Ligand-binding Site Characterization of Cell-free Expressed Human Cannabinoid 1 Receptor Using Mass Spectroscopy

Abstract

Cannabinoid 1 receptor (CB1), a type A G-protein coupled receptor (GPCR), modulates a number of key neurotransmitter pathways including the GABAnergic and dopaminergic systems. Therefore, drugs acting on the CB1 receptor are considered to have enormous pharmacotherapeutic potential in a number of disease conditions. In order to develop high-affinity, selective CB1 receptor ligands, an understanding of the structural organization of the ligand-binding site in the receptorÍs native state is essential. æWe used an E. coli-based, cell-free expression system in combination with Nanodiscs as membrane-mimics for the expression of human CB1 æreceptor (hCB1R). The hCB1R was engineered by a deletion of 102 amino acids at N-terminus and insertion of T4 lysozyme in the intracellular loop3, to improve stability, and addition of a hexa-histidine tag to the C-terminus to assist in purification. Using a novel, modified, radioligand binding assay, the receptor-nanodiscs complex was shown to be functional with an yield of about 500 ng of functional receptor per ml of expression reaction. A cannabinergic ligand (AM841) that covalently binds to C6.47(355) in helix 6 of CB1 was selected as a probe for receptor modification and the following Mass Spectometric (MS) analysis. A MALDI-TOF MS analysis of the trypsin-digested purified receptor treated with AM841, identified a peptide – MDIRLAKTLVLILVVLIIC(AM841)WGPLLAIMVYDVFGKMNK that corresponded to transmembrane helix6, confirming previously published indirect studies. ææThese experiments provide a new approach for the rapid and robust expression of functional CB1 receptors for ligand-assisted cannabinoid receptor binding site(s) characterization studies using different cannabinergic ligands. (Supported by NIH/NIDA Grants DA3801, DA9152.)