The endocannabinoid (eCB) signaling system helps regulate diverse physiological processes. The two principal cannabinoid (CB) receptors, designated CB1 and CB2, are class-A G protein-coupled receptors (GPCRs) stimulated by exogenous natural cannabinoids (e.g., the plant cannabinoid delta9-tetrahydrocannabinol) and eCBs including N-arachidonoyl ethanolamine (""""""""anandamide"""""""") (AEA) and 2-arachidonoylglycerol (2-AG). The tissue distribution and integrated activities of CB receptors and eCB biosynthetic and metabolizing enzymes are key to homeostatic eCB signaling by delimiting spatially and temporally eCB bioactivity. CB1 is predominantly found in the central nervous system, activation of which mediates most CB psychotropic and behavioral effects. At very low levels in brain, CB2 is expressed mainly in the periphery by immunocompetent and hematopoietic cells, osteoclasts, and osteoblasts and mediates immune responses, inflammation, inflammatory and neuropathic pain, and bone remodeling. eCBs are produced on-demand in response to various stimuli and are rapidly inactivated by enzymatic hydrolysis: AEA, primarily by fatty acid amide hydrolase (FAAH), and 2-AG, primarily by monoacylglycerol lipase (MGL). Changes in endocannabinoid signaling accompany various physiological and pathological processes. Hyperactive CB1 transmission has been implicated in a number of disease states including drug addiction, substance abuse disorders, overweight/obesity, and obesity-related cardiometabolic risk (metabolic syndrome). A CB1 antagonist has reached the market as a weight-loss agent, although associated in the clinic with adverse effects. Activation of CB2 by small-molecule agonists may hold therapeutic promise for pain and neuroinflammatory disorders (e.g., Alzheimer's and Huntington's diseases). Such translational applications make a thorough understanding of the mechanism of CB-receptor activation at the molecular level essential to drug discovery aimed at modulating CB receptor transmission for therapeutic gain. In the absence of their crystallographic structures, CB1 and CB2 homology modeling has been conducted based on the X-ray crystal structure of rhodopsin. The general architecture of class-A GPCRs has been characterized by an extracellularly oriented N-terminus, an intracellular carboxyl terminus, and a counterclockwise arrangement of seven hydrophobic transmembrane 1- helices spanning the cell membrane and connected by three extracellular and three cytoplasmic loops. By analogy with rhodopsin, activation of CB2 has been proposed to involve disruption of a salt bridge in transmembrane (TM) helix 3 as well as alterations in the conformation of TM helices 6 and 7. Rhodopsin also contains a cytoplasmic helix 8 (H8) which extends from TM helix 7, but its participation in the activation of other GPCRs, including CB2, is not well established. The applicant hypothesizes that helices 6, 7, and 8 are critical to CB2 activation. Experiments detailed in this proposal are designed to provide supporting experimental evidence that will define the structural changes these helices undergo upon CB2 activation by using a combination of CD and solution and solid-state NMR. Suitably labeled peptides representing TM helix 6 and TM helices 6-7/H8 will be expressed in E. coli, purified, and studied in solution and in defined lipid environments (micelles, phospholipid bilayers). A novel, high-affinity cannabinergic agonist (AM841) previously demonstrated to react specifically and covalently with CB2 cysteine 257 in TM helix 6 and activate the receptor will be used as affinity probe. Molecular modeling will be applied to augment the experimental results. The resulting data will form the basis for future work to elucidate the involvement of other CB2 helical domains in CB activation and inform the design of safe and effective CB2-selective agonists as drug candidates.

Public Health Relevance

The current proposal will provide structural and dynamic information on the transmembrane polypeptides of the human CB2 receptor. Knowledge of the specific orientations and precise distances between identified residues in contact with the ligand, as well as the conformation of the polypeptide-ligand complex, will be helpful in optimizing the binding properties and selection of ligands to the cannabinoid receptor. Therefore, the proposed work is expected to provide significant biomedical findings with therapeutic potential.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Small Research Grants (R03)
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Special Emphasis Panel (ZRG1-MDCN-C (91))
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Hillery, Paul
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Northeastern University
Schools of Pharmacy
United States
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