The Leu/Val6.51 Side Chain of Cannabinoid Receptors Regulates the Binding Mode of the Alkyl Chain of Δ9-Tetrahydrocannabinol

Authors: Claudia Llinas del Torrent, Iu Raïch, Angel Gonzalez, Nil Casajuana-Martin, Jaume Lillo, Joan Biel Rebassa, Carlos Ferreiro-Vera, Verónica Sánchez de Medina, Rafael Franco ⚬, Gemma Navarro, Leonardo Pardo ⚬

Affiliations: †Laboratory of Computational Medicine, Biostatistics Unit, Faculty of Medicine, Universitat Autònoma Barcelona, 08193 Bellaterra, Barcelona, Spain; ‡Department of Biochemistry and Molecular Biomedicine, School of Biology, University of Barcelona, 08028 Barcelona, Spain; §Centro de Investigación en Red, Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, 28031 Madrid, Spain; ∥Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, Universitat de Barcelona, 08028 Barcelona, Spain; ⊥Phytoplant Research S.L.U., 14014 Córdoba, Spain; #Institute of Neuroscience, University of Barcelona (NeuroUB), Av Joan XXIII 27-31, 08028 Barcelona, Spain

License: © 2023 The Authors. Published by American Chemical Society CC BY 4.0 Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/).

Article links: DOI: 10.1021/acs.jcim.3c01054  |  PubMed: 37644761  |  PMC: PMC10523433

Relevance: Relevant: mentioned in keywords or abstract

Full text: PDF (4.9 MB)

Introduction

Cannabinoids are naturally occurring compounds found in the Cannabis sativa plant (more commonly known as marijuana). There are over 180 cannabinoids out of the 1600 chemical compounds that have been isolated from Cannabis, with a characteristic oxygen containing C₂₁ aromatic hydrocarbons.¹ These exogenous cannabinoids can be further classified into 11 subclasses: cannabichromene (CBC), cannabidiol (CBD), cannabielsoin (CBE), cannabigerol (CBG), cannabicyclol (CBL), cannabinol (CBN), cannabinodiol (CBND), cannabitriol (CBT), (−)-Δ⁸-trans-tetrahydrocannabinol (Δ⁸-THC), (−)-Δ⁹-trans-tetrahydrocannabinol (Δ⁹-THC), and miscellaneous-type cannabinoids.² The Δ⁹-THC subclass contains 25 compounds with common structural features such as a dibenzopyran ring and a hydrophobic alkyl chain. This class includes the most abundant phytocannabinoids: (−)-Δ⁹–trans-tetrahydrocannabinol (THC), which is the principal psychoactive constituent of Cannabis, and (−)-Δ⁹-trans-tetrahydrocannabivarin (THCV), which is homologous to THC but has a 3-carbon (propyl chain) instead of a 5-carbon (pentyl chain) in the alkyl chain (Figure ). THCV lacks the psychoactive effects of THC and upregulates energy metabolism, converting it a clinically useful remedy for weight loss, obesity management, and type 2 diabetic patients.^3,4 In addition, THCV can produce beneficial antipsychotic effects.⁵ Endogenous cannabinoids are N-arachidonylethanolamide (anandamide) and 2-arachidonoylglycerol (2-AG) that possess long hydrophobic moieties.⁶

The effects of cannabinoids are primarily mediated through two members of the G-protein-coupled receptor (GPCR) family,⁷ the cannabinoid CB₁ (CB₁R) and CB₂ (CB₂R) receptors. CB₁R is one of the most abundant GPCRs in the central nervous system, whereas CB₂R is mainly expressed in the immune system.⁸ However, other molecular targets for certain cannabinoids, aside from CB₁R and CB₂R, have also been identified.⁹ Some authors propose that THCV is a CB₁R and CB₂R antagonist,¹⁰ whereas others suggest that THC and THCV are partial agonists at both receptors.¹¹ We have recently shown that THC acts as a partial agonist in CB₁R and as an antagonist in CB₂R, whereas THCV acts as an antagonist on both receptors.¹²

Here, we have used the recently released structures of CB₁R¹³⁻¹⁵ and CB₂R¹⁶⁻¹⁸ in their inactive and active, G_i-bound, conformations to delineate the individual signaling contributions of THC and THCV to modulate both receptors. Phe^3.36 and Trp^6.48 have been described as conformational toggle or trigger switches involved in the initial agonist-induced receptor activation in CB₁R,^15,19 CB₂R¹⁶ and other GPCRs.²⁰⁻²² However, these amino acids are conserved in CB₁R and CB₂R (44% sequence identity between receptors), thus they cannot explain the different pharmacological profile of THC and THCV. In this manuscript, a combination of molecular dynamics (MD) simulations and site-directed mutagenesis have permitted to propose residue at position 6.51, which is Leu at CB₁R and Val at CB₂R, as an additional player capable to selectively recognize the alkyl chain of these ligands, further supporting the yin-yang functional relationship already described for CB₁ and CB₂ receptors.¹⁶ This knowledge could be of great use to facilitate the future design of selective drugs in the endocannabinoid system.

Materials and Methods

Initial CB₁R and CB₂R Models

The CB₁R-AM841-G_i (PDB id 6KPG) and CB₂R-AM12033-G_i (6KPF) cryo-EM structures¹⁸ were used in docking studies and MD simulations. Missing residues 55–180 of α_i in the CB₁R-AM841-G_i structure were built from the structure of G_i (6CRK);²³ and missing residues 55–181 and 233–239 of α_i in the CB₂R-AM12033-G_i structure were built from the CB₂R-WIN55,212–2-G_i structure (6PT0),¹⁷ using AutoModel class²⁴ of MODELER v10.1.²⁵ Protonation states were assigned with the PDB 2PQR tool²⁶ using PROPKA to predict the pK_a values of ionizable groups in the proteins at pH 6.5.²⁷ Disulfide bonds between cysteines were built using the tleap module of Ambertools19. Internal water molecules were added to CB₁R and CB₂R using HomolWat.²⁸ THC and THCV were docked into the orthosteric binding cavity of CB₁R and CB₂R and JWH-133 into CB₂R by using AM841 in 6KPG and AM12033 in 6KPF structures as a reference. Thus, the alkyl chains of THC, THCV, and JWH-133 were initially modeled in the L-shape conformation above Trp279^5.43 and Trp194^5.43 of CB₁R and CB₂R, respectively, as observed in the cryo-EM structures. These systems were oriented by the Orientations of Proteins in Membranes (OPM) database,²⁹ and embedded in a lipid bilayer box, constructed using PACKMOL-memgen,³⁰ containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol (CHL) (10:1 POPC:CHL ratio), water molecules (TIP3P), and monatomic Na⁺ and Cl^– ions (0.15 M). The resulting systems comprise between 225 and 250k atoms in a box of ∼120 Å × 120 Å × 140 Å (see the Supporting Information in the Zenodo repository for detailed values).

Molecular Dynamics Simulations

MD simulations of these models were performed with GROMACS2018.5.³¹ The amber14sb-ildn force field was used for the protein, solvent, and ions,³² a GROMACS adaptation of lipid14 for lipids,³³ and the general Amber force field (GAFF2) with HF/6-31G*-derived RESP atomic charges for THC, THCV, and JWH-133.³⁴ Molecular systems were subjected to 5000 steps of energy minimization, using the steepest descent algorithm, PME electrostatics, with the Verlet cutoff scheme. This was followed by a 25 ns equilibration protocol consisting of six steps, in which positional restraints are progressively removed, from all heavy atoms to only helix Cα carbons being restricted, meanwhile gradually reducing the applied forces, from 1000 kJ mol^–1 nm^–2 to 0 kJ mol^–1 nm^–2. After equilibration, three replicas of a 1 μs unrestrained MD trajectory were generated at a constant temperature of 300 K using separate v-rescale thermostats for the receptor, ligand, lipids, and solvent molecules. Initial velocities were randomly generated for each replica from a Maxwell distribution, using different random seeds. A time step of 2.0 fs was used for the integration of equations of motions using the leapfrog algorithm. Bonds involving hydrogen atoms were kept frozen by using the LINCS algorithm. Lennard-Jones interactions were computed using a cutoff of 1.1 nm under the Verlet cutoff scheme for neighbor searching, and the electrostatic interactions were treated using PME with the same real-space cutoff under periodic boundary conditions. Center of mass motion was removed from all systems. The Berendsen pressure control algorithm was chosen for equilibration and Parrinello–Rahman for production MDs. For complete details, see the Supporting Information in the Zenodo repository.

MD Analysis and Data Visualization

The analysis of the trajectories was performed using MDAnalysis;³⁵ visualization and image rendering were performed with PyMOL³⁶ and VMD,³⁷ and graphical representations were obtained with the Seaborn Package.³⁸

CB₁R and CB₂R Mutants

Mutations were produced using the QuikChange Site-Directed Mutagenesis Kit. The cDNA for hCB₁R and hCB₂R, cloned into pcDNA3.1, was amplified using sense and antisense primers harboring the triplets for the desired point mutation (Pfu turbo polymerase was used). The nonmutated DNA template was digested for 1 h with DpnI. PCR products were used to transform XL1-blue supercompetent cells. Finally, positive colonies were tested by sequencing to select those expressing the correct DNA sequence.

cAMP Determination Assays

Determination of cAMP levels in HEK-293T cells transiently expressing CB₁R or CB₂R (1 μg of cDNA) or the mutant version of the receptor was performed by using the Lance-Ultra cAMP kit (PerkinElmer). Two hours before initiating the experiment, the medium was substituted by a serum-free medium. Then, transfected cells were dispensed in white 384-well microplates at a density of 4000 cells per well and incubated for 15 min at room temperature with compounds, followed by 15 min incubation with forskolin, and 1 h more with homogeneous time-resolved fluorescence (HTRF) assay reagents. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Labtech). Data analysis was made based on the fluorescence ratio emitted by the labeled cAMP probe (665 nm) over the light emitted by the europium cryptate-labeled anti-cAMP antibody (620 nm). A standard curve was used to calculate cAMP concentration. Forskolin-stimulated cAMP levels were normalized to 100%.

Pure Cannabinoids

Δ⁹-THC and Δ⁹-THCV substances were provided by Phytoplant Research S.L.U. Δ⁹-THC and Δ⁹-THCV were purified from the Moniek (CPVO/20160114) and Raquel (CPVO/20180114) varieties, respectively, using countercurrent chromatography as previously described.³⁹ The purity of both cannabinoids was set at >95%.

Results

THC Adopts Two Distinct Binding Modes in CB₁R But Not in CB₂R

To understand the different molecular signatures of THC and THCV, at CB₁R and CB₂R, we first performed three replicate runs of unbiased 1 μs MD simulations of these compounds bound to the CB₁R-G_i and CB₂R-G_i complexes (see the Methods section). We have used G_i-bound active states, instead of inactive structures, despite its higher computational cost, because agonists alone are not capable to stabilize the fully active conformation in the absence of the G protein, as shown by NMR experiments.⁴⁰ Similarly, MD simulations of agonist bound to the inactive state of the receptor are not capable of reaching active-like conformations in the absence of the G protein. Moreover, MD simulations of active, G protein-bound, conformations have permitted to identify additional cavities to accommodate hydrophobic chains of ligands in sphingosine-1-phosphate⁴¹ and muscarinic⁴² receptors, which were not identified in similar simulations of inactive structures.

THC and THCV were docked into these structures with the hydrophobic alkyl tail in the L-shape conformation, as observed in the cryo-EM structures of structurally similar ligands (see the Methods section). Root-mean-square deviations (rmsd) of the ligand heavy atoms show that THC visited during the MD simulations two different poses in the binding pocket of CB₁R but not in CB₂R (Figure c). In CB₁R, THC adopts the initial L-shape conformation, in which the hydrophobic alkyl tail occupies a cavity between TMs 3 and 5 (Figure a), and an I-shape conformation, in which the alkyl tail occupies an intracellular cavity between TMs 3 and 6 (Figure b). The structure–function of the alkyl chain of THC has been reviewed,⁴³ and this dual orientation is consistent with previous studies by others.^15,44 The different conformation of the pentyl chain of THC is achieved by a change of a single dihedral angle in the chain, from – anticlinal (dihedral 1–2–3–4 around −90°) in the L-shape to + anticlinal (around 90°) in the I-shape (Figure g). Heatmaps showing when THC adopts the – anticlinal (dihedral <0°) or + anticlinal (>0°) conformation correlates with large (I-shape) and small (L-shape) rmsd values, respectively (Figures c and 2e). Notably, the pentyl chain of THC in CB₂R rarely adopts the + anticlinal conformation (Figure e), thus no large rmsd values could be observed from the initial L-shape conformation during the MD simulations (Figure c). The shorter 3-carbon propyl chain of THCV has fewer steric constraints and can visit the + anticlinal conformation in both CB₁R and CB₂R simulations (Figures f–h).

It is not clear why the five-carbon pentyl chain of THC can also adopt the I-shape conformation, in which the alkyl tail occupies an intracellular cavity between TMs 3 and 6, in CB₁R but not in CB₂R. This intracellular cavity is delineated by the amino acid at position 6.51 (Figure a), which is Leu at CB₁R and Val at CB₂R (Figure b). The probability to undergo a side chain conformational change in Val is smaller than in Leu,⁴⁵ due to the β-branched side chain that is shorter than the γ-branched side chain of Leu. Val is generally found with the γ-carbons flanking the small Hα in the trans (t, χ₁ = 180°) rotamer conformation, whereas Leu can adopt the more stable trans (t, χ₁ = 180°) and less stable gauche+ (g+, χ₁ = −60°) rotamer conformations, as observed in the dynameomics rotamer library.⁴⁶Figure c shows the histogram distributions of the χ₁ dihedral angle of Leu^6.51 along the MD simulations of CB₁R. These panels illustrate that the three-carbon propyl chain of THCV maintains Leu^6.51 in the more stable t conformation during the simulation time, whereas the five-carbon pentyl chain of THC in the I-shape conformation triggers or stabilizes the g+ conformation of Leu^6.51 in CB₁R, opening the access to the intracellular cavity between TMs 3 and 6 (Figure d). In contrast, the conformation of the bulky, β-branched, and more rigid side chain of Val^6.51 in CB₂R cannot be modified by the pentyl chain of THC (not shown), closing the access to the intracellular cavity.

The CB₁R^L6.51V and CB₂R^V6.51L Mutations Reverse the Pharmacology of THC

To experimentally validate the proposed different conformations of THC in CB₁R and CB₂R, we mutated Leu^6.51 to Val in CB₁R (CB₁R^L6.51V) and Val^6.51 to Leu in CB₂R (CB₂R^V6.51L), and we measured cAMP production in HEK-293T cells (Figure e). The nonselective CP-55940 agonist (100 nM) decreased, as expected for a G_i-coupled receptor, cAMP formation induced by forskolin (500 nM), in a statistically significant manner, in wild-type CB₁R and CB₂R and mutant CB₁R^L6.51V and CB₂R^V6.51L. In contrast, THC (10 μM) can significantly decrease forskolin-induced cAMP accumulation in CB₁R but not in CB₂R. We used high concentrations of THC to evaluate the greatest attainable response (ceiling effect). These results suggest that, in cAMP measurements, THC acts as a weak partial agonist only in CB₁R. Remarkably, the pharmacological profile of THC changes in the mutant receptors. THC can significantly decrease forskolin-induced cAMP accumulation in CB₂R^V6.51L but not in CB₁R^L6.51V. Moreover, cAMP accumulation induced by THC is not statistically different between CB₁R and CB₂R^V6.51L. These experimental results, together with computational simulations, suggest that the residue at position 6.51, which is Leu at CB₁R and Val at CB₂R, is an additional element in the mechanism of receptor activation (see the Discussion section).

JWH-133 Activates CB₂R via the Substituted Methyl Groups

JWH-133 is a potent CB₂R agonist, with little affinity for CB₁R.⁴⁷ The structure of JWH-133 is like THC and THCV with a 4-carbon butyl chain, instead of the 3-carbon propyl chain of THCV or the 5-carbon pentyl chain of THC (Figure ). A significant difference between JWH-133 and either THC or THCV is the methyl substitutions on the chain (Figure ). Branching close to the aromatic ring might restrict the dimethylbutyl chain conformation of JWH-133. Thus, it seems reasonable to study the molecular properties of JWH-133, as a full agonist, in complex with CB₂R-G_i to challenge our proposed molecular models of THC and THCV (see section 3.1). Consequently, we performed simulations similar to those with THC and THCV to evaluate the binding mode of JWH-133 in CB₂R (see the Methods section). The alkyl chain of JWH-133 always adopts the L-shape conformation in the + anticlinal conformation, filling the cavity between TMs 3 and 5 (Figure a). The dimethyl moiety of the dimethylbutyl chain mediates hydrophobic interactions with Phe^3.36 and Val^6.51, during the simulation time (Figure c). To experimentally validate the key role of Val^6.51 in JWH-133-induced CB₂R activation, we measured the level of production of cAMP in CB₁R^L6.51V and CB₂R^V6.51L mutant receptors expressed in HEK-293T cells (Figure b). JWH-133 (100 nM) was unable to decrease forskolin-induced cAMP formation in CB₁R but was statistically significantly lower in CB₂R, as expected for a CB₂R selective agonist. Substitution of Leu^6.51 with Val in CB₁R does not facilitate activation of CB₁R^L6.51V by JWH-133. However, substitution of the single Val^6.51 amino acid with Leu in CB₂R makes JWH-133 unable to activate CB₂R^V6.51L. This points to both the bulky, β-branched, and rigid Val^6.51 in CB₂R and the bulky, branched dimethyl group of the dimethylbutyl chain of JWH-133 as key elements for CB₂R activation (see the Discussion section). It was recently shown that the CB₂R^V6.51L mutation also impeded HU308 and CP-55940, both containing the branched dimethyl group in the alkyl chain, to activate the G protein at CB₂R.⁴⁸

Discussion and Conclusions

Among the ∼350 GPCRs for nonsensory functions, ∼35 are activated by hormone-like signaling molecules derived from lipid species with long hydrophobic chains.^6,49 Some of these receptors possess distinctive structural signatures relative to other class A GPCRs such as the N-terminus and ECL-2 folding over the binding site,^50,51 which causes the entry of the ligand to the orthosteric site through a tunnel formed between TMs 1 and 7;^52,53 or lacking the highly conserved Pro^5.50, part of the PIF motif that transmits the signal from the orthosteric ligand binding site to the G protein binding site.^54,55 In PIF-containing GPCRs, the interaction of agonists with TM 5 triggers an inward movement of TM 5 at P^5.50, a rotation of TM 3 at I^3.40, and an outward movement of TM 6 at F^6.44.^56,57 In GPCRs lacking P^5.50, agonists can alter the rotamer of the amino acid at position 3.36²⁰ to trigger the rotation of TM 3 at I^3.40 and outward movement of TM 6 at F^6.44.⁴¹ For instance, in the active crystal structure of S1P₃ bound to the endogenous agonist sphingosine-1-phosphate (S1P),⁵⁸ the long hydrophobic side chain of d18:1 S1P binds in an extended conformation (I-shape) between TMs 4 and 5 (Figure a). S1P triggers conformational changes of Leu122^3.36 from t to g+, among others (see quartet core in⁵⁸), to accommodate the alkyl chain. Similar results were observed in S1P bound to S1P₁⁵⁹ and S1P₂⁶⁰ (Figure a). In the cryo-EM structure of active LPA₁ bound to the lysophosphatidic acid (LPA),⁶¹ the alkyl chain of LPA cannot extend to the cleft between TMs 4 and 5 as S1P, due to the presence of the bulky Trp210^5.43 in LPA₁ (S1P_1–3 possess the less bulky Cys206^5.43, Val194^5.43, or Cys200^5.43), blocking the access. LPA adopts an U-shaped conformation bending backward and triggering the g+ conformation of Leu132^3.36 (Figure b). The long acyl chain of the ONO-0740556 agonist, a more rigid and potent LPA analog, binds LPA₁ in a different bent conformation than LPA⁶² (Figure c). In this case, the aromatic ring of ONO-0740556 triggers the g+ conformation of Leu132^3.36. The lack of side chain in Gly274^6.51 (LPA_1–3 possess Gly at this key position) permits LPA₁ to have a small pocket in front of Leu132^3.36 encaging the terminus of LPA or the phenyl ring of ONO-0740556.^61,62

CB₁R and CB₂R possess Trp279^5.43 and Trp194^5.43, respectively, thus blocking the TMs 4 and 5 cleft created in S1P binding to S1P_1–3; and contain Phe200^3.36 and Phe117^3.36, respectively, as conformational toggle or trigger switch involved in the initial agonist-induced receptor activation. Notably, the indazole ring of MDMB-Fubinaca triggers the active g+ conformation of Phe200^3.36 in CB₁R by an aromatic–aromatic interaction (Figure c),¹⁵ and the aromatic core of WIN 55,212–2 also forms aromatic–aromatic interactions with Phe117^3.36 in g+ of CB₂R (Figure c).¹⁷ In other known structures of active CB₁R and CB₂R bound to agonists, the bulky and branched dimethyl groups of the alkyl chain of AM841 and AM12033 bridge Phe200^3.36 in g+ and Leu359^6.51 of CB₁R and Phe117^3.36 in g+ and Val261^6.51 of CB₂R (Figure d), respectively.¹⁸ The conformational change in Phe^3.36, from pointing toward TM 6 in t to pointing toward TM 7 in g+, permits Trp^6.48 to move toward TM 5 for receptor activation.

These data indicate that the hydrophobic alkyl chain of the signaling molecule is key in the process of ligand-induced receptor activation. Thus, in this paper, we have studied the conformation of the alkyl chain of THC, THCV, and JWH-133 bound to CB₁R and CB₂R and calculated their distances to Phe^3.36 along the MD simulations. Among them, the distances between the terminal methyl group of the alkyl chain of THC and the centroid of the aromatic ring of Phe^3.36 and either the δ- or γ- carbon of Leu/Val^6.51 are important to highlight (see Figures c and 4d). They fluctuated from >5 Å to <5 Å in CB₁R and are always >5 Å in CB₂R, indicating a dual orientation of the alkyl chain in CB₁R, which either occupies a cavity above Trp^5.43 (between TMs 3 and 5) in an L-shape conformation, or an intracellular cavity between Phe^3.36 and Trp^6.48 (TMs 3 and 6) in an I-shape conformation (Figure d). The main achievement of this work is the discovery that THC in CB₁R, but not in CB₂R, can adopt this I-shape conformation. The intracellular cavity between Phe^3.36 and Trp^6.48 is also delineated by the amino acid at position 6.51, which is the γ-branched, flexible Leu side chain in CB₁R and the β-branched, model rigid Val side chain in CB₂R (Figure b). We have shown that the five-carbon pentyl chain of THC can trigger the conformational change of Leu^6.51 from t, blocking the access of the chain to the intracellular cavity, to g+, opening the access (Figures c and 3d). This opening of the chain access to the intracellular cavity is not feasible with the rigid Val^6.51 side chain of CB₂R. The binding mode of THC in the I-shape conformation positions the alkyl chain between Phe^3.36 in the active g+ conformation and Trp^6.48 that is involved in the initial mechanism of agonist-induced receptor activation. Thus, these computational results are compatible with our experiments, showing that THC acts as a partial agonist in CB₁R and as an antagonist in CB₂R (Figure e).¹² In agreement with our computational results, THC could not activate the mutant CB₁R^L6.51V receptor and activated the mutant CB₂R^V6.51L receptor as efficiently as wild type CB₁R (Figure e). We have recently shown that the alkyl chain of cannabidiol, in the allosteric binding mode, also expands toward the intracellular cavity modulating the conformation of Phe^3.36.⁶³

The predicted binding mode of the dimethylbutyl chain conformation of the full agonist JWH-133 at CB₂R is always in the L-shape conformation, filling the cavity between TMs 3 and 5 (Figure a). However, the branched dimethyl moiety of the ligand chain mediates hydrophobic interactions with Phe^3.36 in the active g+ conformation and Val^6.51 (Figure c). Notably, substitution of Val^6.51 for Leu in CB₂R makes JWH-133 unable to activate CB₂R^V6.51L (Figure b). This supports the concept that the branched dimethylbutyl chain conformation of JWH-133 needs a foothold on the rigid Val^6.51 to move Phe^3.36 to the active g+ conformation for receptor activation.

In conclusion, our findings have shown that, in cannabinoid receptors and probably other receptors that recognize signaling molecules derived from lipid species with long hydrophobic chains, the amino acid at position 6.51 defines the size and shape of the cavity near Phe^3.36 and Trp^6.48 and is a key additional player in the mechanism of activation of this type of GPCRs.

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