Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty 
skin pigmentation and endocrine overactivity. CNC is caused by inactivating 
mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit 
(RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA 
dysregulation to endocrine tumorigenesis are poorly understood. In this study, 
we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA 
activity to define the role of RIα in the spatiotemporal organization of the 
cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as 
forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as 
reported by western blots of endogenous PKA targets and the FRET-based global 
PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular 
compartments, we identified similar increases in the response to PGE1 in the 
cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma 
membrane, the response to PGE1 was decreased along with an increase in basal 
FRET ratio. These results were confirmed by western blot analysis of basal and 
PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated 
phosphoprotein. Similar differences were observed between the cytoplasm and the 
plasma membrane in human adrenal cells carrying a RIα inactivating mutation. RIα 
inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial 
membrane and at the plasma membrane, as reported by targeted versions of the 
cAMP indicator Epac1-camps. These results show that RIα inactivation leads to 
multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new 
aspects of signaling dysregulation in tumorigenesis.