Marijuana is one of the most widely used drugs of abuse, and is increasingly being ascribed therapeutic properties. Unfortunately, high-resolution structural knowledge about the marijuana receptor, called CB1, is limited. Such knowledge would aid in the development of compounds to better treat addiction, chronic pain and glaucoma. A major obstacle for obtaining CB1 structural information is the fact that CB1 is a membrane protein and thus difficult to obtain in quantities sufficient for traditional structural methods. Furthermore, like most other members of the G-protein coupled receptor family, CB1 is refractory to traditional purification procedures.
In Aim I of this CEBRA proposal, we will establish conditions for expressing, solubilizing and purifying large amounts of CB1 to set the stage for crystallization studies. On a parallel track, in Aim II we will express, purify and crystallize two separate CB1 domains, the long CB1 N-terminus, and the transmembrane ligand-binding domain of a truncated form of CBI. Finally, in Aim III we will assess the function, structure and dynamics of extracellular loop E-2 in CBI. The goal of these experiments is to test the emerging hypothesis that this loop plays a universally important role in ligand binding kinetics and receptor stability in GPCRs.

National Institute of Health (NIH)
National Institute on Drug Abuse (NIDA)
Research Project (R01)
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Molecular Neuropharmacology and Signaling Study Section (MNPS)
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Rapaka, Rao
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Oregon Health and Science University
Schools of Medicine
United States
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Fay, Jonathan F; Farrens, David L (2013) The membrane proximal region of the cannabinoid receptor CB1 N-terminus can allosterically modulate ligand affinity. Biochemistry 52:8286-94
Fay, Jonathan F; Farrens, David L (2012) A key agonist-induced conformational change in the cannabinoid receptor CB1 is blocked by the allosteric ligand Org 27569. J Biol Chem 287:33873-82
Mansoor, Steven E; Dewitt, Mark A; Farrens, David L (2010) Distance mapping in proteins using fluorescence spectroscopy: the tryptophan-induced quenching (TrIQ) method. Biochemistry 49:9722-31
Farrens, David L (2010) What site-directed labeling studies tell us about the mechanism of rhodopsin activation and G-protein binding. Photochem Photobiol Sci 9:1466-74
Tsukamoto, Hisao; Sinha, Abhinav; DeWitt, Mark et al. (2010) Monomeric rhodopsin is the minimal functional unit required for arrestin binding. J Mol Biol 399:501-11
Tsukamoto, Hisao; Farrens, David L; Koyanagi, Mitsumasa et al. (2009) The magnitude of the light-induced conformational change in different rhodopsins correlates with their ability to activate G proteins. J Biol Chem 284:20676-83
DeWitt, Mark A; Kliegman, Joseph I; Helmann, John D et al. (2007) The conformations of the manganese transport regulator of Bacillus subtilis in its metal-free state. J Mol Biol 365:1257-65
Sommer, Martha E; Farrens, David L; McDowell, J Hugh et al. (2007) Dynamics of arrestin-rhodopsin interactions: loop movement is involved in arrestin activation and receptor binding. J Biol Chem 282:25560-8
Sommer, Martha E; Farrens, David L (2006) Arrestin can act as a regulator of rhodopsin photochemistry. Vision Res 46:4532-46
Fay, Jonathan F; Dunham, Thomas D; Farrens, David L (2005) Cysteine residues in the human cannabinoid receptor: only C257 and C264 are required for a functional receptor, and steric bulk at C386 impairs antagonist SR141716A binding. Biochemistry 44:8757-69