FIGURE 7.

GoLoco-insensitive Gα_il has normal interactions with the effector adenylyl cyclase and the effector-mimetic peptide KB-1753. A, affinity of wild type and N149I Gα_i1 proteins for the Gα-effector mimetic peptide KB-1753 (43) was measured using fluorescence anisotropy. 5 nm FITC-KB-1753 peptide was mixed with increasing amounts of Gα_i1 proteins, and equilibrium fluorescence anisotropy was measured. Data are presented as the mean ± S.E.M. of triplicate determinations. Dissociation constants were determined by nonlinear regression: wild type Gα_i1·GDP (24.1 ± 4 μm), wild type Gα_i1· (294 ± 40 nm), N149I Gα_i1·GDP (13.0 ± 2 μm), N149I Gα_i1· (311 ± 40 nm). B, cells were transiently transfected with cDNA encoding Gα_i1(Q204L,C352G), Gα_i1(N149I,Q204L,C352G), or pcDNA3.1(+) as a vector control, with the adenosine A_2A receptor. Cyclic AMP accumulation was stimulated with 1 μm MECA for 15 min at 37 °C. Data represent the mean ± S.E.M. of four independent experiments in duplicate. ^*, p < 0.05; ^**, p < 0.01 compared with A_2A-R + empty vector transfection under matched stimulation (basal or MECA), one-way analysis of variance followed by Dunnett's post hoc test.