Hydrophobic residues in helix 8 of cannabinoid receptor 1 are critical for structural and functional properties

Abstract

In addition to the heptahelical transmembrane domain shared by all G protein-coupled receptors (GPCRs), many class A GPCRs adopt a helical domain, termed helix 8, in the membrane-proximal region of the C terminus. We investigated the role of residues in the hydrophobic and hydrophilic faces of amphiphilic helix 8 of human cannabinoid receptor 1 (CB1). To differentiate between a role for specific residues and global features, we made two key mutants: one involving replacement of the highly hydrophobic groups, Leu404, Phe408, and Phe412, all with alanine and the second involving substitution of the basic residues, Lys402, Arg405, and Arg409, all with the neutral glutamine. The former showed a very low B(max) based on binding isotherms, a minimal E(max) based on GTPgammaS binding analysis, and defective localization relative to the wild-type CB1 receptor as revealed by confocal microscopy. However, the latter mutant and the wild-type receptors were indistinguishable. Circular dichroism spectroscopy of purified peptides with corresponding sequences indicated that the highly hydrophobic residues are critical for maintaining a strong helical structure in detergent, whereas the positively charged residues are not. Further investigation of mutant receptors revealed that CB1 localization requires a threshold level of hydrophobicity but not specific amino acids. Moreover, mutant receptors carrying two- to six-residue insertions amino-terminal to helix 8 revealed a graded decrease in B(max) values. Our results identify the key helix 8 components (including hydrophobicity of specific residues, structure, and location relative to TM7) determinant for receptor localization leading to robust ligand binding and G protein activation.

Figure 1

CB1 C-terminal sequences and structural representations of helix 8. (A) C-Terminal sequences of the wild-type and two triple mutant receptors; K402Q/R405Q/R409Q and L404A/F408A/F412A. The amino acid numbers corresponding to the sequence of the full-length human CB1 receptor are indicated. The locations of the C terminus of TM7, helix 8, and helix 9 are marked with black bars. The residues mutated to alanine or glutamine are highlighted in red. (B) Helical wheel projection of CB1 helix 8. Hydrophobic and positively charged residues are colored red and blue, respectively. Two serines and an aspartic acid are colored turquoise and light blue, respectively. The hydrophilic and hydrophobic faces of the helix are separated by the solid line, and the residues on which we focused in this study are further indicated by the red and blue arcs, respectively. (C) Schematic view of the amphipathic nature of helix 8 of the C terminus of the human CB1 receptor. The illustration shown here is generated on the basis of our previous structural study of the CB1 C terminus (16). Helix 8 (residues 401–412) is shown as a ribbon. Some key residues are labeled with the amino acid numbers corresponding to the human CB1 sequence. (D) Transparent surface of helix 8 with the side chains highlighted. Hydrophobic and positively charged residues are colored red and blue, respectively.

Figure 2

CD spectra of the CB1 C-terminal peptides in various detergents. The peptide concentration was 50 μM in 10 mM phosphate buffer (pH 7.3). CD spectra of (A) the wild-type, (B) K402Q/R405Q/R409Q, and (C) L404A/F408A/F412Apeptides in the absence (■) and presence of 0.5 (▲, red), 5 (▼, green), or 50 mM DPC (◆, blue). CD spectra of (D) the wild-type, (E) K402Q/R405Q/R409Q, and (F) L404A/F408A/F412A peptides in the absence (■) and presence of 2 (▲, red), 10 (▼, green), or 50 mM SDS (◆, blue). Data represent the averages of four experiments.

Figure 3

CP55940 saturation binding and stimulation of binding of GTPγS to membranes of HEK293 cells expressing the CB1 wild-type and mutant receptors. (A) Saturation binding with [³H]CP55940 to a membrane prepared fromHEK293 cells expressing the wild-type (■) and mutant receptors, K402Q/R405Q/R409Q (△) and L404A/F408A/F412A (●). Each data point represents the mean ± the standard error of the mean of at least three independent experiments performed in duplicate. (B) Stimulation of [³⁵S]GTPγS binding by CP55940 in membrane preparations from HEK293 cells expressing wild-type (■), K402Q/R405Q/R409Q (△), and L404A/F408A/F412A (●) receptors. Data are presented as specific binding of GTPγS to the membranes. Nonspecific binding was assessed in the presence of 10 μM unlabeled GTPγS. Each data point represents the mean ± the standard error of the mean of at least three independent experiments performed in duplicate.

Figure 4

Comparison of the subcellular distribution of the wild-type and CB1 receptors with mutations in helix 8. Localization of GFP-tagged wild-type, K402Q/R405Q/R409Q, and L404A/F408A/F412A receptors (green, left), the late endosome/lysosome marker, LAMP-1 (red, middle), in HEK293 cells, and an overlay of the fluorescence images to show the extent of colocalization (yellow, right). HEK293 cells transiently expressingCB1 receptors were fixed, permeabilized, and stained with antibodies against LAMP-1, as described in Experimental Procedures. Images were chosen as representatives from at least three independent transfections. The scale bar is 15 μm.

Figure 5

Ligand binding and localization of receptors with different combinations of amino acid substitutions in the hydrophobic face of helix 8. (A)Amino acid sequences of the relevant portion of the C terminus of the wild-type or singly or multiply substituted mutant receptors. The amino acid numbers corresponding to the sequence of the humanCB1 receptor are shown, and the C terminus of TM7 and helix 8 are indicated by black bars. The residues substituted with alanine are highlighted in red. Several of the most C-terminal residues are represented by the dashed lines and not shown due to space limitations. (B) Saturation binding with [³H]CP55940 to a membrane prepared from HEK293 cells expressing the wild-type (■) and mutant recptors, L404A(▲), F408A(▼), F412A(◆), L404A/F412A(●), L404F/F408L(□), andL404F/F412L(△). The K_d and B_max values were calculated by nonlinear regression (fitted to a one-site binding model) as described in Experimental Procedures and are reported as the means ± the standard error of the mean. The K_d and B_max values for the wild-type receptor are listed in Table 1. (C) Comparison of the cellular distribution of the wild-type or helix 8mutant receptors. GFP-tagged wild-type and mutant receptors were expressed inHEK293 cells. A day after transfection, cells were fixed and mounted for confocal microscopy. Images were chosen as representatives from at least three independent transfections. The scale bar is 15 μm.

Figure 6

Effect of glutamine insertions in the membrane-proximal region of the CB1 C terminus on ligand binding and localization. (A) Amino acid sequences of the wild-type and mutant receptors. The amino acid numbers corresponding to the sequence of the CB1 receptor are shown, and the C terminus of TM7 and helix 8 are indicated by black bars. The glutamine residues inserted between TM7 and helix 8 are highlighted in red, and helix 8 is shaded in gray. Several of the most C-terminal residues are represented by the dashed lines and not shown due to space limitations. (B) Saturation binding with [³H]CP55940 to the membrane prepared fromtheHEK293 cells expressing the wild-type (■) or insertion mutant receptors, 401(Gln)₂ (●), 401(Gln)₄ (▲), and 401(Gln)₆ (▽). K_d and B_max values were calculated by nonlinear regression (fitted to a one-site binding model) as described in Experimental Procedures and are reported as the means ± the standard error of the mean. Binding data for the wild-type receptor from Table 1 and Figure 5 were included here for the sake of comparison. (C) Comparison of the cellular distribution of the wild-type and mutant CB1 receptors carrying the glutamine insertions. GFP-tagged wild-type and mutant receptors were expressed in HEK293 cells as described in the legend of Figure 5. The scale bar is 15 μm.