Sarah Sutor1, Jörg Heilmann, Roland Seifert. 1. Department of Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, Regensburg Institute of Pharmacology, Medical School of Hannover, Hannover, Germany.
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 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 Gα(i2) 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 Gα(i2) co-expression and fusion system. Fusion of GPCR, most prominently CB₂R, to Gα(i2) , and co-expression with RGS4 altered the pharmacological properties of ligands. CONCLUSIONS: Our data suggest that fusion of CB₂R 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.
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 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 Gα(i2) 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 Gα(i2) co-expression and fusion system. Fusion of GPCR, most prominently CB₂R, to Gα(i2) , and co-expression with RGS4 altered the pharmacological properties of ligands. CONCLUSIONS: Our data suggest that fusion of CB₂R 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.
Authors: Roland Seifert; Andrea Strasser; Erich H Schneider; Detlef Neumann; Stefan Dove; Armin Buschauer Journal: Trends Pharmacol Sci Date: 2012-12-17 Impact factor: 14.819
Authors: Kristina L Leinwand; Ashleigh A Jones; Rick H Huang; Paul Jedlicka; Daniel J Kao; Edwin F de Zoeten; Soumita Ghosh; Ruin Moaddel; Jan Wehkamp; Maureen J Ostaff; Jutta Bader; Carol M Aherne; Colm B Collins Journal: J Crohns Colitis Date: 2017-10-27 Impact factor: 9.071