CAM of PrRP receptor reveals binding & activation site

The prolactin-releasing peptide receptor (PrRPR) and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the ligand binding mode of peptide Gprotein coupled receptors (GPCR). By receptor mutagenesis we identified the conserved aspartate in the upper part of transmembrane helix 6 (D) of the receptor as the first position that directly interacts with arginine 19 of the ligand (R). Permutation of D with R of PrRP20 led to DR, which turned out to be a constitutively active receptor mutant (CAM). This suggests that the mutated residue at the top of transmembrane helix 6 mimics R by interacting with additional binding partners in the receptor. Next, we set up a comparative model of this CAM because no ligand docking is required, and selected a next set of receptor mutants to find the engaged partners of the binding pocket. In an iterative process we identified two acidic residues and two hydrophobic residues that form the peptide ligand binding pocket. As all residues are localized on top or in the upper part of the transmembrane domains we clearly can show that the extracellular surface of the receptor is sufficient for full signal transduction for PrRP, rather than a deep membrane binding pocket. This contributes to the knowledge of the binding of peptide ligands to GPCR and might facilitate the development of GPCR ligands, but also provides new targeting of CAM involved in hereditary diseases. INTRODUCTION Identification of direct receptor-ligand interactions for the approximately 800 identified G protein-coupled receptors (GPCR) is as challenging as it is important for drug discovery (1) as 50% of all currently available drugs target the specific manipulation of GPCR activity (2-3). The PrRP receptor superfamily is expressed in almost all cells/tissues, is involved in a plethora of different signalling pathways, and plays an important role in a large variety of physiological processes. The prolactin-releasing peptide receptor (PrRPR) was originally isolated from rat hypothalamus (4). PrRPR has been detected widely throughout the human and rat brain (5) and most commonly activates the Gq protein-coupled signalling pathway (6). Its eponymous endogenous ligand, the prolactin-releasing peptide (PrRP), was identified in 1998 by a reverse pharmacology approach on the basis of orphan GPCR (7-8). http://www.jbc.org/cgi/doi/10.1074/jbc.M112.349852 The latest version is at JBC Papers in Press. Published on July 9, 2012 as Manuscript M112.349852 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. at V anerbilt U nirsity B om eical & S cice/E ngeering Libaries, on July 2, 2012 w w w .jb.org D ow nladed fom CAM of PrRP receptor reveals binding & activation site 2 PrRP features two equipotent isoforms, PrRP31 (31 residues) and an N-terminally truncated PrRP20 (20 residues) (6,8). PrRP is an RF-amide peptide, consisting of a common carboxy-terminal arginine (R) and an amidated phenylalanine (F) motif and plays a role in energy metabolism, stress responses, circadian rhythm, analgesia, and in anorexigenic effects (7,9). Structure-activity relationship studies of PrRP using N-terminally truncated mutants and alanine substitution within these constructs (10-12) demonstrated the biological significance of the C-terminal R and F residues, and the amidation of the C-terminus. Site-directed mutagenesis is a powerful and widely used tool to study receptor activation. This approach alone can provide insight in the function of GPCR, but it is often used in combination with information provided by other techniques, such as crystallography or molecular modeling, in order to relate receptor function to a tertiary structure (13). The conserved D residue of the Y receptor (YR) family was shown to interact with a specific R of either human pancreatic polypeptide or neuropeptide Y (NPY) in a subtype-specific manner (14-15). The numbering of receptor residues has been performed as suggested by Ballesteros and Weinstein (16). PrRPR shares its phylogenic origin with Y receptors (17), leading to sequence similarities (Figure 1A) and a number of conserved residues, including D (Figure 1C). Furthermore, the ligands of these receptors are structurally similar (18) and share a similar Cterminal sequence (Figure 1B). While the RFamide motif was previously identified as a major requirement for PrRP-induced agonist activity (1011), the critical residues on the receptor remain unknown, and the ligand binding mode is still poorly understood. Here, we describe the first mutagenesis study of the human PrRP receptor (PrRPR). We used the extracellular region to elucidate the binding site and the molecular mechanism of GPCR activation. Considering the relevance of the C-terminal R and F residues of PrRP for receptor binding, we applied the concept of double cycle mutagenesis approach (15,19-20) and identified the first direct contact point between PrRP20 and the PrRPR, consisting of the conserved D and the R residue of PrRP20. To prove the existence of this interaction, we switched the residues involved in the salt bridge formation and created DR PrRPR and DPrRP20. This newly introduced R in the receptor variant DR might serve as surrogate for the absent R of the ligand as it led to a new type of constitutive activity. Given the lack of data of experimentally determined structures of peptide GPCR, we developed a comparative model of the human PrRPR. By combining molecular modelling with double cycle mutagenesis experiments in the framework of this constitutively active mutant (CAM), we conceived an effective strategy to explore structural determinants of ligand recognition on a molecular level. More specifically, we were able to identify Y, W, E, and to some extend F to be involved in receptor activation and ligand binding. This combinatory approach enabled us to clarify the double binding mode of R of the peptide ligand, which has two putative interaction partners within the PrRPR, E and D. The assembled experimental data were used to generate a model of the PrRP/receptor interaction in molecular detail. Furthermore our data describe the binding mode of a peptide ligand to GPCR by solely interacting with residues localized in the extracellular domain or upper part of the TM helices. In our approach we identified a receptor mutant with constitutive activity, which most likely relies on mimicking a direct ligand-receptor interaction. This provides knowledge on the function of an active mode of GPCR and may be applied to other peptide GPCR. EXPERIMENTAL PROCEDURES Peptide synthesis. Rink amide resin (NovaBiochem; Läufelfingen, Switzerland) was used to synthesize PrRP20, APrRP20, DPrRP20, and APrRP20 by automated solid phase peptide synthesis (Syro; MultiSynTech, Bochum, Germany) as previously described, using the orthogonal Fmoc/Bu (9-fluorenylmethoxycarbonyl-tert-butyl) strategy (21). Purification and verification of the peptides was achieved as previously described (Table S1) (22). DNA extraction from SMS-KAN.To obtain genomic DNA from SMS-KAN cells (human neuroblastoma cells, DSMZ, Braunschweig, Germany), approximately 1 million cells were digested overnight at 55°C with 500 μl lysis buffer at V anerbilt U nirsity B om eical & S cice/E ngeering Libaries, on July 2, 2012 w w w .jb.org D ow nladed fom CAM of PrRP receptor reveals binding & activation site 3 (1 M NaCl, 20% SDS, 0.5 M EDTA, 1 M Tris, pH 8.5 was adjusted using hydrochloric acid (HCl)) containing 50 μg proteinase K (Promega, Mannheim, Germany). Genomic DNA was extracted using phenol/chloroform and precipitated from the aqueous phase with isopropanol, washed with ethanol and then dissolved in water. Cloning and mutagenesis of the PrRP receptors in eukaryotic expression vectors. The coding sequence of the human PrRPR was obtained by PCR amplification from the isolated genomic DNA of SMS-KAN cells and cloned into the eukaryotic expression vector pEYFP-N1 (Clontech, Heidelberg, Germany) C-terminally fused to EYFP, using the XhoI and BamHI restriction site to result in the construct phPrRPR_EYFP-N1. The correctness of the entire coding sequence was confirmed by DNA sequencing using the dideoxynucleotide (ddNTP) termination method developed by Sanger (23). Plasmids encoding single point mutations (Table III) were prepared by using the QuikChangeTM sitedirected mutagenesis method (Stratagene, CA, USA) with the desired mutagenic primers. For intermolecular double-cycle mutagenesis approaches, the single alanine mutated receptor constructs were investigated, using single alanine modified PrRP20 analogs. Plasmids encoding double mutations containing YA, WA, EA, ER; WA, DA, FA or QA as a second mutation, respectively, were prepared by using the QuikChangeTM site-directed mutagenesis approach with the DR or DA construct as template. In addition, all PrPR receptor constructs were also generated N-terminally fused to the coding sequence of the hemagglutinin (HA)-tag. The entire coding sequence of each resulting receptor mutant was proven by sequencing. Cell culture. Cell culture material was supplied by PAA Laboratories GmbH (Pasching, Austria). Culture of COS-7 (African green monkey, kidney), HEK293 (human embryonic kidney), and SMS-KAN cells was done as recommended by the supplier (DSMZ, Braunschweig, Germany). Briefly, cells were grown as monolayers at 37°C in a humidified atmosphere of 5% CO2 and 95% air. COS-7 cells were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% (v/v) heat-inactivated fetal calf serum (FCS), 100 units/ml penicillin and 100 μg/ml streptomycin and HEK293 cells were grown in DMEM / Ham’ F12 (1:1) without Lglutamine containing 15% (v/v) heat-inactivated FCS as previously described (15,24). SMS-KAN cells were maintained in nutrient mixture Ham’s F12 / Dulbecco’s modified Eagle medium (1:1) with 15% (v/v) FCS, 4 mM glutamine, and 0.2 mM non-essential amino acids (25). Fluorescence microscopy. HEK293 cells (1.2x10) were seeded into 8-well chamber slides (ibidi, Munich, Germany). The transient transfection of HEK293 cells were performed using 0.1 μg to 1μg vector DNA and 1 μl LipofectaminTM 2000 transfection reagent (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer’s instructions. The nuclei were visualized with Hoechst 33342 (1 μg/ml; Sigma Aldrich, Taufkirchen, Germany) for 10 min after 1h of starving with OPTI-MEM I Reduced Serum Medium (Invitrogen GmbH, Karlsruhe, Germany). Fluorescence images were obtained using an ApoTome Imaging System with an Axio Observer microscope (Zeiss, Jena, Germany). All investigated receptors were correctly integrated in the membrane as confirmed by live-cell microscopy (Figure S1A). Quantification of receptor cell surface localisation by cell surface ELISA. To quantify plasma-membrane receptors, a cell surface ELISA was performed using an antibody directed against the native 15 N-terminal amino acids of the PrRPR. 50 000 HEK293 cells were grown in 96well plates and transfected with the PrRP wt receptor or its mutants after reaching 75-85% of confluence. The cells were starved with OPTIMEM I (30 min) 17 hours post-transfection and fixed in 4% paraformaldehyde (30 min). For immune-staining, cells were blocked with 2% BSA and permeabilized with 0.5% Triton X-100, 2% BSA in Dulbecco’s Modified Eagle’s Medium for 1 hour (37°C) to determine total receptor amounts, whereas surface expressed receptors were quantified without permeabilization. Incubation was performed with the primary antibody (1:2000 dilutions) for 2 hours (25°C) and followed by 1.5 hour (25°C) incubation with the secondary antibody (1:5000). Receptors were at V anerbilt U nirsity B om eical & S cice/E ngeering Libaries, on July 2, 2012 w w w .jb.org D ow nladed fom CAM of PrRP receptor reveals binding & activation site 4 detected by using rabbit anti-N-terminus (GPR10 antibody [N1], GTX108137, GeneTex) followed by horseradish peroxidase-conjugated goat antirabbit IgG (sc-2004, Santa Cruz, Heidelberg, Germany). The results were fully confirmed in a second independent ELISA set up, using a peroxidise-conjugated anti-HA-antibody (1:1000 dilutions, 12CA5, Roche, Mannheim, Germany) versus the N-terminally fused HA-tag of the generated PrRPR constructs (data not shown). Quantification of the bound peroxidase was performed as described and analysis performed with the GraphPad Prism 5.03 program (14). Values are presented as mean values ± s.e.m. of four individual experiments, measured in triplicates. Radioligand binding studies. For radioligand binding studies, 1.5  10 COS-7 cells were seeded into 25 cm flasks. At 60-70% confluency, cells were transiently transfected using 4 μg vector DNA and 15 μl of MetafecteneTM (Biontex Laboratories GmbH, Martinsried/Planegg, Germany). Approximately 24 h after transfection binding assays were performed on intact cells using N [propionylH] hPrRP20. Binding was determined with 1 nM N [propionylH] hPrRP20 in the absence (total binding) or in the presence (non-specific binding) of 1 μM unlabeled hPrRP20, respectively, as described previously (26-27). Our former evaluated protocol (28) was used to obtain N [propionylH] hPrRP20 by selective labelling with a specific activity of 3.52 TBq/mmol and resulting in a Kd-value of 0.58 nM. Specific binding of each PrRP receptor mutant was compared to specific binding of the PrRP wt receptor. IC50-values and the Kd-value were calculated with GraphPad Prism 5.03 (GraphPad Software, San Diego, USA), fitted to a one-site competition or a one-site binding model, respectively. Triplicates were measured in at least two independent experiments for the determination of IC50-values, whereas one experiment in triplicate was made for Kd-value estimation. Signal transduction assay. Signal transduction (inositol phosphate, or IP, accumulation) assays were performed as previously described with minor modifications (22). The time of incubation was increased to 3 h for the double mutants of PrRPR and reduced to 1h for measurement of concentration-response curves. To test for constitutive activity, COS-7 cells were incubated without agonist for 1 h, 3 h, and 6 h at 37°C. Each ligand-receptor interaction was analyzed with the GraphPad Prism 5.03 program by establishing the corresponding data set from different experiments. All signal transduction assays were repeated at least twice independently and measured in duplicate. The global curve fitting function of GraphPad Prism 5.03 was asked to determine given EC50-ratios. The statistical significance of relevant samples was computed by using the unpaired student’s t-test, based on the means, values with P < 0.05 were considered to be significant. Multiple sequence alignment. ClustalW (29) was used to align the primary sequence of the PrRPR with the sequences of mammalian Y and PrRP receptors. Next, the transmembrane regions of six GPCR of known structure (see below) were structurally aligned with Mustang (30). The profiles resulting from these first two steps were then aligned to one another with ClustalW, and the human PrRPR sequence alignment used for modelling was taken from this final profile-profile alignment. The C-terminal 310 residues of the PrRPR primary sequence were threaded onto the 3D coordinates of six available GPCR experimental structures; PDBIDs: 1U19 (31), 3CAP (32), 3DQB (33), 2RH1 (34), 2VT4 (35), 3EML (36). Construction of the comparative models. Extracellular loop regions were reconstructed using kinematic loop closure (37) and cyclic coordinate descent (CCD) (38) as implemented in the ROSETTA v3 software suite. The models were refined with the ROSETTA v3 all-atom energy function. Energetically favourable models were grouped into 15 structurally similar groups by kmeans clustering, and the lowest scoring models of each cluster were analysed. Models based on the template PDB 3DQB had the lowest energy and were used to inform the mutagenesis studies. Model refinement and peptide docking. The comparative model constructed in light of the new mutagenesis data was generated using the original multiple sequence alignment. To model at V anerbilt U nirsity B om eical & S cice/E ngeering Libaries, on July 2, 2012 w w w .jb.org D ow nladed fom CAM of PrRP receptor reveals binding & activation site 5 the PrRPR/ligand complex, an iterative peptide docking and loop remodeling procedure was performed: Energetically favorable changes in orientation were determined using the ROSETTAMEMBRANE all-atom energy function (39). The PrRP8-20 model was docked into the putative binding site of the receptor while allowing remodeling of ELs 1, 2, and 3. Using the ROSETTADOCK protocol (40), translational movements of the peptide of up to 4Å were allowed in three dimensions and the peptide was allowed to rotate along its x, y, and z-axes by up to 10°. Loop regions were constructed using cyclic coordinate descent (CCD) (38). The conformational search was enhanced by conducting the modeling in the presence of loose distance restraints where models that placed D, E, W, and Y within 10Å of R of the peptide were more energetically favorable than those that did not. The PrRP8-20 model was generated by de novo folding the peptide using ROSETTANMR with sparse NMR chemical shift and distance data (41). Of 19,241 PrRP/receptor complex docked models, the top ten by total score were analyzed. Two of these models were considered structurally redundant, leaving eight unique models that agree with the experimental data presented herein (Figure 8). RESULTS R of the endogenous ligand PrRP20 interacts with the D of PrRPR. Based on the data of the NPY/YR system (14-15), we hypothesized D to be the interaction partner of R in the PrRP/PrRPR system. To test this hypothesis, charge and size prerequisites in position D were elucidated by systematic substitution to DA, DE, DN, DR, and DK (Table 1). The expected impact on function was confirmed by the right-shifted concentrationresponse curve of DA, compared to the wildtype (wt) receptor after stimulation with PrRP20 (Figure 1D). The increased EC50-value (26 nM) of the DA mutant confirms the importance of the D side chain. In addition, the results obtained for the other D single mutants support the hypothesis of an ionic interaction; DE behaves similarly to wt, the oppositely charged DK shows strong effects in potency and the bulkier, more positively charged DR is not tolerated (Table 1). The impact of the substitutions increases as follows: E10 μM). Along with the experiments testing DPrRP20 stimulation of wt PrRPR, we demonstrate an approximately equal repulsive effect of R to ER or D to D (Figure 7D). This strengthens our hypothesis of a dual binding mode of R to E and D. Comparative model of PrRP/receptor complex provides structural information on mode of binding. Using the R/E and R/D contacts as restraints, a de novo-folded model of PrRP8-20 based on reported NMR data (18) was docked into an ensemble of comparative models of the PrRPR. The conformation of the EL regions was constructed simultaneously with ligand docking to accurately capture conformational changes induced by the peptide. Details of the modeling procedures are given in the Materials and Methods and Supplemental Information. The lowest-energy ROSETTA model features salt bridges between D, E, and R. W and Y form π-stacking interactions that may be indicative of a “toggle-switch” mechanism (Figure 8A) (43). F appears to further apart from R but might contribute to the positioning of TMH 6 via intra-molecular interactions and is in distance for π-stacking interactions with the F of PrRP20. Additional interactions between peptide and receptor hold the peptide in an optimal binding conformation deeply buried in the upper TMH segments and supported by the ELs from above. DISCUSSION We have evolved a strategy to interrogate detailed molecular mechanisms of GPCR activation by combining reciprocal, double cycle, and intramolecular double mutagenesis with computational modelling. We apply this technique effectively to PrRPR and its CAM, DR PrRPR, identifying distinct receptor residues involved in activation and/or ligand binding. This is the first comprehensive mutational study of the extracellular and transmembrane regions of the PrRPR. The double cycle mutagenic approach suggests the interaction (direct or indirect) between residues D and R and provides a first anchor point for receptor/ligand investigations. Interacting residues can be characterized by reciprocal mutagenesis, as shown before in an intramolecular study with the DR/RD swap in the gastrin-releasing peptide receptor (44) or the D/N residues of the thyrotropin (TSH) receptor (45). By applying this method to the PrRP/PrRPR system, the salt bridge of D to R was verified, and more importantly, by generating the DR receptor, we identified the first CAM of the PrRPR. Up to now, numerous CAM were generated and investigated in a plethora of previous studies, emphasizing the increasing importance of CAMs. For example, CAM of the human angiotensin II type 1 receptor with NGly (46), the ß1B (47)/ ß 2-adrenergic receptor (48-50), the cannabinoid receptor 1 (51), muscarinic m1 (52) and m5 receptors (53) among others, have been found. Interestingly, more than sixty naturally occurring CAM GPCR are known so far (54) and are often related to human disorders (55). Consequently, GPCR activated in an agonist-independent manner are of emerging importance for drug development (3). CAM more readily undergo transition between active and inactive conformations due to removed conformational constraints of the inactive form (56). Because DR in PrRPR is located at the top of TMH6, we hypothesize that this helix is involved in receptor activation via an inward movement of the upper helical region (Figure 4D). Similarly to the PrRPR DR CAM, mutantat V anerbilt U nirsity B om eical & S cice/E ngeering Libaries, on July 2, 2012 w w w .jb.org D ow nladed fom CAM of PrRP receptor reveals binding & activation site 9 induced receptor activity was observed in the SY/TP double mutant of m5 muscarinic receptors (57). These data indicate that the top of TMH6 is directly involved in the switch between the active and the inactive state of several GPCR and that the interaction with the ligand stabilizes the receptor in this active conformation – a notion that supports the “global toggle switch model” (58-60). This model suggests that activation results from an inward movement of the extracellular ends of TMHs 6 and 7 toward TMH3, concomitant with a movement of the intracellular part of the TMHs in the opposite direction, which enables signaling via G-protein coupling. PrRPR represents an excellent model system to further investigate this hypothesis and gain insights to receptor activating mechanisms. Previous work on the TSH receptors showed the effects of spatially distant double mutants on constitutive activity (61-62). However, we focus on the investigation of the molecular vicinity surrounding D, as we suggest that specific inter-residue interactions of the generated CAM occur. To take advantage of the DR CAM to elucidate the mechanism of ligand binding and PrRPR activation, we established an effective combination of intramolecular double and intermolecular reciprocal mutagenic approaches to study PrRPR activation by wt PrRP20, APrRP20, and DPrRP20. With guidance from the PrRPR comparative model, seven possible interacting residues were considered (Figure 5A), and the double mutants EA/DR, WA/DR, YA/DR, and FA/DR revealed an involvement of these residues in receptor activation. Importantly, these receptor mutants were significantly activated by DPrRP20 but not by wt PrRP20 (Fig. 5B), proving that the receptor mutants were not miss-folded and that D on the ligand is still able to interact with DR. CAM are thought to mimic, at least partially, the active conformation of the wt receptor and to spontaneously adopt a structure able to activate Gproteins (63). Therefore, we hypothesize that in DPrRP20, residue D takes over the role of the destroyed intra-molecular interaction of the double mutants, reactivating the “silenced” CAM. The conformation of a basally silenced GPCR might impair its intrinsic capacity for signaling compared to the wt receptor. Notably, further mutations within EL2/TMH5 had no considerable impact on receptor potency, in contrast to all three positions identified via intramolecular interactions (Table 2). This demonstrates the precision and usefulness of the modeling-guided double mutational approach to identify interacting residues in close proximity to the ligand. In contrast, the WA/DR control turned out to be deficient in signaling. This is expected and in agreement with the high conservation of W/W in most peptide GPCR, e.g. in the NPY receptor system (14). Furthermore, W is located in the structurally relevant WxGFmotif, which is suggested to be a key component in the activation mechanism in many GPCR in the rhodopsin family (64). Recent investigations on TMH2 of the CAM NG hAT1 suggested TMH2 to pivot, bringing the top of TMH2 closer to the binding pocket (65). Our results obtained for the conserved Y on top of TMH2 do not support such a spatial approach to D and thus to the binding pocket. This reflects the divergence of GPCR activation and accentuates that the detailed mode of activation is not a common mechanism. The results obtained from studies of the EA mutation lead to the conclusion that this residue is predominantly responsible for ligand binding. Our initial double cycle mutagenic experiments at D support a more complex double binding role for R of PrRP20, which appears to be in contact with two sites on PrRPR. Accordingly, we suggest E to be the second binding partner for peptide residue R (Figure 7D). The extensive mutagenic studies of residue E strongly indicate the participation in binding to R and the constitutive activity of DR supports the hypothesis of a second R-specific interaction site in PrRPR that can be satisfied by the DR but not the DK mutant. A similar dual binding mode for arginine was recently reported for gonadotropin-releasing hormone (GnRH) receptor (66). This has been supported by other studies, where substitution of R to lysine, citruline (Cit), α-amino-4-guanidino-butyric acid (Agb), or α-amino-3-guanidino-propionic acid (Agp) on the peptide lead to reduced binding affinities (12). Interestingly, the tight ensemble of models that is in agreement with the experimental data presented herein exhibits variability in ELs 1 and 2 while still maintaining the contacts between D and E with R. Given this structural variability in our models, we emphasize that the at V anerbilt U nirsity B om eical & S cice/E ngeering Libaries, on July 2, 2012 w w w .jb.org D ow nladed fom CAM of PrRP receptor reveals binding & activation site 10 presented approach is an iterative process, where initial models can be used to guide experimental design, and the resulting data allow for model refinement. The current PrRP/receptor model can only be considered valid in the light of the functional data. However, it provides insight into possible structural mechanisms of peptide/receptor interactions and receptor activation. WA and YA also showed lowered ligand potency, but both mutants revealed a strongly decreased ability to transmit signals compared to the wt receptor (Table 2). This effect may result from intramolecular structural alteration due to the lack of aromaticity at the YA site. Mutational studies reported for the nearby Y residue in both cannabinoid receptors (CB1 and CB2) revealed that the aromaticity at this position is crucial (67). The PrRP/receptor model places W in close proximity to Y (Figure 8A). In this model, the residues form stacking interactions, but this remains to be proven experimentally. We speculate that, due to the effects observed for potency and efficacy, W and Y are related to receptor activation. In contrast, FA mutant reveals full wt efficacy accompanied with reduced potency. From the docked modeling data, we speculate that this residue contributes to the correct conformation of the binding pocket and might interact with the F of the PrPR20. Evolutionary and structural studies revealed that the PrRPR belongs to the family of RF-amide peptide receptors, consisting of five discovered groups: the neuropeptide FF (NPFF) group, the prolactin-releasing peptide (PrRP) group, the gonadotropin-inhibitory hormone (GnIH) group, the kisspeptin group, and the 26RFa group (68-70). However, further phylogenic investigations revealed that the PrPRR shares an ancient receptor with the NPY receptors (17). The human PrRPR possesses high sequence identity with the human NPY2R, particularly in the upper and middle regions of TMH 4, TMH 5, and TMH 6. It is suggested that the PrRPR family began co-evolving with ancestral PrRP/C-RFamide peptide with a redundant NPY binding receptor (17). This explains the importance of the conserved D residue and in turn, might have been responsible for the development of a double binding mode for R in the PrRPR/PrRP system. It could be speculated that other RF-amide receptors evolved similar binding modes for the crucial arginine within the RF-amide motif, especially for the closely related 26RF-amide receptor. In contrast, for the well investigated Yreceptor family, a double binding mode was not identified, neither for R at Y2/Y5R nor for R at Y1/Y4R (14-15). However, the second interaction might occur via the second arginine 33 or 35, respectively. Regarding medical and physiological implications, the expression of CAM can entail oncogenic effects, such as tumor formation in nude mice (71). A variety of diseases are known to be triggered by elevated basal activity, including autosomal dominant hypocalcaemia (72) and ovarian hyperstimulation syndrome (73). Our findings provide insight into the harmful potential of CAM and demonstrate the need for applicable drugs that are able to diminish mutation-induced receptor activity. We are confident that our technique is a promising tool to investigate residues relevant for ligand binding and receptor activation because a CAM is used as a template. Our approach paves the way for obtaining specific structure/function information on a molecular level, which is of indispensible value, as no crystal structure for a peptide GPCR currently exists. This method will hopefully contribute to the elucidation of the structural mechanisms of harmful CAM and help to develop and increase the number of inverse-agonist drugs that target these receptors.