Functional Characterization of PRKAR1A Mutations Reveals a Unique Molecular Mechanism Causing Acrodysostosis but Multiple Mechanisms Causing Carney Complex.

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.

Clinical and pathophysiological implications of chromosomal alterations in adrenocortical tumors: an integrated genomic approach.

PURPOSE: Diagnosing malignancy of adrenocortical tumors (ACT) and predicting prognosis in carcinomas are often challenging. Transcriptome markers have recently emerged, providing promising clinical relevance and improved pathophysiological knowledge. Whether tumoral chromosomal alterations provide similar information is not known. The aim was to evaluate the diagnostic and prognostic value of chromosomal alterations in ACT and to identify genes associated with benign and malignant tumorigenesis. EXPERIMENTAL DESIGN: Chromosomal alterations of 86 adenomas and 52 carcinomas were identified by comparative genomic hybridization arrays and/or quantitative PCR. RESULTS: A larger proportion of the genome is altered in carcinomas compared with adenomas (44 vs. 10%, P = 2.10(-10)). In adenomas, the 9q34 region, which includes the steroidogenic factor 1 locus, is commonly gained and associated with an overexpression of steroidogenic factor 1 (SF-1). In carcinomas, recurrent gains include chromosomes 5, 7, 12, 16, 19, and 20 and recurrent losses chromosomes 13 and 22. Filtering the genes from these regions according to their expression profile identified genes potentially relevant to adrenocortical tumorigenesis. A diagnostic tool was built by combining DNA copy number estimates at six loci (5q, 7p, 11p, 13q, 16q, and 22q). This tool discriminates carcinomas from adenomas in an independent validation cohort (sensitivity 100%, specificity 83%). In carcinomas, the number of chromosomal alterations was not associated with survival (Cox P = 0.84). A prognostic tool based on tumor DNA was designed with a clustering strategy and validated in an independent cohort. CONCLUSIONS: Chromosomal alterations in ACT discriminate carcinomas from adenomas and contain prognostic information. Chromosomal alterations alter the expression of genes important for tumorigenesis.

Recurrent PRKAR1A mutation in acrodysostosis with hormone resistance.

The skeletal dysplasia characteristic of acrodysostosis resembles the Albright's hereditary osteodystrophy seen in patients with pseudohypoparathyroidism type 1a, but defects in the α-stimulatory subunit of the G-protein (GNAS), the cause of pseudohypoparathyroidism type 1a, are not present in patients with acrodysostosis. We report a germ-line mutation in the gene encoding PRKAR1A, the cyclic AMP (cAMP)-dependent regulatory subunit of protein kinase A, in three unrelated patients with acrodysostosis and resistance to multiple hormones. The mutated subunit impairs the protein kinase A response to stimulation by cAMP; this explains our patients' hormone resistance and the similarities of their skeletal abnormalities with those observed in patients with pseudohypoparathyroidism type 1a.

[Structure and functions of the angiotensin II AT1 receptors during evolution]. / Structure et fonctions des récepteurs AT1 de l'angiotensine II au cours de l'évolution.

Angiotensin II AT1 receptor is a G protein coupled receptor, which transduces the physiological effects (vasoconstriction, aldosterone secretion) f this vasoactive peptide. On an evolutionary point of view, this receptor has appeared early in the development of vertebrates, since it is present in cartilagenous fish. It has been duplicated in rodents without any consequence on its functions. It is unlikely that the angiotensin AT2 receptor, whose functions are still debated, has diverged from a common ancestral angiotensin receptor with the AT1 receptor. Numerous activating or inactivating point mutations have been identified by site-directed mutagenesis of the AT1 receptor sequence. However, such natural mutations do not appear to be frequent in the genesis of human diseases or in the diversity of phenotypic traits.

Mechanisms of cell-surface rerouting of an endoplasmic reticulum-retained mutant of the vasopressin V1b/V3 receptor by a pharmacological chaperone.

Cell-surface expression and biological functions of several intracellular-retained G protein-coupled receptors are restored by membrane-permeable ligands called pharmacological chaperones. We have previously demonstrated that a mutation of the hydrophobic motif 341FNX2LLX3L350 in the C terminus of the human pituitary vasopressin V3 receptor (MUT V3R) led to it being retained in the endoplasmic reticulum (ER). Here, we establish the precise role of this motif and investigate whether SSR149415, a non-peptide V3R antagonist, behaves as a pharmacological chaperone for the ER-retained MUT V3R. The absence of the mutated receptor in the plasma membrane is linked to its prolonged association with the molecular chaperone calnexin in the ER and to its intensive degradation by the ubiquitin-proteasomal machinery. However, this is not because of a lack of oligomerization, as demonstrated by the presence of MUT V3R homodimers in the ER. Treatment with SSR149415 restores expression of the mutated receptor on the cell surface and its correct maturation, resulting into the functional recovery of its signaling properties. SSR149415 acts by stabilizing a native-like conformation of the V3R, reducing its association with calnexin and, thus, favoring a secretory pathway rather than the proteasomal degradation pathway. In conclusion, the FN(X)2LL(X)3L sequence is an important motif for the V3R conformation, and the misfolding resulting from its mutation alters the receptor export but can be reverted by SSR149415.

Steroidogenesis in aldosterone-producing adenoma revisited by transcriptome analysis.

CONTEXT: Primary aldosteronism (PAL) is the most frequent cause of secondary arterial hypertension. In PAL, aldosterone production is chronic, excessive, and autonomous. OBJECTIVE: The objective of this study was to identify the angiotensin-II independent alterations of steroidogenesis responsible for PAL. DESIGN: Genomewide gene expression was compared in two tissues differentiated for aldosterone production, both nonstimulated by circulating angiotensin II and differing in their autonomy to produce aldosterone: aldosterone-producing adenoma (APA) and its adjacent dissected zona glomerulosa (ZG). SETTING: The setting of this study was the Comete Network. PATIENTS: Patients with APA were studied. INTERVENTION: Transcriptome comparison was made of one APA and its adjacent ZG by serial analysis of gene expression; validation by in situ hybridization was performed for 19 genes in 11 samples. OUTCOME: The study outcome was genes differentially expressed in APA and adjacent ZG. RESULTS: Activation of steroidogenesis in PAL is restricted to the overexpression of the enzymes producing aldosterone-specific steroids, aldosterone synthase and also 21-hydroxylase, suggesting that upstream precursor production is not limiting. Increased expression of high-density lipoprotein receptor, adrenodoxin and P450 oxidoreductase suggests that these systems provide cholesterol and electrons to the mitochondrial steroidogenic enzymes. As for acute stimulation of aldosterone production, an activation of calcium signaling is suggested by concordant overexpression of calcium-binding proteins or effectors. Calcium activation may result from an abnormal activity of G(q) protein-coupled receptors. This calcium activation may be the starting point of the other gene expression changes observed in APA. Finally, other differentially expressed genes include three genes encoding unidentified proteins. CONCLUSION: This work provides an original and integrated view of the mechanisms of aldosterone production in PAL.