3D Structural Model of the G-Protein-Coupled Cannabinoid CB 2 Receptor Xiang-Qun Xie, 1,2 * Jian-Zhong Chen 1 and Eric M. Billings 2 1 Institute of Materials Science; and Department of Pharmaceutical Science, School of Pharmacy; University of Connecticut, Storrs, Connecticut 2 Bioinformatics Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland ABSTRACT The potential for therapeutic speci- ficity in regulating diseases and for reduced side effects has made cannabinoid (CB) receptors one of the most important G-protein-coupled receptor (GPCR) targets for drug discovery. The cannabinoid (CB) receptor subtype CB2 is of particular interest due to its involvement in signal transduction in the immune system and its increased characterization by mutational and other studies. However, our un- derstanding of their mode of action has been limited by the absence of an experimental receptor struc- ture. In this study, we have developed a 3D model of the CB2 receptor based on the recent crystal struc- ture of a related GPCR, bovine rhodopsin. The model was developed using multiple sequence align- ment of homologous receptor sub-types in humans and mammals, and compared with other GPCRs. Alignments were analyzed with mutation scores, pairwise hydrophobicity profiles and Kyte-Doolittle plots. The 3D model of the transmembrane segment was generated by mapping the CB2 sequence onto the homologous residues of the rhodopsin struc- ture. The extra- and intracellular loop regions of the CB2 were generated by searching for homologous C  backbone sequences in published structures in the Brookhaven Protein Databank (PDB). Residue side chains were positioned through a combination of rotamer library searches, simulated annealing and minimization. Intermediate models of the 7TM helix bundles were analyzed in terms of helix tilt angles, hydrogen-bond networks, conserved resi- dues and motifs, possible disulfide bonds. The amphi- pathic cytoplasmic helix domain was also corre- lated with biological and site-directed mutagenesis data. Finally, the model receptor-binding cavity was characterized using solvent-accessible surface ap- proach. Proteins 2003;53:307–319. © 2003 Wiley-Liss, Inc. Key words: cannabinoid receptor subtype 2 (CB2); G-protein-coupled receptor (GPCR); 3D structure; homology; sequence align- ment; computer modeling INTRODUCTION Cannabinoid (CB) receptor subtype CB2 was expressed in high quantities in human spleen and tonsils, 1,2 and was identified as a member of the rhodopsin-like family of seven-transmembrane (7TM) G-protein-coupled receptors (GPCRs). The CB2 receptor has been identified as a potential target for therapeutic immune intervention as its involvements in signal transduction processes in the immune system. Knowledge of the 3D structure of CB receptors 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 studies are also restricted due to the large protein size and slow correlation time. So far, only one GPCR, bovine rhodopsin, has been obtained at high-resolution (2.8 Å) X-ray crystallographic structure. 3 Studies of the CB2 receptor to date have been limited by the lack of a three dimensional structure leaving many unanswered questions about the molecular level interactions between the receptor and its ligands as well as the nature of the receptor active site(s). 4 Biochemical, pharmacological and computational inves- tigations have provided insights into the cannabinoid receptors and their interaction with cannabimimetic li- gands. Shire et al. 5 first demonstrated that the residues important for CB2 subtype specificity were located some- where within the region of fourth transmembrane domain (TM4), extracellular loop 2 (e2) and fifth transmembrane domain (TM5) (TM4-e2-TM5). The same region was previ- Abbreviations: GPCR, G-protein-coupled receptor; CB1, cannabi- noid receptor subtype 1; CB2, cannabinoid receptor subtype 2; CNS, central nervous system; CD, circular dichroism; DMSO-d6, dimeth- yl-d 6 sulfoxide; DPC, dodecylphosphocholine; TM, transmembrane domain; BR, bacteriorhodopsin; e2, extracellular loop 2; CB2r_Human, human cannabinoid CB2 receptor of Homo sapiens; CB2r_mouse, mouse cannabinoid CB2 receptor of Mus musculus; CB1r_Felca, cat cannabinoid CB1 receptor of Felis silvestris catus; CB1r_Rat, rat cannabinoid CB1 receptor of Rattus norvegicus; CB1r _Human, hu- man cannabinoid CB1 receptor of Homo sapiens; 5H2A_Rat, rat 5- hydroxytryptamine 2a receptor of Rattus norvegicus; DADR_Rat, rat D(1A) dopamine receptor of Rattus norvegicus; A2AA_Rat, rat al- pha-2a adrenergic receptor of Rattus norvegicus; ACM1_Rat, rat muscarinic acetylcholine receptor m1 of Rattus norvegicus; SCR, structurally conserved regions; MM, molecular mechanic simulation; MD, molecular dynamic simulation; KD, Kyte-Doolittle method; NOE, nuclear Overhauser effect. Grant sponsor: National Institutes of Health; Grant numbers: DA11510 and DA15770. *Correspondence to: Xiang-Qun (Sean) Xie, U-3136, IMS, Univer- sity of Connecticut, Storrs, CT 06269-3136. E-mail: xie@uconn.edu. Received 31 October 2002; Accepted 12 May 2003 PROTEINS: Structure, Function, and Genetics 53:307–319 (2003) © 2003 WILEY-LISS, INC.