Mutagenesis and computer modeling studies of a GPCR conserved residue W5.43(194) in ligand recognition and signal transduction for CB2 receptor

Abstract

W5.43(194), a conserved tryptophan residue among G-protein coupled receptors (GPCRs) and cannabinoid receptors (CB), was examined in the present report for its significance in CB2 receptor ligand binding and adenylyl cyclase (AC) activity. Computer modeling postulates that this site in CB2 may be involved in the affinity of WIN55212-2 and SR144528 through aromatic contacts. In the present study, we reported that a CB2 receptor mutant, W5.43(194)Y, which had a tyrosine (Y) substitution for tryptophan (W), retained the binding affinity for CB agonist CP55940, but reduced binding affinity for CB2 agonist WIN55212-2 and inverse agonist SR144528 by 8-fold and 5-fold, respectively; the CB2 W5.43(194)F and W5.43(194)A mutations significantly affect the binding activities of CP55940, WIN55212-2 and SR144528. Furthermore, we found that agonist-mediated inhibition of the forskolin-induced cAMP production was dramatically diminished in the CB2 mutant W5.43(194)Y, whereas W5.43(194)F and W5.43(194)A mutants resulted in complete elimination of downstream signaling, suggesting that W5.43(194) was essential for the full activation of CB2. These results indicate that both aromatic interaction and hydrogen bonding are involved in ligand binding for the residue W5.43(194), and the mutations of this tryptophan site may affect the conformation of the ligand binding pocket and therefore control the active conformation of the wild type CB2 receptor. W5.43(194)Y/F/A mutations also displayed noticeable enhancement of the constitutive activation probably attributed to the receptor conformational changes resulted from the mutations.

Fig. 1. The expression of CB2 receptor mutants in transfected HEK293 cells was analyzed by Western blot

From left to right: CB2 WT (wild type), W194A, W194Y, W194F.

Fig. 2. Cannabinoid ligands (a. CP55940; b. WIN55212-2; c. SR144528) bind to CB2 receptor wild type (●) and mutant W194Y (□) using membranes prepared from transfected HEK293 cells as described

Data are mean ± S.E.M. of three independent experiments performed in duplicate or triplicate. Curves were generated as described in Materials and Methods.

Fig. 3. Comparison of CB2 WT and W194Y mutant receptors in agonist-induced inhibition of forskolin-stimulated cAMP accumulation

EC₅₀ values were 5.91±4.87 nM for CB2 WT and 45.48±5.86 nM for W194Y treated with CP55940 (a), 7.49±3.85 nM for CB2 WT and 21.79±1.65 nM for W194Y mutant treated with WIN55212-2 (b). Data are mean ± S.E.M. of three independent experiments in triplicate.

Fig. 4. Effect of cannabinoids on forskolin-stimulated cAMP accumulation in CB2 WT transfected HEK293 cells

HEK293 cells expressing CB2 WT was assayed for cAMP accumulation in response to a gradient concentrations forskolin (10-10⁵ nM) alone (○) and in the presence of 0.3 μM of CP55940 (▲), WIN55212-2 (△), or SR144528 (*). The results were expressed as % of the LANCE signal representing basal cAMP accumulation, measured in the absence of forskolin. Data shown represent the mean ± S.E.M. of at least three independent experiments performed in triplicate.

Fig. 5. Effect of CB2 inverse agonist SR144528 on constitutive activation of CB2 WT and mutant receptors

HEK293 cells expressing CB2 WT (a), W194Y (b), W194F(c) and W194A(d) mutant receptors were assayed for cAMP accumulation in response to a gradient concentrations forskolin (10-10⁵ nM) in the presence (▲) or absence (○) of the inverse agonist SR144528 (0.3 μM). The results were expressed as % of the LANCE signal representing basal cAMP accumulation, measured in the absence of forskolin. Data shown represent the mean ± S.E.M. of at least three independent experiments performed in triplicate.

Fig. 6. 3D model of the putative interaction of cannabinoid ligands with CB2 receptor determined by Surflex-Dock and MD/MM calculations

(A) The predicted binding pocket surrounded by known binding residues. (B) The agonists WIN55212-2 (green color) and CP55940 (orange) are located in the amphipathic CB2 receptor binding pocket. As illustrated, WIN55212-2 interacts with W194 and Y190 via hydrogen bonding and also forms significant π-π stacking interactions between the naphthyl ring and the aromatic microdomain clustered by W194, F197 and W258.(C) The CB2 antagonist SR144528 (purple) has similar π-π stacking interactions but possesses a different H-bonding interaction with Ser165. A different docking pose was also observed for SR144528 (D).