Structure-Activity Relationship in the Leucettine Family of Kinase Inhibitors.

The protein kinase DYRK1A is involved in Alzheimer's disease, Down syndrome, diabetes, viral infections, and leukemia. Leucettines, a family of 2-aminoimidazolin-4-ones derived from the marine sponge alkaloid Leucettamine B, have been developed as pharmacological inhibitors of DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). We report here on the synthesis and structure-activity relationship (SAR) of 68 Leucettines. Leucettines were tested on 11 purified kinases and in 5 cellular assays: (1) CLK1 pre-mRNA splicing, (2) Threonine-212-Tau phosphorylation, (3) glutamate-induced cell death, (4) autophagy and (5) antagonism of ligand-activated cannabinoid receptor CB1. The Leucettine SAR observed for DYRK1A is essentially identical for CLK1, CLK4, DYRK1B, and DYRK2. DYRK3 and CLK3 are less sensitive to Leucettines. In contrast, the cellular SAR highlights correlations between inhibition of specific kinase targets and some but not all cellular effects. Leucettines deserve further development as potential therapeutics against various diseases on the basis of their molecular targets and cellular effects.

Synthesis and biological evaluation of indole-2-carboxamides bearing photoactivatable functionalities as novel allosteric modulators for the cannabinoid CB1 receptor.

5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ORG27569, 1) is a prototypical allosteric modulator for the cannabinoid CB1 receptor. Based on this indole-2-carboxamide scaffold, we designed and synthesized novel CB1 allosteric modulators that possess photoactivatable functionalities, which include benzophenone, phenyl azide, aliphatic azide and phenyltrifluoromethyldiazrine. To assess their allosteric effects, the dissociation constant (KB) and allosteric binding cooperativity factor (α) were determined and compared to their parent compounds. Within this series, benzophenone-containing compounds 26 and 27, phenylazide-containing compound 28, and the aliphatic azide containing compound 36b showed allosteric binding parameters (KB and α) comparable to their parent compound 1, 7, 8, and 9, respectively. We further assessed these modulators for their impact on G-protein coupling activity. Interestingly, these compounds exhibited negative allosteric modulator properties in a manner similar to their parent compounds, which antagonize agonist-induced G-protein coupling. These novel CB1 allosteric modulators, possessing photoactivatable functionalities, provide valuable tools for future photo-affinity labeling and mapping the CB1 allosteric binding site(s).

Mechanisms of Biased ß-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor.

Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins ß-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of ß-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of ß-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying ß-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and ß-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of ß-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control ß-arrestin-mediated signaling.

Computational Prediction and Biochemical Analyses of New Inverse Agonists for the CB1 Receptor.

Human cannabinoid type 1 (CB1) G-protein coupled receptor is a potential therapeutic target for obesity. The previously predicted and experimentally validated ensemble of ligand-free conformations of CB1 [Scott, C. E. et al. Protein Sci. 2013 , 22 , 101 - 113 ; Ahn, K. H. et al. Proteins 2013 , 81 , 1304 - 1317] are used here to predict the binding sites for known CB1-selective inverse agonists including rimonabant and its seven known derivatives. This binding pocket, which differs significantly from previously published models, is used to identify 16 novel compounds expected to be CB1 inverse agonists by exploiting potential new interactions. We show experimentally that two of these compounds exhibit inverse agonist properties including inhibition of basal and agonist-induced G-protein coupling activity, as well as an enhanced level of CB1 cell surface localization. This demonstrates the utility of using the predicted binding sites for an ensemble of CB1 receptor structures for designing new CB1 inverse agonists.

Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1.

G protein-coupled receptors (GPCRs) are the major transducers of external stimuli and key therapeutic targets in many pathological conditions. When activated by different ligands, one receptor can elicit multiple signalling cascades that are mediated by G proteins or ß-arrestin, a process defined as functional selectivity or ligand bias. However, the dynamic mechanisms underlying ß-arrestin signalling remain unknown. Here by studying the cannabinoid receptor 1 (CB1R), we identify ligand-specific endocytic dwell times, that is, the time during which receptors are clustered into clathrin pits together with ß-arrestins before endocytosis, as the mechanism controlling ß-arrestin signalling. Agonists inducing short endocytic dwell times produce little or no ß-arrestin signalling, whereas those eliciting prolonged dwell times induce robust signalling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased ß-arrestin signalling. These studies reveal how receptor activation translates into ß-arrestin signalling and identify a mechanism to control this pathway.

Optimization of chemical functionalities of indole-2-carboxamides to improve allosteric parameters for the cannabinoid receptor 1 (CB1).

5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (1; ORG27569) is a prototypical allosteric modulator for the cannabinoid type 1 receptor (CB1). Here, we reveal key structural requirements of indole-2-carboxamides for allosteric modulation of CB1: a critical chain length at the C3-position, an electron withdrawing group at the C5-position, the length of the linker between the amide bond and the phenyl ring B, and the amino substituent on the phenyl ring B. These significantly impact the binding affinity (KB) and the binding cooperativity (α). A potent CB1 allosteric modulator 5-chloro-N-(4-(dimethylamino)phenethyl)-3-propyl-1H-indole-2-carboxamide (12d) was identified. It exhibited a KB of 259.3 nM with a strikingly high binding α of 24.5. We also identified 5-chloro-N-(4-(dimethylamino)phenethyl)-3-hexyl-1H-indole-2-carboxamide (12f) with a KB of 89.1 nM, which is among the lowest KB values obtained for any allosteric modulator of CB1. These positive allosteric modulators of orthosteric agonist binding nonetheless antagonized the agonist-induced G-protein coupling to the CB1 receptor, yet induced ß-arrestin mediated ERK1/2 phosphorylation.

Molecular basis of cannabinoid CB1 receptor coupling to the G protein heterotrimer Gαißγ: identification of key CB1 contacts with the C-terminal helix α5 of Gαi.

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and Gi heterotrimer (CB1-Gi), guided by the x-ray structure of ß2-adrenergic receptor (ß2AR) in complex with Gs (ß2AR-Gs), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218(3.54), Tyr-224(IC2), Asp-338(6.30), Arg-340(6.32), Leu-341(6.33), and Thr-344(6.36), as potential key contacts with the extreme C-terminal helix α5 of Gαi. Ala mutations of these residues at the receptor-Gi interface resulted in little G protein coupling activity, consistent with the present model of the CB1-Gi complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α5 of Gαi. The model also suggests that unique conformational changes in the extreme C-terminal helix α5 of Gα play a crucial role in the receptor-mediated G protein activation.

Structure-activity relationship study of indole-2-carboxamides identifies a potent allosteric modulator for the cannabinoid receptor 1 (CB1).

The cannabinoid CB1 receptor is involved in complex physiological functions. The discovery of CB1 allosteric modulators generates new opportunities for drug discovery targeting the pharmacologically important CB1 receptor. 5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ORG27569; 1) represents a new class of indole-2-carboxamides that exhibit allostery of CB1. To better understand the SAR, a group of indole-2-carboxamide analogues were synthesized and assessed for allostery of the CB1 receptor. We found that within the structure of indole-2-carboxamides, the presence of the indole ring is preferred for maintaining the modulator's high binding affinity for the allosteric site but not for generating allostery on the orthosteric site. However, the C3 substituents of the indole-2-carboxamides significantly impact the allostery of the ligand. A robust CB1 allosteric modulator 5-chloro-N-(4-(dimethylamino)phenethyl)-3-pentyl-1H-indole-2-carboxamide (11j) was identified. It showed an equilibrium dissociation constant (KB) of 167.3 nM with a markedly high binding cooperativity factor (α = 16.55) and potent antagonism of agonist-induced GTPγS binding.

Distinct roles of ß-arrestin 1 and ß-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1).

The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070-12082). Here, we identify the mediators of CB1 receptor internalization and ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an ORG27569-induced signaling mechanism for CB1 wherein ß-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-ß-arrestin 1 upon ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of ß-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with ORG27569. These results demonstrate for the first time the involvement of ß-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two ß-arrestin isoforms in CB1 signaling and internalization.

Computationally-predicted CB1 cannabinoid receptor mutants show distinct patterns of salt-bridges that correlate with their level of constitutive activity reflected in G protein coupling levels, thermal stability, and ligand binding.

The cannabinoid receptor 1 (CB1), a member of the class A G-protein-coupled receptor (GPCR) family, possesses an observable level of constitutive activity. Its activation mechanism, however, has yet to be elucidated. Previously we discovered dramatic changes in CB1 activity due to single mutations; T3.46A, which made the receptor inactive, and T3.46I and L3.43A, which made it essentially fully constitutively active. Our subsequent prediction of the structures of these mutant receptors indicated that these changes in activity are explained in terms of the pattern of salt-bridges in the receptor region involving transmembrane domains 2, 3, 5, and 6. Here we identified key salt-bridges, R2.37 + D6.30 and D2.63 + K3.28, critical for CB1 inactive and active states, respectively, and generated new mutant receptors that we predicted would change CB1 activity by either precluding or promoting these interactions. We find that breaking the R2.37 + D6.30 salt-bridge resulted in substantial increase in G-protein coupling activity and reduced thermal stability relative to the wild-type reflecting the changes in constitutive activity from inactive to active. In contrast, breaking the D2.63 + K3.28 salt-bridge produced the opposite profile suggesting this interaction is critical for the receptor activation. Thus, we demonstrate an excellent correlation with the predicted pattern of key salt-bridges and experimental levels of activity and conformational flexibility. These results are also consistent with the extended ternary complex model with respect to shifts in agonist and inverse agonist affinity and provide a powerful framework for understanding the molecular basis for the multiple stages of CB1 activation and that of other GPCRs in general.

Profiling two indole-2-carboxamides for allosteric modulation of the CB1 receptor.

Allosteric modulation of G-protein coupled receptors (GPCRs) represents a novel approach for fine-tuning GPCR functions. The cannabinoid CB1 receptor, a GPCR associated with the CNS, has been implicated in the treatment of drug addiction, pain, and appetite disorders. We report here the synthesis and pharmacological characterization of two indole-2-carboxamides:5-chloro-3-ethyl-1-methyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-a) and 5-chloro-3-pentyl-N-(4-(piperidin-1-yl)phenethyl)-1H-indole-2-carboxamide (ICAM-b). Although both ICAM-a and ICAM-b enhanced CP55, 940 binding, ICAM-b exhibited the strongest positive cooperativity thus far demonstrated for enhancing agonist binding to the CB1 receptor. Although it displayed negative modulatory effects on G-protein coupling to CB1, ICAM-b induced ß-arrestin-mediated downstream activation of extracellular signal-regulated kinase (ERK) signaling. These results indicate that this compound represents a novel class of CB1 ligands that produce biased signaling via CB1.

Molecular basis for dramatic changes in cannabinoid CB1 G protein-coupled receptor activation upon single and double point mutations.

There is considerable interest in determining the activation mechanism of G protein-coupled receptors (GPCRs), one of the most important types of proteins for intercellular signaling. Recently, it was demonstrated for the cannabinoid CB1 GPCR, that a single mutation T210A could make CB1 completely inactive whereas T210I makes it essentially constitutively active. To obtain an understanding of this dramatic dependence of activity on mutation, we used first-principles-based methods to predict the ensemble of low-energy seven-helix conformations for the wild-type (WT) and mutants (T210A and T210I). We find that the transmembrane (TM) helix packings depend markedly on these mutations, leading for T210A to both TM3+TM6 and TM2+TM6 salt-bridge couplings in the cytoplasmic face that explains the inactivity of this mutant. In contrast T210I has no such couplings across the receptor explaining the ease in activating this mutant. WT has just the TM3+TM6 coupling, known to be broken upon GPCR activation. To test this hypothesis on activity, we predicted double mutants that would convert the inactive mutant to normal activity and then confirmed this experimentally. This CB1 activation mechanism, or one similar to it, is expected to play a role in other constitutively active GPCRs as well.

Allosteric modulator ORG27569 induces CB1 cannabinoid receptor high affinity agonist binding state, receptor internalization, and Gi protein-independent ERK1/2 kinase activation.

The cannabinoid receptor 1 (CB1), a member of the class A G protein-coupled receptor family, is expressed in brain tissue where agonist stimulation primarily activates the pertussis toxin-sensitive inhibitory G protein (G(i)). Ligands such as CP55940 ((1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3- hydroxypropyl)cyclohexan-1-ol) and Δ(9)-tetrahydrocannabinol are orthosteric agonists for the receptor, bind the conventional binding pocket, and trigger G(i)-mediated effects including inhibition of adenylate cyclase. ORG27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)ethyl]amide) has been identified as an allosteric modulator that displays positive cooperativity for CP55940 binding to CB1 yet acts as an antagonist of G protein coupling. To examine this apparent conundrum, we used the wild-type CB1 and two mutants, T210A and T210I (D'Antona, A. M., Ahn, K. H., and Kendall, D. A. (2006) Biochemistry 45, 5606-5617), which collectively cover a spectrum of receptor states from inactive to partially active to more fully constitutively active. Using these receptors, we demonstrated that ORG27569 induces a CB1 receptor state that is characterized by enhanced agonist affinity and decreased inverse agonist affinity consistent with an active conformation. Also consistent with this conformation, the impact of ORG27569 binding was most dramatic on the inactive T210A receptor and less pronounced on the already active T210I receptor. Although ORG27569 antagonized CP55940-induced guanosine 5'-3-O-(thio)triphosphate binding, which is indicative of G protein coupling inhibition in a concentration-dependent manner, the ORG27569-induced conformational change of the CB1 receptor led to cellular internalization and downstream activation of ERK signaling, providing the first case of allosteric ligand-biased signaling via CB1. ORG27569-induced ERK phosphorylation persisted even after pertussis toxin treatment to abrogate G(i) and occurs in HEK293 and neuronal cells.

The cannabinoid type-1 receptor carboxyl-terminus, more than just a tail.

The cannabinoid type-1 (CB(1)) receptor is a G protein-coupled receptor that binds the main active ingredient of marijuana, Δ(9)-tetrahydrocannabinol, and has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. In the two decades since the discovery of CB(1), studies at the molecular level have centered on the transmembrane core. This interest has now expanded as we discover that other regions of CB(1), including the CB(1) carboxyl-terminus, have critical structures that are important for CB(1) activity and regulation. Following the recent description of the three dimensional structure of the full-length CB(1) carboxyl-terminal tail [Biopolymers (2009) vol. 91, pp. 565-573], several residues and structural motifs including two α-helices (termed H8 and H9) have been postulated to interact with common G protein-coupled receptor accessory proteins, such as G-proteins and ß-arrestins. This discourse will focus on the CB(1) carboxyl-terminus; our current understanding of the structural features of this region, evidence for its interaction with proteins, and the impact of structure on the binding and regulatory function of CB(1) accessory proteins. The involvement of the carboxyl-terminus in the receptor life cycle including activation, desensitization, and internalization will be highlighted.

Ligand Binding Sensitivity of the Extracellular Loop Two of the Cannabinoid Receptor 1.

The cannabinoid receptor one (CB1) is a class A G-protein-coupled receptor thought to bind ligands primarily within its helical bundle. Evidence suggests, however, that the extracellular domain may also play a role. We have previously shown that the C-terminus of the extracellular loop 2 of CB1 is important in binding some compounds; receptors with mutations in this region (F268W, P269A, H270A, and I271A) bound some agonists with severely reduced affinity relative to the wild-type receptor. In the present work, we examine the impact of these mutations on binding a chemically diverse set of ligands. The receptors, F268W and I271A, exhibited a greater sensitivity to binding the inverse agonists/antagonists SLV319, AVE1625, NESS0327 relative to P269A and H270A, suggesting that the Pro and His are not involved in binding those compounds. In contrast, binding of the agonists, BAY593074 and WIN55212-2, was diminished in all four receptors, suggesting the conformational unit contributed by all four residues is important. A more marked loss in binding was observed for agonists of the nonclassical (CP55940) and classical (HU-210, JWH061, JWH179) cannabinoid classes and for a silent antagonist derivative (O-2050), pointing to the critical nature of this region for binding both the bicyclic/tricyclic core and the alkyl chain of these derivatives. However, moving the location of the alkyl chain on a series of pyrazole analogues shows it can be better accommodated in certain locations (O-1255) than others (O-1302, O-1690) and underscores the involvement of residues F268 and I271.

Hydrophobic residues in helix 8 of cannabinoid receptor 1 are critical for structural and functional properties.

In addition to the heptahelical transmembrane domain shared by all G protein-coupled receptors (GPCRs), many class A GPCRs adopt a helical domain, termed helix 8, in the membrane-proximal region of the C terminus. We investigated the role of residues in the hydrophobic and hydrophilic faces of amphiphilic helix 8 of human cannabinoid receptor 1 (CB1). To differentiate between a role for specific residues and global features, we made two key mutants: one involving replacement of the highly hydrophobic groups, Leu404, Phe408, and Phe412, all with alanine and the second involving substitution of the basic residues, Lys402, Arg405, and Arg409, all with the neutral glutamine. The former showed a very low B(max) based on binding isotherms, a minimal E(max) based on GTPgammaS binding analysis, and defective localization relative to the wild-type CB1 receptor as revealed by confocal microscopy. However, the latter mutant and the wild-type receptors were indistinguishable. Circular dichroism spectroscopy of purified peptides with corresponding sequences indicated that the highly hydrophobic residues are critical for maintaining a strong helical structure in detergent, whereas the positively charged residues are not. Further investigation of mutant receptors revealed that CB1 localization requires a threshold level of hydrophobicity but not specific amino acids. Moreover, mutant receptors carrying two- to six-residue insertions amino-terminal to helix 8 revealed a graded decrease in B(max) values. Our results identify the key helix 8 components (including hydrophobicity of specific residues, structure, and location relative to TM7) determinant for receptor localization leading to robust ligand binding and G protein activation.

Dual role of the second extracellular loop of the cannabinoid receptor 1: ligand binding and receptor localization.

The seven transmembrane alpha-helices of G protein-coupled receptors (GPCRs) are the hallmark of this superfamily. Intrahelical interactions are critical to receptor assembly and, for the GPCR subclass that binds small molecules, ligand binding. Most research has focused on identifying the ligand binding pocket within the helical bundle, whereas the role of the extracellular loops remains undefined. Molecular modeling of the cannabinoid receptor 1 (CB1) extracellular loop 2 (EC2), however, suggests that EC2 is poised for key interactions. To test this possibility, we employed alanine scanning mutagenesis of CB1 EC2 and identified two distinct regions critical for ligand binding, G protein coupling activity, and receptor trafficking. Receptors with mutations in the N terminus of EC2 (W255A, N256A) were retained in the endoplasmic reticulum and did not bind the agonist (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940) or the inverse agonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). In contrast, the C terminus of EC2 differentiates agonist and inverse agonist; the P269A, H270A, and I271A receptors exhibited diminished binding for several agonists but bound inverse agonists SR141716A, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and 4-[6-methoxy-2-(4-methoxyphenyl)benzofuran-3-carbonyl]benzonitrile (LY320135) with wild-type receptor affinity. The F268A receptor involving substitution in the Cys-X-X-X-Ar motif, displayed both impaired localization and ligand binding. Other amino acid substitutions at position 268 revealed that highly hydrophobic residues are required to accomplish both functions. It is noteworthy that a F268W receptor was trafficked to the cell surface yet displayed differential binding preference for inverse agonists comparable with the P269A, H270A, and I271A receptors. The findings are consistent with a dual role for EC2 in stabilizing receptor assembly and in ligand binding.

Structural analysis of the human cannabinoid receptor one carboxyl-terminus identifies two amphipathic helices.

Recent research has implicated the C-terminus of G-protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C-tail has proven a formidable task. Here, a peptide corresponding to the full-length C-tail of the human CB1 receptor (residues 400-472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an alpha-helical conformation in negatively charged and zwitterionic detergents (48-51% and 36-38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self-association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled ((15)N- and (13)C-) C-terminus in dodecylphosphocholine (DPC) identified two amphipathic alpha-helical domains. The first domain, S401-F412, corresponds to the helix 8 common to G protein-coupled receptors while the second domain, A440-M461, is a newly identified structural motif in the distal region of the carboxyl-terminus of the receptor. Molecular modeling of the C-tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions.

A cannabinoid receptor 1 mutation proximal to the DRY motif results in constitutive activity and reveals intramolecular interactions involved in receptor activation.

Activation of a G-protein-coupled receptor involves changes in specific microdomain interactions within the transmembrane region of the receptor. Here, we have focused on the role of L207, proximal to the DRY motif of the human cannabinoid receptor 1 in the interconversion of the receptor resting and active states. Ligand binding analysis of the mutant receptor L207A revealed an enhanced affinity for agonists (three- to six-fold) and a diminished affinity for inverse agonists (19- to 35-fold) compared to the wild-type receptor, properties characteristic of constitutive activity. To further examine whether this mutant adopts a ligand-independent, active form, treatment with GTPgammaS was used to inhibit G protein coupling. Under these conditions, the L207A receptor exhibited a 10-fold increase in affinity for the inverse agonist SR141716A, consistent with a shift away from an enhanced precoupled state. Analysis of the cellular activity of the L207A receptor showed elevated basal cyclic AMP accumulation relative to the wild type that is inhibited by SR141716A, consistent with receptor-mediated Gs precoupling. Using toxins to selectively abrogate Gs or Gi coupling, we found that CP55940 nonetheless induced only a Gi response suggesting a strong preference of this ligand-bound form for Gi in this system. Molecular dynamics simulations reveal that the single residue change of L207A impacts the association of TM3 and TM6 in the receptor by altering hydrophobic interactions involving L207, the salt bridge involving the Arg of the DRY motif, and the helical structure of TM6, consistent with events leading to activation. The structural alterations parallel those observed in models of a mutant CB(1) receptor T210I, with established constitutive activity (D'Antona, A.M., Ahn, K.H. and Kendall, D.A., 2006. Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization. Biochemistry, 45, 5606-5617).

Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization.

Human cannabinoid receptor 1 (CB(1)) has attracted substantial interest as a potential therapeutic target for treating obesity and other obsessive disorders. An understanding of the mechanism governing the transition of the CB(1) receptor between its inactive and active states is critical for understanding how therapeutics can selectively regulate receptor activity. We have examined the importance of the Thr at position 210 in CB(1) in this transition, a residue predicted to be on the same face of the helix as the Arg of the DRY motif highly conserved in the G protein-coupled receptor superfamily. This Thr was substituted with Ile and Ala via mutagenesis, and the receptors, T210I and T210A, were expressed in HEK 293 cells. The T210I receptor exhibited enhanced agonist and diminished inverse agonist affinity relative to the wild type, consistent with a shift toward the active form. However, treatment with GTPgammaS to inhibit G protein coupling diminished the affinity change for the inverse agonist SR141716A. The decreased thermal stability of the T210I receptor and increased level of internalization of a T210I receptor-GFP chimera were also observed, consistent with constitutive activity. In contrast, the T210A receptor exhibited the opposite profile: diminished agonist and enhanced inverse agonist affinity. The T210A receptor was found to be more thermally stable than the wild type, and high levels of a T210A receptor-GFP chimera were localized to the cell surface as predicted for an inactive receptor form. These results suggest that T210 plays a key role in governing the transition between inactive and active CB(1) receptor states.