Session: SAT 199-223-Disorders of Bone & Calcium Homeostasis: Case Reports
Poster Board SAT-223
To further document the functional consequences of PRKAR1A mutations on cAMP/PKA signaling and the molecular basis of the phenotype observed in the patients, 5 novel mutations of the cAMP domain B (Q285R, G289E, A328V, R335L, Q372X) were characterized. These 5 mutations were all de novo and identified in unique ADOHR patients.
Studies were performed using western blot analysis (WB), BRET (bioluminescence resonance energy transfer) technology and Cre-luciferase reporter assays (Cre-Luc RA) after transient expression of mutant proteins in HEK293 cells.
All 5 PRKAR1A mutant proteins are expressed (WB). As shown by BRET saturation experiments, the affinity of mutant proteins for PRKAC1A was similar (BRET50 = 0.42 to 0.57) to that observed for WT protein (BRET50 = 0.42). This BRET signal is reduced after addition of a cAMP analog for both the WT and mutant PRKAR1A proteins, reflecting the dissociation between the catalytic and regulatory subunits; however, the dose–response curve for all mutant proteins was significantly shifted to the right (EC50 = 2 to 70nM) as compared to WT PRKAR1A (EC50 = 1nM), indicating a decreased sensitivity to cAMP of the mutant proteins. Cre-Luc RA showed significantly lower CRE–luciferase stimulation with forskolin for mutant as compared to WT PRKAR1A (P<0.01). Interestingly the extent to which the signals for both BRET analysis and Cre-Luc RA were altered varied as a function of the mutations.
These studies further indicate that PRKAR1A mutations identified in ADOHR are dominant gain of function mutations, acting as dominant negative for PKA function and partially inactivating the catalytic subunit. The mutation/phenotype correlations are under investigation.
YR and CLS contributed equally to the work
Nothing to Disclose: YR, CL, AL, CS, ELC
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