Purification &Structural Analysis/Cannabinoid Receptor

DESCRIPTION: (provided by the applicant) Structural information about the "marijuana receptor" is limited. This is unfortunate since marijuana is one of the most widely used street drugs, and is increasingly being ascribed therapeutic properties. More detailed knowledge about the structure of this receptor would greatly assist the design of both activating and inhibiting drugs, and facilitate treatment regimes. A major reason for the lack of structural information about the marijuana receptor (the neuronal cannabinoid receptor, called CB1 is the fact that CB1 is a G-protein coupled receptor (GPCR). The GPCR proteins are a large family of membrane receptor proteins that are involved in transmitting signals across membranes. Unfortunately, these proteins are usually expressed at low levels, and like most membrane proteins, are difficult to purify and manipulate. Aided in part by our experience purifying and carrying out physical studies of rhodopsin (the only GPCR for which a high resolution crystal structure is known) we propose to express and purify large amounts of the CB1 receptor. Access to large quantities of pure CB1 will allow us to try crystallizing CB1, and will also enable us to begin studying dynamic changes that may occur in the CB1 structure upon ligand binding. These latter studies will involve using fluorescence and EPR spectroscopy to determine if CB1 activation involves a conformational change at one of its transmembrane helices, helix F. Such a movement has previously been observed in rhodopsin and the beta-adrenergic receptor, and if detected in CB1, will suggest these disparate GPCRs may share a universal mechanism of activation.