Cholesterol as a modulator of cannabinoid receptor CB2 signaling

Abstract

Signaling through integral membrane G protein-coupled receptors (GPCRs) is influenced by lipid composition of cell membranes. By using novel high affinity ligands of human cannabinoid receptor CB₂, we demonstrate that cholesterol increases basal activation levels of the receptor and alters the pharmacological categorization of these ligands. Our results revealed that (2-(6-chloro-2-((2,2,3,3-tetramethylcyclopropane-1-carbonyl)imino)benzo[d]thiazol-3(2H)-yl)ethyl acetate ligand (MRI-2646) acts as a partial agonist of CB₂ in membranes devoid of cholesterol and as a neutral antagonist or a partial inverse agonist in cholesterol-containing membranes. The differential effects of a specific ligand on activation of CB₂ in different types of membranes may have implications for screening of drug candidates in a search of modulators of GPCR activity. MD simulation suggests that cholesterol exerts an allosteric effect on the intracellular regions of the receptor that interact with the G-protein complex thereby altering the recruitment of G protein.

Figure 1

Benzothiazole-based CB₂ ligands. (A) Structure of benzothiazole-based CB₂ ligands. (B) Synthesis scheme for ligands, Reagents and conditions: a. 2-bromoethanol 90 °C; b. BOP, 2,2,3,3-tetramethylcyclopropane carboxylic acid. c. Acetyl Chloride.

Figure 2

Affinity and functional effects of novel MRI ligands on CB₂ receptor in CHO cell membranes. (a) binding affinities (nM) and E_max (% and nM) of [³⁵S]-GTPγS binding to CHO membranes (PerkinElmer, Cat. No ES111-M400UA) expressing hCB₂. (b) [³⁵S]-GTPγS binding to membranes as a factor of ligand concentration. Binding of [³⁵S]-GTPγS was determined as described in “Methods”. Non-specific binding was defined as 0% activity. The assay of GTPγS non-specific binding contains non-radioactive GTPγS.

Figure 3

GEF of CB₂ expressed in: (a) E. coli membranes and (b) CHO membranes (Millipore EMD). 2 μg of total protein per assay. Each point represents an average of four independent measurements (n = 4). Ligands were added at a concentration of 2 μM to ensure saturation of the receptor, and G protein was added as described in “Methods”. The dotted line indicates the rates of activation of G protein in the absence of a ligand. The rates of activation with CP-55,9040 are set to 100%, and rates of activation in the presence of SR-144,528 to 0%.

Figure 4

GEF on CB₂ expressed in different cell lines. An amount of 2 μg of membrane protein per assay was used. Each data points represents an average of four independent measurements (n = 4) with error rates indicated by the bars. 100% represents full activation in the presence of 2 μM of CP-55,940, and 0%–with 2 μM SR-144,528.

Figure 5

GEF analysis of three membrane preparations with or without supplementation with exogenous G protein. (a) Expi293F membranes; (b) CHO membranes; (c) Sf9 membranes. Bars represent an average of four independent measurements (n = 4). Dotted lines represent the rates of activation of G protein in the absence of a ligand.

Figure 6

Effect of treatment with MβCD/Chol of E. coli membranes on activation of CB₂ by synthetic ligands. (a) Membranes expressing CB₂ were treated with 20 mM MβCD/Chol for 1 h at 4 °C, washed with PBS, and the activation of CB₂ in the presence of ligands determined by GEF; (b) control, untreated E. coli membranes expressing CB₂. Each data point represents an average of four independent measurements (n = 4) with standard deviation indicated by vertical bars.

Figure 7

Activation of G protein on CB₂ receptor reconstituted in liposomes. Liposome composition: 75% POPC, 25% POPG and cholesterol content relative to total phospholipids as indicated, or total lipids extracted from bovine brain, as indicated. Proteins were reconstituted at a protein-to-lipid ratio in the range 1:850 to 1:1100 mol/mol. (a–d) CB₂ purified from E. coli cells. (e,f) CB₂ purified from Expi293F GNTI^- cells. Average values of four independent measurements are plotted (n = 4). Dotted lines represent the rates of activation of G protein in the absence of a ligand.

Figure 8

Depictions of ligand binding site and preferentially affected residues. (a) Representative snapshot of MRI-2659 in orthosteric binding site. MRI-2646 and MRI-2659 share common backbone position; (b) Cryo-EM structure (PDB: 6PT0). Those residues whose simulation average RMSD from the cryo-EM structure changed by > 1 Å as a function of 0% vs 40 mol% cholesterol are highlighted by spheres. Such residues from free and ligand-bound conditions (MRI-2594, MRI-2646, MRI-2659) are all shown. Note proximity to G_α subunit (grey). A number of these residues reside in the extracellular loops; the functional relevance is not known.

Figure 9

Difference in Trp258^6.48 rotamer angle related to presence of cholesterol. (a) Trp258^6.48 χ¹ rotamer angle distributions with 0% and 40 mol% cholesterol. (b) Difference in mean Trp258^6.48 χ¹ rotamer angle between no cholesterol vs 40 mol% cholesterol conditions; green (negative change) dashed line. TM domains are annotated, with up and down arrows indicating direction of helix, with up from intracellular to extracellular.

Figure 10

Difference in per-residue root-mean-square deviation (RMSD) between simulation and antagonist-bound cryo-EM structure. Trp258^6.48 highlighted in red, residues with > 1 Å difference highlighted in orange (positive change) or green (negative change) dashed line. TM domains are annotated, with up and down arrows indicating direction of helix, with up from intracellular to extracellular.

Figure 11

Membrane cholesterol dependent protean agonism.