RGS4 negatively modulates NOP receptor-driven inhibition of D1-stimulated cAMP production in HEK293T cells
The crosstalk between NOP and RGS4 was first investigated in HEK273 cells by measuring the inhibition of cAMP production stimulated by D1 receptor agonist SKF-38393 (40 nM) as a biochemical readout (Fig. 1). The NOP receptor endogenous ligand, N/OFQ, and the small molecule NOP agonist, AT-403, were tested in cells transfected with the NOP receptor alone or with RGS4. In NOP-transfected cells (Fig.1), N/OFQ inhibited cAMP production in a concentration-dependent manner. N/OFQ showed a pIC50 of 9.43 (10.15-8.70) and a maximal inhibition of cAMP production (Emax) of 61%. Co-transfection of RGS4 with the NOP receptor caused a rightward shift of the concentration-response curve of N/OFQ (Fig.1A) with a significant reduction of pIC50 to 8.66 (9.62-7.69) (df=12, t=3.56) and Emax to 45%. To investigate whether the NOP receptor might be regulated by other RGS proteins, the response of N/OFQ in the presence of RGS19 was assessed (Fig. 1A). In fact, RGS19 is structurally very similar to RGS4 and was reported to interact with the NOP receptor (Xie et al. , 2005). When NOP receptor and RGS19 were co-transfected, the N/OFQ curve was shifted to the right (Fig.1), with a significant reduction of N/OFQ potency (pIC50 8.71, 9.88-7.55) (df=12, t=3.093) and a non-significant reduction of N/OFQ Emax to 39%.
Whether RGS4 and RGS19 also modulate the effect of AT-403 was next investigated. AT-403 (Fig. 1B) inhibited the D1-stimulated cAMP production in a concentration-dependent manner, showing slightly higher potency (pIC50=9.92, 10.78-9.06) and efficacy (75%) than N/OFQ. RGS4 co-transfection caused a rightward shift of the AT-403 curve (Fig. 2), with a significant reduction of potency (pIC50=9.22, 9.77-8.66) (df=11, t=2.71) and efficacy (50%, df=11 t=3.79). Co-transfection of RGS19 also shifted to the right the AT-403 curve (Fig. 1B), leading to a significant reduction (df=11 t=4.10) of AT-403 potency (pIC50=8.93, 9.92-7.93) and efficacy (44%, df=11, t=3.24).