Mass spectrometry-based proteomics of human cannabinoid receptor 2: covalent cysteine 6.47(257)-ligand interaction affording megagonist receptor activation

Abstract

The lack of experimental characterization of the structures and ligand-binding motifs of therapeutic G-protein coupled receptors (GPCRs) hampers rational drug discovery. The human cannabinoid receptor 2 (hCB2R) is a class-A GPCR and promising therapeutic target for small-molecule cannabinergic agonists as medicines. Prior mutational and modeling data constitute provisional evidence that AM-841, a high-affinity classical cannabinoid, interacts with cysteine C6.47(257) in hCB2R transmembrane helix 6 (TMH6) to afford improved hCB2R selectivity and unprecedented agonist potency. We now apply bottom-up mass spectrometry (MS)-based proteomics to define directly the hCB2R-AM-841 interaction at the amino-acid level. Recombinant hCB2R, overexpressed as an N-terminal FLAG-tagged/C-terminal 6His-tagged protein (FLAG-hCB2R-6His) with a baculovirus system, was solubilized and purified by immunochromatography as functional receptor. A multiplex multiple reaction monitoring (MRM)-MS method was developed that allowed us to observe unambiguously all seven discrete TMH peptides in the tryptic digest of purified FLAG-hCB2R-6His and demonstrate that AM-841 modifies hCB2R TMH6 exclusively. High-resolution mass spectra of the TMH6 tryptic peptide obtained by Q-TOF MS/MS analysis demonstrated that AM-841 covalently and selectively modifies hCB2R at TMH6 cysteine C6.47(257). These data demonstrate how integration of MS-based proteomics into a ligand-assisted protein structure (LAPS) experimental paradigm can offer guidance to structure-enabled GPCR agonist design.

Figure 1

(a) Chemical structure of AM-841. (b) Saturation-binding assay of [³H]CP-55940 radioligand to FLAG-hCB2R-6His in membranes from Sf-21 cells overexpressing this receptor. Membrane incubation with AM-841 prior to [³H]CP-55940 binding (▲) reduced the receptor B_max by ~ 75% relative to the B_max in membranes not exposed to AM-841 (■). Data are means ± SEM of at least two independent experiments performed in duplicate. (c) Western blot analysis of the of the purified FLAG-hCB2R-6His preparation with anti-FLAG antibody detection. M, monomer; D, dimer; O, oligomers.

Figure 2

LC-MS/MS analysis of hCB2R TMH6 model peptide, either not exposed to AM-841 (panel a) or incubated with AM-841 prior to analysis (panel b). Mass shifts demonstrating covalent labeling of the peptide’s cysteine (Cys) residue by AM-841 are indicated.

Figure 3

Multiplex MRM-MS analysis of FLAG-hCB2R-6His tryptic digests. (a) Spectra for each of the seven hCB2R TMHs are shown, with the respective GRAVY hydrophobicity score indicated. A propensity for one missed cleavage in TMH6 is demonstrated. (b) Incubation of purified FLAG-hCB2R-6His with AM-841 reduces the TMH6-associated signal intensity by ~ 75% (red spectrum) with respect to the signal intensity of TMH6 (blue spectrum) from naïve receptor not exposed to AM-841, whereas the spectral intensities of the other TMHs remained unchanged after holoreceptor incubation with AM-841 (as exemplified by TMH1).

Figure 4

High-resolution Q-TOF MS/MS spectra of the hCB2R TMH6 peptide with one missed cleavage (LAKTLGLVLAVLLICWFPVLALMAHSLATTLSDQVK) obtained from analysis of tryptic digests of (a) purified FLAG-hCB2R-6His itself (monoisotopic m/z = 977.3175⁴⁺) or (b) purified FLAG-hCB2R-6His that had been incubated with AM-841 (monoisotopic m/z = 1074.3757⁴⁺). The mass difference is equivalent to the mass of AM-841.

Figure 5

High-resolution MS/MS fragmentation spectra of the hCB2R TMH6 peptide with one missed cleavage (LAKTLGLVLAVLLICWFPVLALMAHSLATTLSDQVK) obtained from analysis of tryptic digests of (a) purified FLAG-hCB2R-6His itself in which C6.47(257) was carbamidomethylated [Cys(257)-CAM] or (b) purified FLAG-hCB2R-6His that had been incubated with AM-841. The mass shift at the y-22 ion demonstrates covalent labeling of the hCB2R y-22 cysteine residue, C6.47(257), by AM-841.

Figure 6

Schematic illustration of the reaction between AM-841 and the -SH moiety of hCB2R C6.47(257). R′ and R″ denote the portions of the TMH6 peptide chain flanking the reactive C6.47(257) residue.