Orthosteric ligand binding to CB₁ in the presence of allosteric modulators. Left panels show binding assays using [³H]CP55940 against (A) CP55940 (i.e., orthosteric agonist shown for comparison), (B) LDK1285, (C) LDK1288, (D) LDK1305, and (E) PSNCBAM1. Right panels show binding assays using [³H]SR141716A against (F) SR141716A (i.e., orthosteric inverse agonist shown for comparison), (G) LDK1285, (H) LDK1288, (I) LDK1305, and (J) PSNCBAM1. Each data point represents the mean ± S.E. of three independent experiments performed in duplicate.

Internalization of CB₁ upon treatment with LDK1285, LDK1288, LDK1305, or PSNCBAM1. (A) Cells expressing CB₁T210A-GFP were incubated with either vehicle alone (0.03% DMSO) or 10 μM of the indicated compound for 1, 2, or 3 hours before fixation. The 0-hour time point indicates untreated cells expressing CB₁T210A-GFP. Images are representative of three independent transfections. Scale bar, 15 μm (white bar; see bottom-right image). (B) Internal CB₁T210A-GFP was quantified as described in Materials and Methods. Percent internal fluorescence is expressed as the mean ± S.E. fold change over vehicle (n = 3 independent experiments). Statistical significance was assessed using one-way analysis of variance and Dunnett’s multiple comparisons test; *P < 0.05; ***P < 0.001. (C) Membrane fluorescence, derived from the experiment in frame A, is expressed as the mean ± S.E. fold change over vehicle (n = 3 independent experiments). Orange line indicates LDK1285; gray, LDK1288; yellow, LDK1305; blue, PSNCBAM1. Statistical significance was assessed using one-way analysis of variance and Dunnett’s multiple-comparisons test; ^††P < 0.01 in comparison with PSNCBAM1. (D) Cells expressing CB₁-GFP and β-arrestin 2-mcherry were treated with PSNCBAM1 (5 μM; blue curve) or CP55940 (0.5 μM; gray curve) or PSCNBAM1 and CP55940 (5, 0.5 μM, respectively; black curve). β-arrestin-2 recruitment to CB₁ was measured by TIRF microscopy and is displayed as percent change in fluorescence intensity over time. CP55940 t_1/2 = 36 seconds; CP55940 plus PSNCBAM1 t_1/2 = 42 seconds. (E) Cells expressing CB₁-GFP and β-arrestin 2-mcherry were treated with LDK1288 (5 μM; orange curve) or CP55940 (0.5 μM; red curve) or LDK1288 and CP55940 (5, 0.5 μM, respectively; black curve). β-arrestin 2 recruitment to CB₁ was measured by TIRF microscopy and is displayed as percent change in fluorescence intensity over time. CP55940 t_1/2 = 33 second; CP55940 plus LDK1288 = 36 second.

Effect of CP55940, LDK1285, LDK1288, or LDK1305 on ERK1/2 phosphorylation over time. HEK293 cells expressing CB₁ were untreated (DMSO only) or treated with (A) CP55,940 (0.5 μM), (B) LDK1285 (10 μM), (C) LDK1288 (10 μM), or (D) LDK1305 (10 μM), for 2, 5, 10, 15, or 20 minutes. (E) Cells expressing CB₁ and either β-arrestin 1 siRNA or control siRNA were treated with DMSO vehicle only, LDK1285, LDK1288, or LDK1305 (all at 10 μM) for 5 minutes. Cell lysates were separated by SDS-PAGE and analyzed by Western blots probed for phospho-ERK1/2 (pERK1/2). The total level of ERK1/2 was detected for comparison. Note that the two bands correspond to the predominant isoforms of ERK, ERK1 (p44) and ERK2 (p42). Cells not expressing CB₁ and treated with LDK1288 showed no kinase phosphorylation relative to vehicle alone treatment (data not shown). Quantification of at least three independent experiments is displayed in the right-hand column and expressed as mean S.E.-fold increase above the basal level of phosphorylation.

Coupling partner dependency of MEK phosphorylation induced by LDK1285, LDK1288, or LDK1305. HEK293 cells expressing CB₁ and β-arrestin 1 siRNA, CB₁ and β-arrestin 2 siRNA, or CB₁ and control siRNA, or expressing CB₁ alone and pretreated with PTX were either untreated (DMSO only) or treated with (A) LDK1285 (10 μM), (B) LDK1288 (10 μM), or (C) LDK1305 (10 μM) for 5 minutes. Cell lysates were separated by SDS-PAGE and analyzed by Western blots probed for phospho-MEK (pMEK). The total level of MEK was detected for comparison. Quantification of at least three independent experiments is displayed in the right-hand column and expressed as mean S.E.-fold increase above the basal level of phosphorylation.