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. 1999 Jan 1;19(1):180–192. doi: 10.1523/JNEUROSCI.19-01-00180.1999

Fig. 2.

Fig. 2.

Pertussis toxin, βARK1 PH domain peptide expression, and transducin α (Gt) expression block Gi-coupled receptor potentiation of Gs-stimulated AC4. A, HEK 293 cells stably expressing AC4 were incubated overnight with vehicle (−PTx) or 200 ng/ml pertussis toxin (+PTx). The following day, cells were treated with vehicle or 10 μm isoproterenol (iso) in the presence or absence of 500 nm somatostatin (som). Relative cAMP accumulation was determined as described in Materials and Methods. The data are the means ± SD of triplicate assays. B, HEK 293 cells were transiently transfected with RSV-β-galactosidase and the 5-HT7Areceptor, along with either pCDNAIII or the βARK1 PH domain and either pCEP4 or pCEP4-AC4 as described in Materials and Methods. Cells transfected with the βARK1 PH domain are denoted PH domain. AC4-transfected cells with or without the PH domain were treated with 10 μm serotonin in the presence or absence of 500 nm somatostatin. 5-HT-stimulated activity did not differ significantly between AC4-transfected and AC4- and PH domain-transfected cells; the ratio value for AC4 cells in response to 5-HT alone was 2.088 ± 0.389 and for AC4 and PH domain cells was 3.565 ± 0.688. C, Cells were transiently transfected with RSV-β-galactosidase and the 5-HT7Areceptor, along with either pCDNAIII or transducin α and either pCEP4 or pCEP4-AC4 as described in Materials and Methods. Cells transfected with transducin α are denoted Gt. Cells were treated with 1 μm serotonin in the presence or absence of 500 nm somatostatin. 5-HT-stimulated activity did not differ significantly between AC4-transfected and AC4- and Gt-transfected cells; the ratio value for AC4 cells in response to 5-HT alone was 0.630 ± 0.292 and for AC4 and Gt cells was 0.622 ± 0.091. ForB and C, relative cAMP accumulation was determined as described in Materials and Methods. The data are expressed as percent cAMP accumulation in the absence of somatostatin, with this level being set as 100%. The fold stimulation over the basal activity was similar between transfections with or without the PH domain or transducin α. The data are the means ± SD of triplicate assays and were subtracted for endogenous (pCEP4-transfectant) cAMP accumulation and corrected for transfection efficiency using β-galactosidase.