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

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.

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.

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.

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.