Dual role of the second extracellular loop of the cannabinoid receptor 1: ligand binding and receptor localization

Abstract

The seven transmembrane alpha-helices of G protein-coupled receptors (GPCRs) are the hallmark of this superfamily. Intrahelical interactions are critical to receptor assembly and, for the GPCR subclass that binds small molecules, ligand binding. Most research has focused on identifying the ligand binding pocket within the helical bundle, whereas the role of the extracellular loops remains undefined. Molecular modeling of the cannabinoid receptor 1 (CB1) extracellular loop 2 (EC2), however, suggests that EC2 is poised for key interactions. To test this possibility, we employed alanine scanning mutagenesis of CB1 EC2 and identified two distinct regions critical for ligand binding, G protein coupling activity, and receptor trafficking. Receptors with mutations in the N terminus of EC2 (W255A, N256A) were retained in the endoplasmic reticulum and did not bind the agonist (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP55940) or the inverse agonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). In contrast, the C terminus of EC2 differentiates agonist and inverse agonist; the P269A, H270A, and I271A receptors exhibited diminished binding for several agonists but bound inverse agonists SR141716A, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), and 4-[6-methoxy-2-(4-methoxyphenyl)benzofuran-3-carbonyl]benzonitrile (LY320135) with wild-type receptor affinity. The F268A receptor involving substitution in the Cys-X-X-X-Ar motif, displayed both impaired localization and ligand binding. Other amino acid substitutions at position 268 revealed that highly hydrophobic residues are required to accomplish both functions. It is noteworthy that a F268W receptor was trafficked to the cell surface yet displayed differential binding preference for inverse agonists comparable with the P269A, H270A, and I271A receptors. The findings are consistent with a dual role for EC2 in stabilizing receptor assembly and in ligand binding.

Fig. 1.

Schematic diagram, sequence comparison, and molecular models of the EC2 loop region of CB1. A, schematic diagram of the EC2 loop of the human CB1 receptor. The tryptophan residue highlighted in green is highly conserved among rhodopsin-like G protein-coupled receptors. The residues that are critical for receptor trafficking are highlighted in yellow. The cluster of residues critical for CP55940 binding identified here are shown in blue. The residues most sensitive to binding multiple agonists are shaded in darker blue. The residues involved in the disulfide bond of the EC2 loop are shown in red with a black-bar linker. The lipid bilayer is represented by the beige rectangle. The residue number indicated corresponds to the position of the residue in the linear sequence. B, amino acid sequence alignment of the EC2 region (yellow) and flanking residues (unshaded) from a variety of cannabinoid and other rhodopsin-like G protein-coupled receptors; hCB1, human CB1 receptor; mCB1, mouse CB1 receptor; rCB1, rat CB1 receptor; hCB2, human CB2 receptor; mCB2, mouse CB2 receptor; rCB2, rat CB2 receptor; hADRB2, human β2-adrenergic receptor; hACM3, human muscarinic acetylcholine receptor M3; bRho, bovine rhodopsin; hV1aR, human vasopressin 1a receptor; hOPRD, human δ-type opioid receptor; tADRB1, turkey β1-adrenergic receptor; hD2DR, human dopamine D2 receptor. The CB1 EC2 region and flanking residues were defined based on the crystal structures of the β2-adrenergic receptor (Cherezov et al., 2007). Green and red residues are indicated as described at the top (A). The phenylalanine of the Cys-X-X-X-Ar motif is highlighted in blue. C, illustration of molecular model of the human CB1 receptor from an expanded extracellular view. The molecular model of the human CB1 receptor has been derived from the X-ray crystal structure of the β2-adrenergic receptor. The TM helices are colored as: TM1 (blue), TM2–TM3 (cyan), TM4–TM5(green), and TM6–TM7(yellow/orange). The residues from EC1 are cyan (His178 and Phe189); EC2 are fuchsia (Trp255, Asn256, Phe268, Pro269, and Ile271). D, a putative binding pocket for CP55940 (gray) within the model of the human CB1 receptor. The TM helices are colored as in C. Several key contact residues for CP55940 are illustrated (fuchsia), including the previously proposed contact points Lys192 and Ser383.

Fig. 2.

Comparison of inverse agonist [³H]SR141716A binding for wild-type and the CB1 mutant receptors that displayed diminished binding to CP55940. Homologous competition binding assays using the CB1 receptor inverse agonist SR141716A were performed on membrane preparations from HEK293T cells expressing the wild-type (■), W255A (▿), P269A (▵), H270A (▾), and I271A (♢) receptors. Each data point represents the mean ± S.E.M. of at least three independent experiments performed in duplicate.

Fig. 3.

Cellular distribution of the wild-type and mutant CB1 receptors in HEK293T cells. C-terminally GFP-tagged wild-type and mutant receptors were transiently expressed in HEK293T cells. Forty-eight hours after transfection, cells were fixed and mounted for confocal microscopy as described under Materials and Methods. Subcellular distribution of the receptors was assessed by detecting GFP fluorescence. The data are representative images of at least five independent experiments. Empty vector = pcDNA3.1 vector alone (i.e., not carrying the gene for CB1) used to transfect cells as a control. Scale bar, 15 μm.

Fig. 4.

Comparison of ligand binding for wild-type and the CB1 receptors with mutations at residue 268. Homologous competition binding assays using CB1 receptor agonist CP55940 (A) and inverse agonist SR141716A (B) were performed on membrane preparations from HEK293T cells expressing the wild-type (■), F268Y (●), and F268W (▵) receptors. Each data point represents the mean ± S.E.M. of at least three independent experiments performed in duplicate.

Fig. 5.

Comparison of the subcellular distribution of the wild-type and CB1 receptors with mutations at residue 268. A, subcellular localization of GFP-tagged wild-type and CB1 mutant receptors. HEK293T cells were transiently transfected with GFP-tagged CB1 wild-type or the F268Y, F268W, or F268N receptors. Subcellular distribution of the receptors was assessed by detecting GFP fluorescence. The data are representative images of at least five independent experiments. Scale bar, 15 μm. B, colocalization of GFP-tagged wild-type, F268A, and F268N receptors with the ER marker, PDI, in HEK293T cells. HEK293T cells transiently expressing wild-type, F268A, and F268N receptors were fixed, permeabilized, and stained with antibodies against PDI as described under Materials and Methods. Green, GFP fluorescence from receptors (left); red, PDI fluorescence (middle); yellow, colocalization of the receptors and PDI (right). Images were chosen as representatives from at least three independent transfections. Scale bar, 15 μm.

Fig. 6.

Competitive displacement of [³H]SR141716A with various ligands by the F268W, P269A, H270A, and I271A mutant receptors. AM251 (A), LY320135 (B), CP55940 (C), and HU-210 (D) were used for displacing 4 nM [³H]SR141716A in membrane preparations from HEK293T cells expressing wild-type (■), F268W (●), P269A (▵), H270A (▿), and I271A (♢) receptors. Each data point represents the mean ± S.E.M. of at least three independent experiments performed in duplicate.