Title

CB1 cannabinoid receptor: Structural studies using receptor purification and covalent ligands

Date of Completion

January 2006

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The CB1 cannabinoid receptor, a member of the family A G-protein coupled receptors, is recognized as a therapeutic target for the potential treatment of numerous diseases and is currently receiving considerable attention from the pharmaceutical community. The different classes of CB1 selective ligands have quite diverse structures and yet all interact with the same receptor protein. The detailed three-dimensional structure of cannabinoid receptor 1 (CB1) has yet to be determined. This information, however, is crucial for the development of medications. In order to elucidate the structural characteristics of the CB1 cannabinoid receptor, the first step is to obtain purified receptor protein in sufficient quantities. This has proven to be notoriously difficult for this class of membrane bound proteins. ^ In this study, we discuss our successes in the expression of the CB1 cannabinoid receptor at high levels, the purification of the receptor and whole sequence coverage by mass spectrometry (Section 2). The purified receptor was utilized in experiments aimed at elucidating structural characteristics of CB1 that regulate ligand binding and efficacy of classical cannabinoids (Section 3) and endocannabinoids (Section 4). The ligand-based strategy that we have developed to study these aspects of ligand binding and receptor function utilizes mass spectrometric analysis, site mutagenesis studies, saturation binding assays and functional assays. ^ Using these techniques we found that AM841, a covalent classical cannabinoid, could activate the receptor more efficiently than its noncovalent analogs that have similar reversible-binding affinity for the CB1 receptor. The information presented in this thesis will assist in constructing improved CB1 receptor computer models and further the understanding of the structure and functions of the CB1 receptor, and will contribute to the development of more effective cannabinergic medicines using target-based drug design to provide therapeutic benefits.^