Structure of the Human Cannabinoid Receptor

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DESCRIPTION (provided by applicant): The main objective of this proposal is to learn about the structure of the helical bundle, which makes up the integral membrane portion and the ligand-binding site of the human cannabinoid receptor (CB1). Understanding the helix-helix interactions within the bundle will help to elucidate the residues involved in the ligand-binding pocket, information that is crucial for further development of therapeutics. The aims of the proposal are to identify transmembrane domains that drive the assembly of CB1 from fragments, and to identify helical regions involved in these interactions. Receptors will be generated that incorporate sites for recognition by specific proteases, expressed in HEK 293 cells and proteolytically cleaved to produce large fragments. Retention of the native form will be determined by ligand binding and G protein activation assays and will serve to identify stabilizing interhelical associations between the fragments. The interacting surfaces will be more precisely mapped via cysteine disulfide crosslinking. These studies will improve our understanding of how interacting transmembrane segments are arranged in the helical bundle in order to orchestrate receptor assembly, ligand binding and receptor activation. This information can then be applied to the development of selective therapeutics and drugs to antagonize the ill effects of marijuana.