR1 recruits arrestin3 to the membrane upon stimulation with ligand, but this interaction seems to be transient: While arrestin3 stays at the membrane, GPR1 internalizes into intracellular vesicles. Receptors that dissociate from arrestin at the membrane before internalization are classi ed as class A, which, in general, rapidly recycle back to the membrane after activation [32,33]. Next, we examined the ability of truncated chemerin proteins and peptides to activate GPR1. ChemS157 and ChemF156 display the same activity, indicating that these two proteins have a same activity pro le for CMKLR1 and GPR1.
Peptides derived from the respective C-termini of ChemS157 and ChemF156 show no decreased activity, chemerin-9 (chemerin 149-157 ) displays the same potency as the corresponding full-length protein ( Table 1). The minimal activation sequence starts at Y 149 , removal of this residue and the following F 150 leads to a signi cant loss of activity, similar to results obtained at CMKLR [34]. In line with previous results, this demonstrates that the C-terminus of chemerin is the dominant part of the protein that binds to GPR1 [2,18]. A peptide lacking the C-terminal S 157 still showed no loss of activity, but further truncation of the sequence by removing F 156 almost completely abolished activity. Chemerin 149-155 (chemerin-7) induced only marginal arrestin-recruitment starting at 1 µM. The same residue is critical for the activation of CMKLR1 [34]. Hence, chemerin species lacking the last phenylalanine display no activity at either GPR1 or CMKLR1. Total levels of chemerin increase with the body-mass index, and the ratio of the different chemerin forms (prochemerin, ChemS157, ChemF156, and ChemA155) differ between lean and obese subjects [35,36]. Interestingly, a previous study performed in mice demonstrated a higher bioactivity ratio (active chemerin/total chemerin) in mice fed a high-fat diet when determined by GPR1 activation, but not measured by CMKLR1 activation [37].
The chemerin C-terminus is responsible for the activation of GPR1 and CMKLR1. Based on the high sequence homology of these two receptors, we hypothesized that they might share a common binding mode. Based on our previous results on the binding mode of chemerin-9 at CMKLR1, we selected several residues in the ECLs of GPR1 for investigation. We exchanged Y 2.63 , F 2.68 , F 4.76 , F 4.79, and E 6.58 for alanine and examined the in uence of these mutations in a nanoBRET-based binding assay (Fig. 3). As expected, all mutations had a signi cant impact on ligand binding, con rming that chemerin-9 occupies approximately the same sites in GPR1 and CMKLR1. An essential interaction in the binding mode of chemerin-9 at CMKLR1 is between the ultimate phenylalanine in the ligand and a hydrophobic pocket formed by the ECL2 of the receptor. To investigate whether this interaction occurs in GPR1 as well, we performed 2D mutagenesis experiments. In CMKLR1, positions 4.67 and 4.69 consist of alanine and leucine, respectively. Exchanging these for a valine and a phenylalanine decreased the EC 50 shift between chemerin-9 and [L 8 ]-chemerin-9 by tightening the hydrophobic pocket formed by ECL2 [27]. Now, we reversed this experiment by introducing the V 4.67 A_F 4.69 L mutants into GPR1. Indeed, while [L 8 ]-chemerin-9 is as active as chemerin-9 at the wild type GPR1, a small shift at the V 4.6 7A_F 4.69 L mutant (Fig. 4) can be found. Thus, the ECL2 represents the binding site for F 8 in both receptors. The fact that the ECL2 forms a tighter hydrophobic pocket in GPR1 than in CMKLR1 can be exploited for the rational design of selective ligands: As a proof of concept, [L 8 ]-chemerin-9 displays a high potency at GPR1 (EC 50 = 1.4 nM), but does not induce arrestin recruitment at CMKLR1 (Fig. 5). It is important to note, however, that [L 8 ]-chemerin-9 does induce G protein-signaling at CMKLR1, although with a 20-fold loss of activity compared to chemerin-9 [27]. Conversely, ligands with larger side chains in position 8 may be suitable for the design of selective ligands targeting CMKLR1.
We previously showed that cyclic chemerin-9 derivates activate CMKLR1 with high potency, and the same holds true for GPR1 (Fig. 6) [27]. This highlights that the binding mode of the chemerin C-terminus is highly related at both receptors. It also demonstrates that cyclization is a suitable approach to stabilize rationally designed ligands for GPR1.
Activation of GPR1 by chemerin-9 requires a speci c conformation of the ligand. Surprisingly, however, two Tam-labeled scrambled peptides that are both unable to activate GPR1 are still internalized by GPR1 (Fig. 7). This discrepancy between ligand-induced arrestin recruitment on the one hand, and e cient receptor-mediated uptake on the other, is a strong indication for an activation-independent internalization mechanism [38]. Additionally, the fact that the intracellular concentration of the wild-type and both scrambled peptides continues to increase during extended periods indicates that receptors recycle back to the membrane after internalization. This hypothesis is supported by our ndings that GPR1 co-localizes with rab4, even in the absence of ligand (Fig. 8), as rab4-positive vehicles are characterized by rapid recycling [39]. Ultimately, we used an approach to speci cally label cell surface receptors by a peptidetemplated acyl transfer [31]. This technique allowed to follow a speci c receptor population with high spatiotemporal resolution [30]. We demonstrate by this approach as well that receptors spontaneously internalize without ligand stimulation (Fig. 9). The fact that the ratio of internalized to membrane-bound receptors quickly reaches an equilibrium further supports the idea that internalized GPR1 rapidly recycles back to the membrane. These results demonstrate that any molecule that binds to GPR1 is scavenged and brought into the cell by the receptor, regardless of whether it induces GPR1 activation. The receptor then quickly recycles back to the membrane, making it perfectly adapted to decrease extracellular concentrations of chemerin. Importantly this constitutive internalization is not accompanied by constitutive activity [40].
The ligand-binding modes are highly conserved between CMKLR1 and GPR1. Therefore, any agonist or antagonist of CMKLR1 is likely to be bound and internalized by GPR1. There are few examples of cell types that express both GPR1 and CMKLR1: Macrophages generally express CMKLR1, but resident alveolar macrophages additionally display GPR1 mRNA expression [41]. Interestingly, chemerin is described to mediate anti-in ammatory functions in a murine lung model of lung disease [24]. Chemerin plays an essential role in adipose tissue, and CMKLR1 expression was previously demonstrated in adipocytes [19]. GPR1 expression was found in the stromal vascular fraction of adipose tissue, and hence adipose tissue macrophages may be another population of immune cells that have GPR1 and CMKLR1 at their disposal [42].
Our results that both scrambled chemerin-9 peptides are internalized by GPR1 to varying degrees may indicate that this receptor scavenges not only chemerin but also related peptides or proteins. Atypical chemokine receptors show many similarities to GPR1. They are essential for the regulation of chemokine activity and lack classical G protein signaling [43]. Meyrath et al. previously published a study demonstrating that the atypical chemokine receptor 3 (ACKR3, formerly CXCR7) not only binds chemokines, but also a broad range of opioid peptides [44]. Considering that GPR1 is expressed in the brain, a similar role for this receptor is possible [1]. Indeed, a previously published study states that the neuropeptide FAM19A1 may be an additional ligand for GPR1 [45].
The closest homologs of the chemerin receptors GPR1 and CMKLR1 are the receptors for the chemotactic protein complement 5 a, namely C5a receptor 1 (C5aR1) and C5a receptor 2 (C5aR2) [46]. They show similar characteristics, with C5aR1 being a classical, G i -coupled receptor that mediates chemotaxis of immune cells, and C5aR2 being a constitutively internalizing scavenging receptor that does not signal through G proteins [47]. In contrast to GPR1, however, C5aR2 does not co-localize with rab4 and seems to recycle at a slower rate [48]. GPR1, C5aR2, and the atypical chemokine receptors all display mutated DRY motifs in TM3. However, a study on the C5aR2 showed that mutation of this motif is not responsible for the lack of G protein coupling and a similar study on ACKR2 (formerly known as D6) found the same [49,50]. Not surprisingly, mutation of the altered DRY motif (DHY) in GPR1 did not restore G protein signaling [42]. The molecular basis for the lack of G protein signaling, therefore, remains unclear.
Taken together, our results demonstrate that the C-terminus of chemerin displays a common binding mode at the two receptors GPR1 and CMKLR1.
Differences in the ECL2 of these receptors have been exploited to design the rst GPR1-selective ligand and may be useful for the rational design of CMKLR1selective ligands as well. A cycle of ligand-independent internalization followed by rapid recycling makes GPR1 perfectly adapted as a scavenging receptor, and our results indicate that this role of GPR1 may not be restricted to chemerin.  . Bovine arrestin-3 was fused to Rluc8 and cloned into pcDNA3 vector for BRET studies. Primers for PCR were bought from Biomers (Ulm, Germany). Furimazine was purchased from Promega (Madison, WI, USA).

Peptide Synthesis
All peptides were synthesized by 9-uorenylmethoxycarbonyl/tert-butyl (Fmoc/ t Bu) solid-phase peptide synthesis strategy on a scale of 15 µmol on a Wang resin preloaded with the rst amino acid, or on a 2-chlorotrityl chloride resin. All reactions were performed at rt unless stated otherwise. All standard amino acids were coupled using a Syro II peptide synthesizer (MultiSynTech, Bochum, Germany). Coupling reactions were performed twice with 8 equiv of the respective, Fmoc-protected amino acid activated in situ with equimolar amounts of oxyma and DIC in DMF for 30 min. Fmoc-removal was achieved by reaction with 40 % (v/v) piperidine in DMF for 3 min and 20% (v/v) piperidine in DMF for 10 min, the resin was washed with DMF and DCM after every reaction. Fmoc-D-hCys(Trt)-OH and Fmoc-PEG(4)-OH were manually coupled to the peptide by reaction with 5 equiv and equimolar amounts of HOBt and DIC in DMF overnight. N-terminal Tam-labeling was achieved by reaction with 2 equiv Tam, 1.9 equiv HATU, 2 equiv DIPEA in DMF for 2 h.
[N-C]-c(chemerin-9) was synthesized on a 2-chlorotrityl chloride resin, the rst amino acid was coupled to the resin by reaction with 1.5 equiv Fmoc-Ser(tBu)-OH, 6 equiv DIPEA in DCM overnight. All following amino acids were coupled as described above. After protected cleavage from the resin with 10% (v/v) acetic acid, 10% (v/v) tri uoroethanol in DCM for 2 h at rt, a lactam bond between the N-and C-terminus was formed by incubation with 5 equiv HOBt, DIC in DCM for 72 h at rt.

Protein Expression
Full-length chemerinS157 was produced as a His 10 -fusion protein in E.coli BL21 (DE3) as described previously [51]. In brief, the plasmid DNA (His 10 -chemerinS157 in pET16b) was transformed into E.coli. Bacteria were grown in LB medium supplemented with 0.1 mg/mL ampicillin. Upon reaching OD 600 = 0.8, expression was induced with 1 mM isopropyl β-D-thiogalactopyranoside (IPTG). After 6 h expression at 37° C, cells were harvested by centrifugation. Resuspended in base buffer (0.5 M NaCl, 25 mM Tris/HCl, pH 7.8), the cells were lysed using a FastPrep-24 bead beating lysis system (MP Biomedicals, Irvine, USA). After washing with base buffer supplemented with 2 M urea, inclusion bodies were solubilized in base buffer containing 8 M urea.
The solubilized protein was puri ed by immobilized metal a nity chromatography and refolded by stepwise dialysis employing decreasing urea concentrations and a cysteine/cystamine redox pair. Purity and identity were con rmed by SDS-PAGE, RP-HPLC, and MALDI ToF MS (Ultra ex III, Bruker).

Mutagenesis
All mutations were introduced into an hGPR1-eYFP pVitro2 plasmid kindly provided by Stefan Schultz [51]. All PCR reactions were carried out using Phusion polymerase. The SigP-Cys-E3-hGPR1-eYGP construct was cloned by PCR utilizing the primers shown in Table 2 by introducing the Cys-E3 tag and the signal peptide in two sequential PCR reactions. The Nluc-GPR1-eYFP construct was cloned by overlap extension PCR using the primers display in Table 2, exploiting the MluI and XbaI restriction sites. The secNluc-pNL1.3 vector was purchased from Promega. Point mutations were introduced into the Nluc-GPR1-eYFP construct using the QuickChange mutagenesis protocol with primers carrying the respective mutations. The success of any mutagenesis or cloning reactions was veri ed by Sanger sequencing.

Characterization of Arrestin Recruitment
Arrestin recruitment was characterized by measuring the bioluminescence resonance energy transfer (BRET) ratio between luciferase-tagged arrestin3 and either GPR1-eYFP or CMKLR1-eYFP as described previously [52]. . For concentration-response curves, cells were stimulated with agonist or blank, and luminescence was measured after 10 min. BRET signal was calculated as the ratio of uorescence divided by luminescence, netBRET signal was calculated by subtracting the BRET signal of unstimulated wells from the respective samples.

Ligand BRET
Ligand binding was characterized using a NanoBRET approach with Tam-labeled ligands and hGPR1-eYFP N-terminally modi ed with a NanoLuc [53]. COS-7 cells in 25 cm 2 cell culture asks were transfected with 4000 ng of the respective Nluc-GPR1-eYFP construct using MetafectenePro according to the manufacturer's protocol. One day post-transfection, cells were detached using trypsin/EDTA, resuspended in 10 mL phenol red-free DMEM/Ham's F12, 15% FBS, seeded into solid-black 96 well plates (100 µL/well), and grown overnight. Before the assay, the medium was replaced by 100 µL BRET buffer. Cells were stimulated with Tam-labeled peptides, and 50 µL of furimazine in BRET buffer was added. Measurements were performed using a Tecan Spark plate reader (Tecan, Männerdorf, Switzerland), measuring Tam-emission (550-700 nm), and NanoLuc luminescence (430-470 nm). BRET and netBRET signals were calculated as described above.
Next, cells were transfected with 900 ng of the respective GPR1-eYFP plasmid and, where applicable, 100 ng of either rab4-CFP, rab11-CFP, or mCherry-arrestin3. Transfection was achieved using Lipofectamine 2000 according to the manufacturer's protocol. One day post-transfection, uorescence microscopy experiments were performed on an AxioVision Observer.Z1 microscope equipped with an ApoTome imaging system (Zeiss, Jena, Germany). Before the experiment, cells were starved in OptiMEM reduced serum medium containing Hoechst 33342 for 30 min. To observe arrestin recruitment, cells were stimulated with 1 µM chemerin-9 in OptiMEM for the indicated period. To observe peptide uptake, cells were stimulated with 1 µM Tam-chemerin-9 in OptiMEM, which was replaced with acidic wash (50 mM glycine, 100 mM NaCl, pH 3) in HBSS after the indicated time, followed by two washing steps with OptiMEM. Microscopy was carried out in OptiMEM; the exposure time was held constant whenever changes over time were observed.

Peptide-Templated On-Surface Labeling
Peptide-templated acyl transfer for selective labeling of membrane receptors was carried out as described previously [30]. Because E3-tagged GPR1 was not expressed in the membrane, the endothelin B receptor N-terminal signal peptide was attached. This signal peptide improves transport to the membrane and is cleaved upon successful membrane integration [54]. HEK293 cells were seeded out and transfected with SigP-Cys-E3-GPR1-eYFP in pVitro2 as described above and incubated in Hoechst 33342 in OptiMEM for 30 min, followed by 10 min incubation in 20 mM HEPES in HBSS, pH 7 (labeling buffer). Labeling of cell surface receptors was achieved by 5 min incubation with 150 nM Tam-K3 peptide probe in labeling buffer supplemented with 0.1 mM TCEP. The cells were washed by incubation with 200 mM NaHCO 3 in DPBS w/o Ca 2+ and Mg 2+ , pH 3 for 1.5 min, followed by two washing steps with labeling buffer. Microscopy studies were nally carried out in labeling buffer as described above. To prevent receptor internalization, the cells were cooled on ice, followed by labeling and washing with ice-cold solutions and microscopy at rt. The Tam-K3 peptide was synthesized as described before [31]. The fraction of Tam-uorescence in the membrane was determined by quantifying total and intracellular uorescence for each cell individually in imageJ. The uorescence fraction in the membrane was de ned as the difference between intracellular and total uorescence divided by total uorescence.

Peptide Uptake using a High Content Imaging System
To quantify receptor-mediated peptide uptake, the intracellular accumulation of Tam-uorescence was observed at different time points. HEK293 cells stably transfected with GPR1-eYFP were seeded into µclear, black 96 well plates (100,000 cells/well) coated with poly D-lysine and grown overnight. Before the experiment, cells were incubated with Hoechst 33342 in OptiMEM, which was replaced with pure OptiMEM after 30 min. Cells were stimulated with 1 or 10 µM of the respective, Tam-labeled peptide for the speci ed periods, followed by washing with acidic wash (50 mM glycine, 100 mM NaCl, pH 3) in HBSS.
Microscopy was carried out in OptiMEM using an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices, San José, United States), using the appropriate lters for the respective uorophores. The uorescence intensity per cell was automatically analyzed for each well by a module detecting the nuclei (5-30 µm in diameter and 100 gray levels above background) and the granules by Tam-peptide uorescence (2-5 µm in diameter, 70 gray levels above background).

Statistical Analysis
Linear and nonlinear regression, statistical analysis, and calculation of mean, SEM or SD was carried out using GraphPad Prism 8 except for BRET kinetics, where mean ± 95% CI values were plotted using seaborn and matplotlib in python 3. Linear regression of ΔΔG vs log(EC 50 ) values was calculated in python 3 using the scipy stats module. The applied statistical tests, including sample sizes, are given in the respective gure legends.

Peptide Docking
To include structurally diverse templates for peptide docking, the homology models were clustered based on C α RMSD, and chemerin-9 was docked into the ten best scoring models by total score from each of the three largest clusters using Rosetta FlexPepDock ab initio. [61] One sided restraints for the identi ed binding residues were applied to keep the peptide in the binding pocket, and a loop conformation of the peptide was enforced by a distance restraint between the peptide N-and C-terminus. An interaction between chemerin-9 residue F 8 and CMKLR1 residues V 4.67 and F 4.69 was enforced by a distance restraint. In total, 25,000 models were produced. After clustering, the best scoring models from the cluster that best represented the experimental data were energy minimized using Rosetta FastRelax. The 20 best scoring nal models by interface score ΔG separate were analyzed using a per residue energy breakdown. A detailed description of the modeling and docking process is given in the Supplementary Information.  ChemS157 over time, data points represent mean of three technical replicates with shaded 95% con dence interval (CI). b) GPR1-eYFP and Arr3-mCherry, before and 15 min after stimulation with 1 µM ChemS157. GPR1 internalizes without arrestin3, which stays at the membrane. Cell nuclei were stained with Hoechst 33342; scale bar = 10 µm.

Figure 3
Identi cation of the ligand-binding pocket at GPR1. a) Mutagenesis data obtained in a nanoBRET binding assay revealed several conserved, mostly aromatic residues in the extracellular loops and upper TMs to be involved in ligand binding. Concentration-response curves of Nluc-tagged GPR1 and mutants stimulated with Tam-EG(4)-chemerin-9 are shown. Data points represent mean ± SEM from at least two independent experiments performed in triplicates. b) Close-up view of the binding pocket in the best scoring GPR1 model with experimentally con rmed key residues shown as sticks.   Binding mode of the Chemerin C-terminus at GPR1. a) Best scoring model by interface score ΔG separated, with the ligand shown in red, the receptor in gray.
Residues of the binding pocket are highlighted as sticks. b) Contact map of the 20 best scoring modes by interface score ΔG separated, with darker colors indicating stronger interactions. Only receptor residues with <-1 REU are included. c) The cyclized derivates [N-C]-c(chemerin-9) and [4][5][6][7][8][9]-c(chemerin-9) retain activity in an arrestin recruitment assay, con rming the loop-like conformation of the chemerin-9 terminus. d) The predicted binding energies for several receptor residues strongly correlate with the logarithmic EC50 shifts of the respective alanine mutants, demonstrating that the models are in good agreement with the available experimental data. HEK293 cells stably expressing GPR1 internalize Tam-EG(4)-chemerin-9 and the scrambled peptides Tam-EG(4)-scrC9 and Tam-EG(4)-scr2C9, but not Tam-EG(4)-chemerin-7. Cells were stimulated with 1 µM of the respective peptide; data points represent mean ± SEM from at least two independent experiments performed in duplicates. a) Intracellular accumulation of the labeled peptides over time. b) After 20 min, the concentration of Tam-EG(4)-chemerin-9 and both scrambled peptides linearly increases over time. The slope of the linear regression for Tam-EG(4)-chemerin-7 does not signi cantly differ from zero, and is signi cantly smaller than the slope for Tam-EG(4)-chemerin-9, Tam-EG(4)-scrC9, and Tam-EG(4)-scr2C9 (p<0.05, one-way ANOVA with Tukey's multiple comparisons post-test). Bars represent mean ± SEM. Figure 8