Impact of fusion to Gα i2 and co-expression with RGS proteins on pharmacological properties of human cannabinoid receptors CB 1R and CB 2R

Abstract

Objectives: G protein coupled receptor (GPCR)-Gα fusion proteins are often employed to investigate receptor/G protein interaction. In this study, the impact of Gα fusion proteins on pharmacology of CBRs, both mediating signals through Gα i proteins, were investigated. Gα i2 was fused to the C-terminus of the CBRs or co-expressed with non-fused Gα i2 in Sf9 cells, always together with Gβ 1γ2. Furthermore, the impact of RGS proteins on CBR signaling in combination with the CBR fusion approach was examined, using RGS4 and RGS19 as paradigms. Methods: CBR ligands were characterized in the steady-state GTPase assay and pharmacological properties of ligands in the different test systems were correlated. Key findings: Fusion of CBRs to Gα i2 enhanced the maximal stimulatory effects of ligands compared to the co-expression system, especially for CB 2R. RGS4, but not RGS19, behaved as a GTPase-activating protein at CBRs in the Gα i2 co-expression and fusion system. Fusion of GPCR, most prominently CB 2R, to Gα i2, and co-expression with RGS4 altered the pharmacological properties of ligands. Conclusions: Our data suggest that fusion of CB 2R to Gα i2 and co-expression with RGS4 impedes with conformational changes. Moreover, our results support the concept of ligand-specific receptor conformations. Finally, this paper describes the most sensitive CBR test system currently available. © 2011 The Authors JPP © 2011 Royal Pharmaceutical Society.