Health & Environmental Research Online (HERO)


Print Feedback Export to File
540944 
Journal Article 
Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor 
Zvonok, N; Yaddanapudi, S; Williams, J; Dai, SJ; Dong, KL; Rejtar, T; Karger, BL; Makriyannis, A 
2007 
Yes 
Journal of Proteome Research
ISSN: 1535-3893
EISSN: 1535-3907 
6 
6 
2068-2079 
English 
The CB1 and CB2 cannabinoid receptors belong to the GPCR superfamily and are associated with a variety of physiological and pathophysiological processes. Both receptors, with several lead compounds at different phases of development, are potentially useful targets for drug discovery. For this reason, fully elucidating the structural features of these membrane-associated proteins would be extremely valuable in designing more selective, novel therapeutic drug molecules. As a first step toward obtaining information on the structural features of the drug-receptor complex, we describe the full mass spectrometric ( MS) analysis of the recombinant human cannabinoid CB2 receptor. This first complete proteomic characterization of a GPCR protein beyond rhodopsin was accomplished by a combination of several LC/MS approaches involving nanocapillary liquid chromatography, coupled with either a quadrupole-linear ion trap or linear ion trap-FTICR mass spectrometer. The CB2 receptor, with incorporated N-terminal FLAG and C-terminal HIS6 epitope tags, was functionally expressed in baculovirus cells and purified using a single step of anti-FLAG M2 affinity chromatography. To overcome the difficulties involved with in-gel digestion, due to the highly hydrophobic nature of this membrane-associated protein, we conducted in-solution trypsin and chymotrypsin digestions of purified and desalted samples in the presence of a low concentration of CYMAL5. This was followed by nanoLC peptide separation and analysis using a nanospray ESI source operated in the positive mode. The results can be reported confidently, based on the overlapping sequence data obtained using the highly mass accurate LTQ-FT and the 4000 Q-Trap mass spectrometers. Both instruments gave very similar patterns of identified peptides, with full coverage of all transmembrane helices, resulting in the complete characterization of the cannabinoid CB2 receptor. Mass spectrometric identification of all amino acid residues in the cannabinoid CB2 receptor is a key step toward the "Ligand Based Structural Biology" approach developed in our laboratory for characterizing ligand binding sites in GPCRs using a variety of covalent cannabinergic ligands. 
cannabinoid receptor 2; CB2; G protein-coupled receptor; GPCR; protein; expression; FLAG affinity purification; membrane protein; in-solution; protein digestion; mass spectrometry; peptide mapping; proteomic; analysis; LTQ-FT; Q-Trap; integral membrane-proteins; escherichia-coli; spectroscopic analysis; baculovirus system; coupled receptors; pichia-pastoris; opioid; receptor; affinity tags; purification; expression