Since the discovery of cannabinoid (CB) receptors, cannabinoid research has witnessed rapid and important developments and renaissance. CB2 derived from human promyelicytic leukemia cell is a subtype of cannabinoid receptors. Unlike its closely related sub-type receptor CB1, which is believed to be responsible for the modulation of Q-type Ca2+ and inwardly rectifying K+ channels in CNS, CB2 receptor is expressed in high quantifies in human spleen and tonsils, and is likely to be involved in the signal transduction processes in immune system, Therefore, CB2 receptor can potentially be a target for immuno-treatments. Thus, knowledge of the 3D structure of CB receptors and the further understanding of ligand-receptor interaction will greatly aid in the rational design of specific CB2 ligands possessing potent therapeutic activities, but devoid from the undesirable side effects. However, its intrinsic membrane protein property makes it difficult to crystallize for x-ray study. Direct NMR study is also restricted due to the large protein size and slow correlation time, whereas NMR study of synthetic polypeptide is limited by available length of peptides, and low signal-to-noise (S/N) of natural abundant peptides. The objective of this proposal is to obtain the purified recombinant CB2 protein segments (transmembrane domains, or helix bundles) that are expressed in Escherichia coli (E. coli) (isotope-enriched media) for structural biology determination by NMR and computer modeling. Such studies will provide valuable experimental data to refine a 3D construct of CB2 receptor. In addition, the proposed studies will determine the structural and conformational information of the CB2 receptor segments that will shed light towards the understanding of receptor binding-activating-signaling mechanisms. Eventually, an experimental-based 3D CB2 structure will be more reliable for rational drug design. Overall, the method proposed here represents a novel combined approach of protein engineering, modem isotope aided NMR, and computer modeling for study of G-protein coupled transmembrane receptors (GPCRs) that is a large family of drug targets (approximately 45 percent of the market drugs). The work accomplished through this proposed research can potentially make a significant contribution to cannabinoid research and NMR structural biology, as well as GPCRs in general.
