FIGURE 4.

Effect of PRKAR1A mutations occurring in acrodysostosis on PKA regulatory and catalytic subunit dissociation. HEK293 cells were co-transfected with a fixed amount of plasmid, resulting in the expression of PRKAR1A-luciferase constructs carrying either the WT PRKAR1A sequence or the indicated mutations with or without a saturating amount of plasmid resulting in the expression of YFP-PRKACA. 48 h later, cells were lysed, and cell lysates were exposed or not to the indicated doses of 8-bromo-cAMP (top), 8-AHA-cAMP (middle), or 8-PIP-cAMP (bottom), and the BRET signal was measured as described under “Materials and Methods.” The signal in the absence of YFP-PRKACA was considered as 0%, and that in the presence of YFP-PRKACA without the cAMP analog was considered as 100%. To evaluate the EC₅₀, pooled data for each mutant relating the cAMP analog concentration and the BRET signal were fitted to a sigmoid curve with variable slope. Values for variants with mutations in domain A are connected by dashed lines. The mean, S.D., and statistical analysis for data from the individual experiments are shown in Table 2.