Title

Molecular characterization of cannabinoid CB2 receptor-ligand interactions using species-selective probes

Date of Completion

January 2006

Keywords

Health Sciences, Pharmacology

Degree

Ph.D.

Abstract

Understanding the molecular interactions between receptor and ligand(s) is an essential component in drug design. The cannabinoid CB2 receptor, a G-protein coupled receptor, is an important target for therapeutics involving inflammation, pain, and immune system disorders. Studying receptor-ligand interactions of the CB2 receptor is the main focus of this dissertation. First, human embryonic kidney cells were transfected with either the human CB2 or mouse CB2 receptor and cell lines were established. Next, ligand-binding efficiencies of different classes of cannabinergic ligands were tested for each cell line. The non-classical cannabinoids, the cannabilactones, showed differential binding between the human and mouse CB2 cell lines; in fact, more than a 10-fold higher binding affinity at the mouse CB2 receptor compared to human CB2, resulting in species-selectivity. In addition, functional activity of the lead cannabilactone; AM1714, showed higher efficacy at the mouse CB2 receptor (measured by cAMP accumulation), in accordance to the binding profile of this ligand. To elucidate the amino acids responsible for species-selectivity, site-directed mutagenesis was applied to the human CB2 receptor at those specific amino acids which differ between the human and mouse receptors within the transmembrane helices (this strategy was also applied to the mouse CB2 receptor only for transmembrane helix 2). Next, binding affinities with the cannabilactone ligands was assessed. It was shown that mutating amino acid position 2:63(93) in both the human and mouse totally disrupts binding with all cannabinergic ligands tested, concluding that this position is critical for overall receptor function. Classical cannabinoid analog, AM4056 binding is improved when mouse CB2 S2.44(74) is mutated to a glycine, the corresponding amino acid in human CB2. This amino acid position is also critical for AM1714 binding in the mouse CB2 receptor because binding affinity was significantly decreased in the mouse CB2 S2.44(74)G, mutant, compared to mouse CB2 wild-type. Lastly, the human CB2 mutant, S2.60(90)N showed an increased binding affinity with cannabilactone analog, AM1950, suggesting a strengthened hydrogen-bonding. In conclusion, transmembrane helix 2 of the CB2 receptor demonstrates a role in CB2 species-selectivity. The molecular characterization of this receptor via site-directed mutagenesis & species-selective probes has proven to be a successful avenue in studying receptor-ligand interactions.^