Kwang H. Ahn

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

Background: CB1 is activated by agonist CP55940 in a G protein-dependent manner. Results: β-Arrestin 2 plays a role in CB1 internalization, whereas β-arrestin 1 is critical for ORG27569-induced ERK1/2, MEK1/2, and c-Src phosphorylation. Conclusion: Allosteric modulator ORG27569 endows CB1 with downstream signaling selectivity. Significance: This work discusses the first case of β-arrestin involvement in CB1-biased signaling. 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 β-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-β-arrestin 1 upon ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of β-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 β-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two β-arrestin isoforms in CB1 signaling and internalization.

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

The seven transmembrane α-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.

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 β-arrestin, a process defined as functional selectivity or ligand bias. However, the dynamic mechanisms underlying β-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 β-arrestins before endocytosis, as the mechanism controlling β-arrestin signalling. Agonists inducing short endocytic dwell times produce little or no β-arrestin signalling, whereas those eliciting prolonged dwell times induce robust signalling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased β-arrestin signalling. These studies reveal how receptor activation translates into β-arrestin signalling and identify a mechanism to control this pathway.

Mechanisms of Biased β-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 β-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 β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-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 β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-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 β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling.

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 modulators 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.

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 β-arrestin mediated ERK1/2 phosphorylation.

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.

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.

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 β‐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.

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 α‐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 (15N‐ and 13C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐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.