Figure 5.

Involvement of RGS4 in neurabin-mediated attenuation of A1R signaling. A, RGS4 inhibitor CCG-4986 reverses neurabin-mediated attenuation of A1R signaling. CHO cells expressing HA-A1R alone or in combination with neurabin were treated with forskolin alone, forskolin plus R-PIA, or forskolin plus R-PIA and CCG-4986 (30 μm). Data are from four independent experiments with cells expressing HA-A1R alone and from 10 independent experiments with cells expressing HA-A1R with neurabin, and expressed as the fold change in cAMP level relative to forskolin alone (defined as onefold). Values are given as the mean ± SEM. *p < 0.05, indicated treatment versus forskolin alone. B, Knockdown of RGS4 expression by antisense oligo diminishes neurabin-mediated attenuation of A1R signaling. CHO cells were cotransfected with HA-A1R and neurabin, and together with antisense oligo against RGS4 mRNA or scrambled oligo control. Reduction in RGS4 mRNA level was confirmed by RT-PCR. Relative cAMP level is presented as the fold change versus forskolin alone (defined as onefold). *p < 0.05, R-PIA stimulated versus forskolin alone; n = 8 for each condition. C, Knockdown of RGS4 expression by siRNA also abolishes the effect of neurabin in attenuation of A1R signaling. CHO cells were cotransfected with HA-A1R and neurabin, and together with siRNA against RGS4 or control siRNA. Reduction in RGS4 mRNA level was confirmed by RT-PCR. *p < 0.05, R-PIA stimulated versus forskolin alone; n = 6–8 for each condition. D, Attenuation of A1R signaling by neurabin and RGS4 in native neurons. Primary cortical neurons cultured from WT and Nrb^−/− mice were treated as indicated. Data were expressed as the fold change in cAMP level relative to forskolin alone (defined as onefold); n = 7–8 for each condition. *p < 0.05, when compared with the relative cAMP level in neurons of either genotype treated with forskolin alone. #p < 0.05, when compared with the relative cAMP level in WT neurons treated with R-PIA and forskolin.