This is a revised application for Mentored Research Scientist Development Award (DA#00355 K01). The proposed research is directed towards understanding the chemical nature and the tertiary structure of the active site(s) of cannabinoid receptors. To date two types of cannabinoid receptors have been identified and characterized. They are 1) CB1, the central (brain) cannabinoid receptor and 2) CB2, the peripheral (spleen) cannabinoid receptor. There are reports suggesting the existence of more receptor subtypes. Besides the discovery of specific receptors for cannabinoids, a number of important findings have been made in the area of cannabinoid research in the last decade or so. Some of these findings are: the discovery of endogenous cannabinoid, and the development of specific cannabinoid receptor antagonists. Despite this rapid progress, the molecular mechanism of action of cannabinoids still remains unknown. In this grant application, the PI proposes novel mono- and bi-functional covalent ligands to probe the active sites(s) of cannabinoid receptors. These probes incorporate either a classical cannabinoid (CC) or an arachidonylethanolamide (AEA) structure and contain reactive group(s) such as an azido (photoactivatable) and an isothiocyanato (electrophilic) group at strategic positions. This latter group is targeted towards the nucleophilic amino acids such as lysine and cysteine present at or near the active site(s) of the receptor. Indeed, recently a lysine residue (Lys192) in the third transmembrane helix has been shown to be important for binding of classical cannabinoids and anandamides. The analogs synthesized will be tested for their affinity for cannabinoid receptors and for their efficacy to label the receptor at or near the active site(s). A select group of promising probes will be radiolabeled with iodine-125. The labeled or radiolabeled receptor protein is then purified and subjected to sequence analysis and LC/MS to identify the amino acid residue(s) involved in the covalent bond formation. This approach will give valuable insights into the tertiary structure of the cannabinoid binding site, ligand orientation at the binding site and the identity of the amino acid residues important for binding. Such information is vital for understanding the molecular mechanism of action of cannabinoids as well as for the rational design of therapeutically useful cannabinoid-based drugs such as analgesics, anti-convulsants, anti-glaucoma and immunomodulatory agents.