Received for publication October 1, 2004.
Revised April 18, 2005.
Accepted for publication April 19, 2005.

CYSTEINE 2.59(89) IN THE SECOND TRANSMEMBRANE DOMAIN OF HUMAN CB2 RECEPTOR IS ACCESSIBLE WITHIN THE LIGAND BINDING CREVICE: EVIDENCE FOR POSSIBLE CB2 DEVIATION FROM A RHODOPSIN TEMPLATE

Abstract

ABSTRACT In this study, the sensitivity of the CB2 receptor to methanethiosulfonate (MTS) derivatives was tested and a native cysteine residue conferring the sensitivity was identified. By incubating HEK293 cells stably transfected with CB2 receptors and MTS derivatives such as MTS ethylammonium (MTSEA), [³H]-HU-243 binding was inhibited. Pretreatment of the CB2 receptor with cannabinoid ligands prevented this inhibition, suggesting that MTSEA modification occurred within the binding crevice. In order to identify the cysteine(s) responsible for the MTSEA sensitivity, ten CB2 mutants were prepared, in which the eight cysteines in transmembrane domains or extracellular loop 2 were mutated to serine or alanine, one at a time or in combination. Five mutants exhibited specific [³H]-HU-243 binding, with K_d and B_max values similar to wild-type CB2. However, five other mutants had no detectable ligand binding and were not detected on cell membranes by Western blot analysis. Among the five mutants with normal binding, only the C2.59(89)S mutant's sensitivity to MTSEA was reduced significantly. These data demonstrate that C2.59(89) is the residue mainly conferring the inhibitory effect of MTSEA on ligand binding. Further, the magnitude of the second-order rate constant (1.14±0.28 M^-1s^-1)for the MTSEA reaction with wild-type CB2 suggests that C2.59(89) resides at the margin of the CB2 binding site crevice. The accessibility of C2.59(89) to MTSEA provides experimental evidence for a possible conformational difference between TMH2 of CB2 versus Rho. Modeling studies undertaken to explore the origin of such differences suggest it is possibly due to the conformational influence of S2.54(84).

Key words: Cannabinoid, Molecular dynamics, Structure-activity relationships and modeling, Receptor binding studies