Mapping the ligand binding site(s) of the cannabinoid receptor

The cannabinoids, including Δ9-tetrahydrocannabinol, the active component of marijuana, are a diverse group of hydrophobic compounds mediating a variety of psychoactive and other physiological effects. These compounds exert most of their effects by binding selectively to a membrane bound receptor. The cannabinoid receptor has been identified as a member of the G protein coupled receptor family based on its structural features and main biochemical characteristics. It has seven transmembrane domains and transmits extracellular signals across the plasma membrane through coupling to the heterotrimeric G proteins. Cannabinoids have been extensively studied in the fields of medicinal chemistry and physiology over the past two decades. However, the molecular mechanism of their actions remains largely unknown. ^ The primary goal of this thesis is to identify the active site of the cannabinoid receptor, and in turn to understand the molecular interactions of the receptor with its ligands. To achieve this goal, a recombinant system expressing the human cannabinoid receptor and methods to evaluate the activity of the receptor were developed. Subsequently, designed mutations of the cannabinoid receptor were used to probe the active site(s) of the receptor. These mutations were designed based on structural analysis, chemical properties of the ligands, and the subtype selectivity of the cannabinoid receptor. Using this approach, we were able to identify the molecular components essential for ligand binding, as a functional domain within the receptor, and separately, at the level of a single amino acid side chain. ^ Studies presented in this thesis provide the following information: (i) The chemical nature of lysine residue 192 of the cannabinoid receptor CB1 is one of the major determinants for the binding activity of CP 55,940, a bicyclic cannabinoid. (ii) Mutations of this residue also affect the ability of the receptor to couple to the Gi protein. (iii) The third transmembrane domain of the receptor plays a role in the subtype selectivity of the aminoalkylindoles. Furthermore, this work sets a foundation for the further development of biochemical and biophysical studies of the cannabinoid receptor.