Cannabinoid 1 (CB₁) receptor arrestin subtype‐selectivity and phosphorylation dependence

Abstract

Background and purpose Arrestin or G protein bias may be desirable for novel cannabinoid therapeutics. Arrestin‐2 and arrestin‐3 translocation to CB1 receptor have been suggested to mediate different functions that may be exploited with biased ligands. Here, the requirement of a recently described phosphorylation motif ‘pxxp’ (where ‘p’ denotes phosphorylatable serine or threonine and ‘x’ denotes any other amino acid) within the CB1 receptor C‐terminus for interaction with different arrestin subtypes was examined. Experimental approach Site‐directed mutagenesis was conducted to generate nine different phosphorylation‐impaired CB1 receptor C‐terminal mutants. Bioluminescence resonance energy transfer (BRET) was employed to measure arrestin‐2/3 translocation and G protein dissociation of a high efficacy agonist for each mutant. Immunocytochemistry was used to quantify receptor expression. Key results The effects of each mutation were shared for arrestin‐2 and arrestin‐3 translocation to CB1 receptor pxxp motifs are partially required for arrestin‐2/3 translocation, but translocation was not completely inhibited until all phosphorylation sites were mutated. The rate of arrestin translocation was reduced with simultaneous mutation of S425 and S429. Desensitisation of G protein dissociation was inhibited in different mutants proportional to the extent of their respective loss of arrestin translocation. Conclusions and implications These data do not support the existence of an ‘essential’ pxxp motif for arrestin translocation to CB1 receptor. These data also identify that arrestin‐2 and arrestin‐3 have equivalent phosphorylation requirements within the CB1 receptor C‐terminus, suggesting arrestin subtype‐selective biased ligands may not be viable and that different regions of the C‐terminus contribute differently to arrestin translocation.

Subtle Structural Modification of a Synthetic Cannabinoid Receptor Agonist Drastically Increases its Efficacy at the CB1 Receptor

“…It has been reported that CB1R agonists have lower potencies for β-arrestin recruitment compared to G protein coupling . As expected, most of the agonists tested in this study demonstrated ∼3-fold weaker potencies for β-arrestin 2 recruitment than for G i1 engagement (Figure and Table ).…”

Synthesis and Functional Evaluation of Synthetic Cannabinoid Receptor Agonists Related to ADB-HEXINACA

“…Altogether, at CB 2 , our data yielded a potency rank order of ADB > MDMB > MMB ∼ MPP > AB > CUMYL > APP. This was not entirely in line with the MPA assay, with as a notable difference the CUMYL-bearing HEXINACA compound (44), which conferred the best potency in the MPA format, while being the second worst in the βarr2 assay, although was still highly potent (<10 nM). However, with the exception of the less active APP-HEXINACA (40), the differences in potency at CB 2 between the other headgroup-bearing HEXINACA compounds were overall relatively limited, in both assay formats.…”

“…While WIN55,212−2 is not an indazole-3-carboxamide like MDMB-FUBINACA, it bears the highest similarity to our investigated compounds, compared to other published CB 2 -agonist structures. Eight ligands were selected for docking: ADB-HEXINACA (5), MDMB-HEXINACA (42), MDMB-5en-HEXINACA (49), MDMB-HEXICA (29), MDMB-HEX7AICA (56), ADB-5en-HEXINACA (46), MPP-HEXINACA (43), and CUMYL-HEXINACA (44), providing a varied subset of compounds to assess the impact of head, core, and tail combinations. It was generally observed that at CB 1 , the ligands' calculated binding poses were equivalent to that of the cognate ligand MDMB-FUBINACA (Figure 7a).…”

“…The rotation of Ser90, and the lack of an equivalent rotation in CB 1 , could account for the improved potency of APP (27, 34, 40, 47, 54, and 61) and MPP (30,37,43,50, 57, and 64) ligands at CB 2 . CUMYL-HEXINACA (44) formed an edge to face π−π interaction with His95. Despite this interaction, lower potency was observed at CB 2 for the CUMYL compounds (31, 38, 44, 51, 58, and 65).…”

“…The ADB (5, 26, 33, 46, 53, and 60) and MDMB (29, 36, 42, 49, 56, and 63) headgroups consistently conferred good potencies across a range of core and tail combinations, exemplified by ADB-5en-HEXINACA (46). The CUMYL (31, 38, 44, 51, 58, and 65) and AB (25,32,39,45,52, and 59) headgroups conveyed a good activity at both receptors, with CUMYL-5en-HEXINACA (51) and CUMYL-HEXINACA (44) displaying some of the highest potencies observed at CB 1 among the compounds examined here.…”