Impact of fusion to Gαi2 and co-expression with RGS proteins on pharmacological properties of human cannabinoid receptors CB1R and CB2R

Abstract

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 Gαi proteins, were investigated. Gαi2 was fused to the C-terminus of the CBRs or co-expressed with non-fused Gαi2 in Sf9 cells, always together with Gβ1γ2. 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 Gαi2 enhanced the maximal stimulatory effects of ligands compared to the co-expression system, especially for CB2R. RGS4, but not RGS19, behaved as a GTPase-activating protein at CBRs in the Gαi2 co-expression and fusion system. Fusion of GPCR, most prominently CB2R, to Gαi2, and co-expression with RGS4 altered the pharmacological properties of ligands. Conclusions Our data suggest that fusion of CB2R to Gαi2 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.