In vitro studies of novel PRKAR1A mutants that extend the predicted RIα protein sequence into the 3'-untranslated open reading frame: proteasomal degradation leads to RIα haploinsufficiency and Carney complex.
BACKGROUND
Carney complex (CNC) is a multiple endocrine neoplasia syndrome due to inactivating mutations in the PRKAR1A gene that codes for type Iα regulatory (RIα) subunit of protein kinase A. Most PRKAR1A mutations are subject to nonsense mRNA decay (NMD) and, thus, lead to haploinsufficiency.


METHODS
Patient phenotyping for CNC features and DNA, RNA, protein, and transfection studies were carried out at a research center.


RESULTS
We describe in unrelated kindreds with CNC four naturally occurring PRKAR1A mutations (1055del4, 1067del4ins5, 1076delTTins13, and 1142del4) that are predicted to escape NMD because they are located in the last coding exon of the gene. The phenotype of CNC was not different from that in other patients with the condition, although the number of patients was small. Each of the mutations caused a frameshift that led to a new stop codon into the 3' untranslated open reading frame, predicting an elongated protein that, however, was absent in patient-derived cells. After site-directed mutagenesis, in vitro transcription, and cell-free translation experiments, the expected size mutant proteins were present. However, when the mutant constructs were transfected in adrenal (NCI-295), testicular (N-TERA), and embryonic (HEK293) cells and despite the presence of the mutant mRNA, Western blot analysis indicated that there were no longer proteins. The subsequent application of proteasome inhibitors to cells transfected with the mutant constructs led to the detection of the aberrant proteins, although a compound that affects protein folding had no effect. The wild-type protein was also decreased in both patient-derived cells and/or tissues as well as in the in vitro systems used in this study.


CONCLUSIONS
This was the first demonstration of proteasomal degradation of RIα protein variants leading to PRKAR1A haploinsufficiency and CNC, adding protein surveillance to NMD in the cellular mechanisms overseeing RIα synthesis. In agreement with the molecular data, CNC patients bearing PRKAR1A defects that extend the open reading frame did not have a different phenotype, although this has to be confirmed in a larger number of patients.
Section on Endocrinology and Genetics, Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Building 10, CRC, Room 1-3330, 10 Center Drive, MSC1103, Bethesda, Maryland 20892, USA.
Yianna
Y
Patronas
Anelia
A
Horvath
Elizabeth
E
Greene
Kitman
K
Tsang
Eirini
E
Bimpaki
Michelle
M
Haran
Maria
M
Nesterova
Constantine A
CA
Stratakis
D000293:Adolescent; D000328:Adult; D056733:Carney Complex / Q000235:genetics* / Q000378:metabolism; D002648:Child; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005838:Genotype; D057809:HEK293 Cells; D057895:Haploinsufficiency; D006801:Humans; D008297:Male; D008875:Middle Aged; D009377:Multiple Endocrine Neoplasia / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D016366:Open Reading Frames; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; C517834:PRKAR1A protein, human; D046988:Proteasome Endopeptidase Complex