The goal of this project is to define the cannabinoid receptor ligand binding site(s). A combination of molecular and biochemical approaches which takes advantage of the expertise of the principal investigators and which contributes in a complementary way to the overall program project will be employed. Clones of the cannabinoid receptor in modified form, and a totally synthetic gene containing silent changes in the DNA to generate restriction site patterns appropriate for manipulation, will be used for expression. Chimeric receptors, constructed by replacement of large segments of the receptor with native or with idealized transmembrane sequences, will allow for screening of the entire molecule and will highlight regions sensitive to ligand interaction. These chimeric proteins will be designed to maintain structural integrity yet reduce sequence homology with potentially key residues of the native receptor. Site-specific mutations, then, will be generated using cassette mutagenesis. These mutations will involve one or a few amino acid replacements to specifically map the functional groups involved in ligand binding and to elucidate the nature of the interaction. Rapid genetic manipulations and screening will be effected using E. coli. Vectors, then will be designed for shuttling of clones between E. coli and baculovirus. The baculovirus system will be employed for expression of the receptor in the context of a lipid bilayer within the intact cell thus optimizing maintenance of receptor structural and conformational integrity. Preparative amounts of wild-type or mutant receptor using both expression systems will be generated for characterization of the receptor active site(s) utilizing high-affinity ligands in conjunction with cross-linking experiments to identify ligand receptor interactions. The basic biochemical and biophysical properties of the recombinant cannabinoid receptor will be defined. The consequences of mutagenesis on cellular compartmentation of the baculovirus-expressed receptor will be examined using anti-receptor antibodies. The relative molecular weight, isoelectric point, hydrophobicity, and ligand binding potential of the expressed receptor will be determined. Definition of these basic properties of the expressed receptor will allow for the design of preparative procedures for the isolation of the receptor under non- denaturation conditions, will serve as a basis for identification of which recombinant constructs can be employed for expression following global and site-specific mutagenesis, and will allow for comparison of the properties of the recombinant receptor to those of the native receptor in brain.
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