Figure 4.

Effects of the peptide encoding region 660–682 of AC6 on Gα_s regulation of adenylyl cyclases. (A) The region of AC6 containing the predicted PKA site used for the synthesis of the peptide is shown as part of the presumed topographical arrangement of mammalian adenylyl cyclases. Sequences of this region are compared between the different adenylyl cyclases from several species. Alignment of sequences was obtained by using the Genetics Computer Group program in a VAX computer. Fully conserved residues are shown in red. Mostly conserved charged residues are shown in blue, and mostly conserved hydrophobic residues are in green. The existence of a motif conserved amongst the different adenylyl cyclases is visually obvious. (B) AC6 containing Hi-5 cell membranes were assayed in the presence of indicated concentrations of activated Gα_s (α_s*) with or without the FLLT peptide. The data best fit a two-site model (8). Indicated constants were obtained from the two-site fit. (C) AC6 containing Hi-5 cell membranes that had been treated in the absence and presence of PKA were assayed for basal and 20 nM α_s*-stimulated activities in the absence and presence of 300 μM FLLT peptide. (D) Comparison of the effect of the FLLT peptide and a modified peptide (S674D-FLLT peptide) where the Ser corresponding to Ser-674 was substituted with an Asp (D) on AC6 containing Hi-5 cell membrane basal and 20 nM α_s*-stimulated adenylyl cyclase activities. Concentrations of both peptides were 300 μM. (E) Effect of varying concentrations of FLLT peptide on stimulation of AC6 by 17.5 nM α_s*. (F) AC2 containing Hi-5 cell membranes assayed in the presence of indicated concentrations of α_s* in the absence and presence of 300 μM FLLT peptide.