Ligand-binding architecture of human CB2 cannabinoid receptor: evidence for receptor subtype-specific binding motif and modeling GPCR activation

Abstract

The extensive physiological influence of transmission through the CB2 cannabinoid receptor makes this G protein-coupled receptor (GPCR) a promising therapeutic target for treating neuropathic pain, inflammation, and immune disorders. However, there is little direct structural information pertaining to either GPCR or CB2-receptor ligand recognition and activation. The present work helps characterize experimentally the ligand-binding interactions of the human CB2 (hCB2) receptor. This study illustrates how our overall experimental approach, "ligand-assisted protein structure" (LAPS), affords direct determination of the requirements for ligand binding to the hCB2 receptor and discrimination among the binding motifs for ligands that activate therapeutically relevant GPCRs.

Figure 1. Chemical Structures of Cannabi-nergic Ligands and Effect of AM-841 on WT and Mutant hCB2 Receptor Ligand Binding

(A) Chemical structures of representative cannabi-nergic ligands used in this study. (B and C) Preincubation with AM-841 eliminates [³H]-CP55940 binding to the WT hCB2 receptor and the hCB2 C7.38(284)S and hCB2 C7.42(288)S mutant receptors, but not to the hCB2 C6.47(257)A or hCB2 C6.47(257)S mutant receptors. Membranes prepared from HEK293 cells expressing either the WT or a mutant hCB2 receptor were preincubated with 9 nM (six-fold the K_i) AM-841 for 1hr at 30°C and then extensively washed to remove unbound, noncovalently associated ligand. The washed membranes were subjected to a saturation binding assay using [³H]-CP55940 as the radioligand. (B) Saturation-binding curves using [³H]-CP55940 for WT and mutant hCB2 receptors preincubated with AM-841 as described above and “control” membranes processed in parallel, but without prior exposure to AM-841. Data represent the means ± SEM of at least 2 independent experiments performed in duplicate. (C) Comparison of the difference in B_max values of each hCB2 receptor with or without preincubation with AM-841. Data shown represent the means ± SEM of at least 2 independent experiments performed in duplicate.

Figure 2. Schematic Representation of the hCB2 Receptor

Amino acids subjected to mutation in this study, C6.47(257), C7.38(284), and C7.42(288), are circled in bold. Alignment of the V6.43/I6.46 groove and the CWXP motif in the CB1 and CB2 receptors is highlighted.

Figure 3. Concentration-Dependent Inhibition of Forskolin-Stimulated cAMP Accumulation in HEK293 Cells Expressing hCB2 WT or Mutant Receptors by Various Agonists

(A) Comparison among AM-841, AM-4056, and WIN55212-2 to inhibit forskolin-stimulated cAMP accumulation in the HEK293 cells expressing WT hCB2 receptor. (B) Comparison of the ability of AM-841 to compete with forskolin-stimulated cAMP accumulation in HEK293 cells expressing either the WT hCB2 receptor or the hCB2 C6.47(257)A or C6.47(257)S mutant receptor.

Figure 4. Illustration of the CB2 R*/AM-841 Binding Site from Modeling Studies

TMHs 1, 4, and 5 have been omitted from this view for simplicity. In the energy-minimized CB2 R*/AM-841complex in which AM-841 is covalently attached to C6.47(257), the carbocyclic ring CH₂OH of AM-841 hydrogen-bonds with S7.39(285) (d = 2.62 Å; O – H- -O angle = 175°), while the phenolic hydroxyl of AM-841 hydrogen-bonds with S6.58(268) (d = 2.61 Å; O – H- -O angle = 176°). Also illustrated here is the formation of a salt bridge between D275 in EC3 and K3.28(109), a residue that is crucial for classical CB binding to the CB1 receptor (Song and Bonner, 1996), but which is not important for binding to the CB2 receptor. In the final, energy-minimized complex illustrated here, K3.28(109) is involved in a salt bridge with D275 of the EC3 loop (d = 2.55 Å; N – H- -O angle = 171°) and in a hydrogen bond with N2.63(93) (d = 2.66 Å; N – H- -N angle = 168°). D275 also forms a hydrogen bond with S274 in EC3 (d = 2.67 Å; O – H- -O angle = 170°) and with S2.60(90) (d = 2.63 Å; O – H- -O angle = 160°), a residue that is accessible from within the binding pocket in the CB2 receptor due to the helix distortion produced by S2.54(84) (Experimental Procedures and Zhang et al., 2005).