Background: ORG27569 is an allosteric modulator of CB1. Results: Although ORG27569 inhibits G protein coupling, it induces CB1 high affinity agonist binding, receptor internalization, and downstream ERK phosphorylation. Conclusion: The ORG27569-induced activation of ERK via CB1 is G i protein-independent. Significance: This is the first case of CB1 allosteric ligand-biased signaling.

Background: CB1 is activated by agonist CP55940 in a G protein-dependent manner. Results: ␤-Arrestin 2 plays a role in CB1 internalization, whereas ␤-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 ␤-arrestin involvement in CB1-biased signaling.

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The seven transmembrane ␣-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,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,, 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.The human cannabinoid receptor 1 (CB1) is a member of the rhodopsin-like G protein-coupled receptor (GPCR) family, members of which comprise seven transmembrane helices (TMs) connected by three intracellular loops and three extracellular loops. In addition to binding ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC), the psychoactive constituent of Cannabis sativa, several endogenous cannabinoid receptor agonists have been identified, including anandamide (arachidonoylethanolamide), 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether (noladin ether). CB1 also binds a variety of synthetic cannabimimetic compounds such as the nonclassic agonist CP55940, a bicyclic analog of ⌬ 9 -THC, the classic agonist HU-210, and the selective inverse agonists SR141716A (also known as rimonabant), AM251, and LY320135. Upon activation, CB1 modulates synaptic transmission, ultimately playing a role in analgesia, anxiety, feeding behavior, and movement disorders (Porter and Felder, 2001;Howlett et al., 2002). Thus, understanding the features of CB1 critical for ligand binding and receptor localization is important for developing its therapeutic potential.Molecular modeling analyses of ligand-receptor interactions involving CB1 are consistent with the idea that the N-(piperidin-1-yl)-5-(4-iodophenyl...

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G protein-coupled receptors (GPCRs) are major transducers of external stimuli and key therapeutic targets in many pathological conditions. When activated by different ligands, one receptor can elicit multiple signaling 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 signaling remain unknown. Here, by studying the cannabinoid 1 receptor (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 signaling. Agonists inducing short endocytic dwell times produce little or no β-arrestin signaling, whereas those eliciting prolonged dwell times induce robust signaling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased β-arrestin signaling. These studies reveal how receptor activation translates into β-arrestin signaling and identify a mechanism to control this pathway.

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 analogs 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.

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.