Fig. 2: Effect of PRKAR1B variants on protein kinase A (PKA) activity and R1β-Cα interaction in HEK293 cells.

(a) PRKAR1B wild-type (WT), A67V and A300T variants, or empty vector (EV) were expressed in HEK293 cells and PKA activity was measured. Both variants showed a lower basal PKA activity (in absence of cAMP, left panel) but only significantly different for A300T compared to the WT. Total PKA activity (in presence of cAMP, right panel) for both variants did not show any differences than the WT. Each experiment was done in duplicate. Data represent mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) with multiple comparisons, **p < 0.01. (b) Results of fluorescence resonance energy transfer (FRET) when R1β-Cα vectors were coexpressed in HEK293 cells. Energy transfer between R1β-Cα was measured before and after photobleaching (0–5 seconds and 6–30 seconds respectively). R1β harboring any of the two variants (p.A67V or p.A300T) appears to bind stronger to the catalytic PKA subunit. Results represent three combined separate experiments. Box plots show median values with upper and lower quartiles, whiskers represent minimal and maximal values. The different groups were compared by Mann–Whitney test, *p < 0.0001.