Delineating the interactions between the cannabinoid CB₂ receptor and its regulatory effectors; β-arrestins and GPCR kinases

Background and purpose: The cannabinoid CB₂ receptor (CB₂ ) is a promising therapeutic target for modulating inflammation. However, little is known surrounding the mechanisms underpinning CB₂ desensitisation and regulation, particularly the role of GPCR kinases (GRKs). Here, we evaluated the role of six GRK isoforms in β-arrestin recruitment to CB₂ . Mutagenesis of several distal C-terminal aspartic acid residues was also performed in an attempt to delineate additional structural elements involved in the regulation of CB₂ .

Experimental approach: In CB₂ -expressing HEK 293 cells, β-arrestin translocation was measured using real-time BRET assays. G protein dissociation BRET assays were performed to assess the activation and desensitisation of CB₂ in the presence of β-arrestin 2.

Key results: Overexpression of GRK isoforms 1-6 failed to considerably improve translocation of either β-arrestin 1 or β-arrestin 2 to CB₂ . Consistent with this, inhibition of endogenous GRK2/3 did not substantially reduce β-arrestin 2 translocation. Mutagenesis of C-terminal aspartic acid residues resulted in attenuation of β-arrestin 2 translocation, which translated to a reduction in desensitisation of G protein activation.

Conclusion and implications: Our findings suggest that CB₂ does not adhere to the classical GPCR regulatory paradigm, entailing GRK-mediated and β-arrestin-mediated desensitisation. Instead, C-terminal aspartic acid residues may act as phospho-mimics to induce β-arrestin activation. This study provides novel insights into the regulatory mechanisms of CB₂ , which may aid in our understanding of drug tolerance and dependence.