Key Determinants of Cannabinoid Receptor One Ligand Binding and Recptor Activation

The cannabinoid receptor 1 (CB1) is a rhodopsin-like G-protein coupled receptor (GPCR) found predominantly in the central nervous system. CB1 regulation is of therapeutic importance as studies correlate receptor activation with alterations in mood, intraocular pressure, intestinal motility, and memory. CB1 agonists, inverse agonists, and antagonists have been developed, although their underlying interactions with the receptor determining their affinity, potency, and efficacy remain, in large part, unclear. This work helped characterize the ligand binding sites of various classes of cannabinoid ligands and domains of the receptor involved in activation. ^ Residues F268, P269, H270, and I271 at the carboxy-terminal end of the CB1 extracellular loop 2 (EC2) have been shown more important for agonist than inverse agonist binding. A variety of ligands were tested to bind receptors with individual mutations at these residues. The carboxy-terminal region of EC2 was determined to also be involved in antagonist binding. Moreover, agonist binding, especially those with long alkyl tails is most sensitive to mutations at F268 or I271. ^ Ligand binding identified CB1 residues F174, F177, L193, and M363 as being integral for CB1 agonist, HU210, binding. L193A and M363A mutant receptors had less of a loss in affinity for Δ9-THC which contains a pentyl alkyl tail, than CP55940 or HU210 which are similar in structure, though contain dimethyl heptyl tails. Molecular modeling in conjunction with the experimental data suggests L193 and M363 may form a hydrophobic pocket for the dimethyl heptyl tails found on some of the most potent cannabinoid agonists. ^ Most rhodopsin-like GPCRs have a transmembrane domain 2 (S/N)LxxxD motif. This study suggests the CB1 (S/N)LxxxD motif is important for receptor activation. Mutating CB1 L159, enhanced agonist affinity and reduced inverse agonist affinity, implying increased constitutive activity. Interestingly, this mutant CB1 L159A receptor was unable to activate inhibitory G-protein (G), the class of G-protein which CB1 predominantly couples to. This suggests the L159A mutation may promote an active state of the receptor which predominantly signals through stimulatory G-protein (Gs) or β-arrestin.