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

Area of Science:

• Pharmacology
• Molecular Biology
• Biochemistry

Background:

• Cannabinoid therapeutics may benefit from biased signaling, where arrestin or G protein pathways are selectively activated.
• Arrestin-2 and arrestin-3 translocation to the cannabinoid receptor 1 (CB1) are implicated in distinct signaling functions.
• A specific phosphorylation motif ('pxxp') in the CB1 receptor's C-terminus was investigated for its role in arrestin interaction.

Purpose of the Study:

• To investigate the role of the 'pxxp' phosphorylation motif in the CB1 receptor C-terminus for arrestin-2 and arrestin-3 interaction.
• To determine if specific phosphorylation sites are essential for arrestin translocation to the CB1 receptor.
• To assess the potential for developing arrestin subtype-selective biased ligands for the CB1 receptor.

Main Methods:

• Site-directed mutagenesis was used to create nine phosphorylation-impaired CB1 receptor C-terminal mutants.
• Bioluminescence resonance energy transfer (BRET) was utilized to measure arrestin-2/3 translocation and G protein dissociation.
• Immunocytochemistry was employed to quantify receptor expression levels.

Main Results:

• The 'pxxp' motifs were found to be partially required for arrestin-2 and arrestin-3 translocation to the CB1 receptor.
• Complete inhibition of arrestin translocation was only observed when all phosphorylation sites were mutated.
• Simultaneous mutation of specific sites (S425 and S429) reduced the rate of arrestin translocation.
• Desensitization of G protein dissociation was proportionally inhibited by mutations that reduced arrestin translocation.

Conclusions:

• The 'pxxp' motif is not essential for arrestin translocation to the CB1 receptor.
• Arrestin-2 and arrestin-3 exhibit equivalent phosphorylation requirements in the CB1 receptor C-terminus.
• The findings suggest that arrestin subtype-selective biased ligands targeting the CB1 receptor may not be feasible.
• Different regions of the CB1 receptor C-terminus contribute differentially to arrestin translocation.