DESIGN, EXPRESSION AND PURIFICATION OF FUNCTIONAL HUMAN CANNABINOID RECEPTOR 2

Cannabinoid receptors (CB1 and CB2) are GPCRs that belong to the rhodopsin-like family. Recent studies have demonstrated a role for CB2 in attenuating bone cancer-induced pain, reducing microglial activation in Alzheimer’s disease, and regulating bone mass in osteoporosis. As such, there is an urgent need for the development of new therapeutic agents targeting CB2. However, structure- or fragment-based in silico drug design is hampered by the absence of 3D structural information on CB2, because of its inherent structural complexity.\ud Recently, it has been shown that specific residues of CB2 (in transmembrane helices III, V, VII and extracellular loop 2) play crucial roles in the binding of the vast majority of cannabinoid ligands. In this report, we performed computer modeling along with site-directed mutagenesis studies and the results suggest that four residues that lie within these regions have a novel importance for receptor recognition of compound XIE95-26, a discovered CB2 inverse agonist in our lab, and known cannabinoid ligands CP-55940 (agonist) and SR144528 (inverse agonist). These mutant CB2 receptors were characterized by western blot, and ligand binding studies. Moreover, functional assays were performed for some mutant CB2 receptor to illustrate the role of altered residues in downstream signaling. Next, we’ve taken further steps to provide a genetically modified functional CB2 for biophysical and biochemical studies with the goal of improving the current purification methodology for the production of chimeric CB2 fusion proteins from the baculovirus system. By introducing a fusion protein and rational modifications to the chimeric receptor, investigators endeavoring to purify other GPCRs have overcome the challenge of obtaining sufficient stable protein for structural studies. Thus, different generations of truncated CB2 receptor were obtained to assess any improvements in receptor expression. Receptor expression and ligand binding capacity were assessed as criteria for pilot scale purification and for detergent selection. The best constructs were subjected to the optimal purification scheme using immobilized metal affinity, anti-M2 affinity, and size exclusion chromatography steps.\ud Collectively, results from this study will provide insights into the structure and functional mechanisms of CB2 and facilitate the design of small molecules that influence CB2 signaling behavior.\u