BACKGROUND AND PURPOSE: Arrestin or G protein bias may be desirable for novel cannabinoid therapeutics. Arrestin-2 and arrestin-3 translocation to CB 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 CB receptor C-terminus for interaction with different arrestin subtypes was examined. EXPERIMENTAL APPROACH: Site-directed mutagenesis was conducted to generate nine different phosphorylation-impaired CB 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 CB 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 CB receptor. These data also identify that Arrestin-2 and arrestin-3 have equivalent phosphorylation requirements within the CB 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.
