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Vol. 55, Issue 3, 605-613, March 1999
Departments of Pharmacology and Toxicology, and
Microbiology and
Immunology, Virginia Commonwealth University, Richmond Virginia
(Q.T., S.D.M., J.A., K.W.N., G.A.C., M.E.A.); and
Department of
Chemistry, Kennesaw State University, Kennesaw, Georgia (D.P.H.,
K.B., M.C.E., P.H.R.)
The human cannabinoid receptors, central cannabinoid receptor
(CB1) and peripheral cannabinoid receptor
(CB2), share only 44% amino acid identity overall, yet
most ligands do not discriminate between receptor subtypes.
Site-directed mutagenesis was employed as a means of mapping the ligand
recognition site for the human CB2 cannabinoid receptor. A
lysine residue in the third transmembrane domain of the CB2
receptor (K109), which is conserved between the CB1 and
CB2 receptors, was mutated to alanine or arginine to
determine the role of this charged amino acid in receptor function. The
analogous mutation in the CB1 receptor (K192A) was found to be crucial for recognition of several cannabinoid compounds excluding (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN 55,212-2). In contrast, in human embryonic kidney (HEK)-293 cells
expressing the mutant or wild-type CB2 receptors, we found no significant differences in either the binding profile of several cannabinoid ligands nor in inhibition of cAMP accumulation. We identified a high-affinity site for
(
)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP-55,940) in the region of helices 3, 6, and
7, with S3.31(112), T3.35(116), and N7.49(295) in the K109A mutant
using molecular modeling. The serine residue, unique to the
CB2 receptor, was then mutated to glycine in the K109A
mutant. This double mutant, K109AS112G, retains the ability to bind
aminoalkylindoles but loses affinity for classical cannabinoids, as
predicted by the molecular model. Distinct cellular localization of the
mutant receptors observed with immunofluorescence also suggests
differences in receptor function. In summary, we identified amino acid
residues in the CB2 receptor that could lead to subtype specificity.
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