title: Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex.
abstract: The main target of cAMP is PKA, the main regulatory subunit of which (PRKAR1A) presents mutations in two genetic disorders: acrodysostosis and Carney complex. In addition to the initial recurrent mutation (R368X) of the PRKAR1A gene, several missense and nonsense mutations have been observed recently in acrodysostosis with hormonal resistance. These mutations are located in one of the two cAMP-binding domains of the protein, and their functional characterization is presented here. Expression of each of the PRKAR1A mutants results in a reduction of forskolin-induced PKA activation (measured by a reporter assay) and an impaired ability of cAMP to dissociate PRKAR1A from the catalytic PKA subunits by BRET assay. Modeling studies and sensitivity to cAMP analogs specific for domain A (8-piperidinoadenosine 3',5'-cyclic monophosphate) or domain B (8-(6-aminohexyl)aminoadenosine-3',5'-cyclic monophosphate) indicate that the mutations impair cAMP binding locally in the domain containing the mutation. Interestingly, two of these mutations affect amino acids for which alternative amino acid substitutions have been reported to cause the Carney complex phenotype. To decipher the molecular mechanism through which homologous substitutions can produce such strikingly different clinical phenotypes, we studied these mutations using the same approaches. Interestingly, the Carney mutants also demonstrated resistance to cAMP, but they expressed additional functional defects, including accelerated PRKAR1A protein degradation. These data demonstrate that a cAMP binding defect is the common molecular mechanism for resistance of PKA activation in acrodysosotosis and that several distinct mechanisms lead to constitutive PKA activation in Carney complex.
authors.affiliation: From the INSERM U970, Université Paris Descartes, Paris Centre de Recherche Cardiovasculaire, 56 Rue Leblanc, 75015 Paris, France, the Service de Biochimie et Génétique Moléculaire and.
authors.forename: Yara
authors.initials: Y
authors.lastname: Rhayem
authors.affiliation: INSERM U1169, Université Paris Sud, Hôpital Bicêtre, 94270 Le Kremlin Bicêtre, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Le Stunff
authors.affiliation: From the INSERM U970, Université Paris Descartes, Paris Centre de Recherche Cardiovasculaire, 56 Rue Leblanc, 75015 Paris, France.
authors.forename: Waed
authors.initials: W
authors.lastname: Abdel Khalek
authors.affiliation: From the INSERM U970, Université Paris Descartes, Paris Centre de Recherche Cardiovasculaire, 56 Rue Leblanc, 75015 Paris, France.
authors.forename: Colette
authors.initials: C
authors.lastname: Auzan
authors.affiliation: Service d'Endocrinologie, Hôpital Cochin, Assistance Publique, Hôpitaux de Paris, 75014 Paris, France, the Institut Cochin, INSERM U1060, Université Paris Descartes, 75014 Paris, France.
authors.forename: Jerome
authors.initials: J
authors.lastname: Bertherat
authors.affiliation: the Service d'Endocrinologie Pédiatrique, Hôpital Bicêtre, Assistance Publique, Hôpitaux de Paris, 94270 Le Kremlin Bicêtre, France, and.
authors.forename: Agnès
authors.initials: A
authors.lastname: Linglart
authors.affiliation: UMR 1149 INSERM, Université Paris Diderot, ERL CNRS 8252, Faculté de Médecine Site Bichat, 75018 Paris, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Couvineau
authors.affiliation: the Service de Biochimie et Génétique Moléculaire and INSERM U1169, Université Paris Sud, Hôpital Bicêtre, 94270 Le Kremlin Bicêtre, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Silve
authors.affiliation: From the INSERM U970, Université Paris Descartes, Paris Centre de Recherche Cardiovasculaire, 56 Rue Leblanc, 75015 Paris, France, the Service de Biochimie et Génétique Moléculaire and eric.clauser@inserm.fr.
authors.forename: Eric
authors.initials: E
authors.lastname: Clauser
mesh_terms: D019943:Amino Acid Substitution; D060807:Bioluminescence Resonance Energy Transfer Techniques; D056733:Carney Complex / Q000201:enzymology* / Q000235:genetics*; D018389:Codon, Nonsense; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004413:Dysostoses / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008607:Intellectual Disability / Q000201:enzymology* / Q000235:genetics*; D020125:Mutation, Missense; D010009:Osteochondrodysplasias / Q000201:enzymology* / Q000235:genetics*; D010281:Parathyroid Hormone / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D013972:Thyrotropin / Q000494:pharmacology; D014158:Transcription, Genetic
chemical_list: D018389:Codon, Nonsense; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; C517834:PRKAR1A protein, human; D010281:Parathyroid Hormone; D005576:Colforsin; D013972:Thyrotropin; D000242:Cyclic AMP
keywords: Acrodysostosis; CRE-luciferase; Carney complex; PRKAR1A; bioluminescence resonance energy transfer (BRET); cell signaling; functional genomics; protein kinase A (PKA); site-directed mutagenesis