Fig. 4.

Expression of tyrosine-deficient Gα_i2 mutant or RGS12 small interfering RNA (siRNA) blocks Gα_i2-RGS12 association and enhances DPDPE-induced inhibition of cAMP. A: cultured smooth muscle cells transfected with control vector, vector containing Gα_i2 mutant (Y69F, Y231F, or Y321F), or RGS12 siRNA were treated with 1 μM DPDPE for 1 min. Activation of cSrc (pSrc) was measured using specific phosphorylated (Tyr⁴¹⁶) Src antibody. Gα_i2 immunoprecipitates were used to measure tyrosine phosphorylation (pGα_i2) using phosphotyrosine antibody and association of Gα_i2 with RGS12 (RGS12-Gα_i2) using RGS12 antibody. Immunoblots are representative of 4 separate experiments. b, Basal; d, DPDPE. Expression of Gα_i2 mutant had no effect on cSrc activation, whereas suppression of RGS12 had no effect on cSrc activation and Gα_i2 phosphorylation. B: cultured smooth muscle cells transfected with control vector, vector containing Gα_i2 mutant (Y69F, Y231F, or Y321F), or RGS12 siRNA were pretreated with 10 μM forskolin for 10 min and then treated with 1 μM DPDPE for 1 min. cAMP levels were measured by radioimmunoassay. Basal levels (2.5 ± 0.25 to 2.7 ± 0.31 pmol/mg protein) of cAMP are similar in control cells and cells expressing Gα_i2 mutant or RGS12 siRNA. Values (means ± SE of 5 experiments) are expressed as pmol/mg protein above basal level. **P < 0.01 compared with control.