Structural and functional characterization of the cannabinoid receptor one: Roles for two non-transmembrane domains, the second extracellular loop and carboxyl terminus

Date of Completion

January 2009


Chemistry, Biochemistry




The cannabinoid receptor one (CB1), a member of the rhodopsin-like G protein-coupled receptor (GPCR) family, has become an important therapeutic target for treating numerous neuropsychiatric and movement disorders. Thus, understanding the structural features of CB1 critical for receptor localization and ligand binding is important for developing its therapeutic potential. The heptahelical transmembrane domains shared by all GPCRs have been extensively studied to understand the mechanism of ligand interactions and receptor activation. However, there is growing evidence that the non-transmembrane domains, the extracellular- and intracellular-loops, and carboxyl terminus (C terminus), also play essential roles in receptor regulation and function. ^ This study focuses on two non-transmembrane domains within the CB1 receptor; the second extracellular loop (EC2) and C terminus. The individual amino acid residues of EC2 were evaluated for their role in ligand binding, G protein coupling activity, and receptor trafficking. The ligand binding data identifies two distinct regions in the N and C terminus of the loop that exhibit diminished agonist binding. Further investigation indicates that the residues in the N terminus play a role in its ER assembly for subsequent trafficking, whereas those in the C terminus of the loop differentiate agonist and inverse agonist binding. ^ To investigate the biological significance of the C terminus of CB1, a peptide corresponding to the entire C terminus was expressed, purified, and characterized using circular dichroism (CD) and nuclear magnetic resonate (NMR) spectroscopy. The structural information indicates high helical content (up to 50.6%) is induced in detergents and identifies two amphipathic helical domains, helix 8 and helix 9. Further analysis of helix 8 demonstrates a direct correlation between the presence of this helical motif including its location relative to the membrane, with localization of the receptor. CD spectroscopy suggests that the highly hydrophobic face of the helix is critical for its formation and that the helical unit, rather than particular amino acids, is responsible for receptor trafficking. The combination of secondary structural analysis, pharmacological evaluation, and global mutagenesis employed here has led to insights that should help clarify the wide variety of data reported for the role of helix 8 in other GPCRs. ^