Functional characterization of PRKAR1A mutants causing acrodysostosis with hormonal resistance

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SAT 199-223-Disorders of Bone & Calcium Homeostasis: Case Reports
Clinical
Saturday, June 15, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SAT-223
Yara Rhayem1, Catherine Le-Stunff2, Agnes Linglart3, Caroline Silve4 and Eric Laurent Clauser*1
1INSERM U970, Paris, France, 2INSERM U986, Hôpital Bicêtre, Bicêtre, France, 3INSERM U986, Service d'Endocrinologie Pédiatrique, Hôpital Bicêtre, Bicêtre, France, 4INSERM U986, Hôpital Bicêtre, Le Kremlin Bicêtre, France
Acrodysostosis is a rare genetic disease characterized by a skeletal dysmorphic syndrome. One genetic cause in patients with acrodysostosis and multihormonal resistance (PTH, PTHRP, TSH) (ADOHR) was recently identified as mutations of the PRKAR1Agene (1), the major regulatory subunit of cAMP dependent protein kinase (PKA), a pivotal actor of the cAMP signaling pathway. PRKAR1A binds to the catalytic subunit (PRKAC1A) inhibiting its enzymatic activity. The binding of cAMP to the domains A and B of PRKAR1A activates the enzyme. To date, 9 PRKAR1A mutations located in cAMP binding domains have been identified associated with ADOHR (2). The functional characterization of 2 of them suggests an inhibitory effect of the PRKAR1A mutants on PKA activity (1,3).

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

(1) Linglart A, et al N Engl J Med 2011; 364:2218-2226. (2) Silve C, et al IBMS BoneKEy 2012; 225:1-7. (3) Nagasaki K, et al J Clin Endocrinol Metab 2012; 97: E1808–E1813.

Nothing to Disclose: YR, CL, AL, CS, ELC

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm

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