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).