Mechanisms of Aberrant PKA Activation by Cα Subunit Mutations.

Somatic mutations in PRKACA, coding for the catalytic α subunit of protein kinase A (PKA), have been recently identified as the most frequent genetic alteration in cortisol-secreting adrenocortical adenomas, which are responsible for adrenal Cushing's syndrome. The mutations identified so far lie at the interface between the catalytic (C) and regulatory (R) subunit of PKA. Detailed functional studies of the most frequent of these mutations (L206R) as well as of another one in the same region of the C subunit (199_200insW) have revealed that these mutations cause constitutive activation of PKA and lack of regulation by cAMP. This is due to interference with the binding of the R subunit, which keeps the C subunit inactive in the absence of cyclic AMP. Here, we review these recent findings, with a particular focus on the mechanisms of action of PRKACA mutations.

Real-time monitoring of GPCR/cAMP signalling by FRET and single-molecule microscopy.

G-protein-coupled receptors (GPCRs), located on the surface of virtually every cell in our organism, mediate the effects of many hormones and neurotransmitters. Although GPCRs have been extensively studied for more than 4 decades using pharmacological and biochemical methods, the recent introduction of optical methods such as fluorescence resonance energy transfer (FRET) and single-molecule microscopy is fostering novel and important discoveries in the field. Here, we review the use of such optical methods, focusing on some recent examples of their application to important and still unresolved questions concerning the spatial organisation and dynamics of GPCR signalling.

Thyroid-stimulating hormone receptor activity after internalization.

The thyroid-stimulating hormone (TSH) receptor (TSHR) belongs to the large family of G-protein-coupled receptors (GPCRs) and is predominantly coupled to G(s). Thus, the effects of TSH are largely mediated by the stimulation of adenylyl cyclase and the ensuing rise of intracellular cyclic AMP (cAMP) concentrations. Like for other GPCRs, a prolonged stimulation of the TSHR leads to its internalization into endosomes followed by its recycling to the cell surface. Until recently, GPCRs were believed to activate "classical" G-protein-dependent pathways only when located on the cell surface and to cease doing so upon agonist-induced internalization. However, our recent findings on the TSHR and similar ones on the parathyroid hormone and sphingosine receptors suggest that internalized GPCRs can continue to signal through G(s)-cAMP in an intracellular compartment. Interestingly, this type of intracellular cAMP signaling differs from that occurring on the cell surface, as it is persistent and apparently leads to specific signaling outcomes. Although further studies are needed to investigate the possible physiological and pathophysiological consequences of GPCR-cAMP signaling in the endocytic compartment, endosomes should no longer be viewed as passive carriers for receptors en route to degradation but rather as specialized intracellular platforms for GPCR signaling.

Absence of primary hypothyroidism and goiter in Slc26a4 (-/-) mice fed on a low iodine diet.

BACKGROUND: Mutations in the SLC26A4 gene, coding for the anion transporter pendrin, are responsible for Pendred syndrome, characterized by congenital sensorineural deafness and dyshormonogenic goiter. The physiological role of pendrin in the thyroid is still unclear and the lack of a thyroid phenotype in some patients with SLC26A4 mutations and in Slc26a4 (-/-) mice indicate the existence of environmental or individual modifiers able to compensate for pendrin inactivation in the thyroid. Since pendrin can transport iodide in vitro, variations in iodide supply have been claimed to account for the thyroid phenotype associated with pendrin defects. AIM: The Slc26a4 (-/-) mouse model was used to test the hypothesis that iodide supply may influence the penetrance and expressivity of SLC26A4 mutations. MATERIALS AND METHODS: Slc26a4 (-/-) and (+/+) mice were fed up to 6 months on a standard or low iodine diet and were evaluated for thyroid structural abnormalities or biochemical hypothyroidism. RESULTS: A 27-fold iodide restriction induced similar modifications in thyroid histology, but no differences in thyroid size, T4 or TSH levels were observed between between Slc26a4 (-/-) and (+/+) mice, either in standard conditions and during iodine restriction. CONCLUSIONS: Iodide restriction is not able to induce a thyroid phenotype in Slc26a4 (-/-) mice. These experimental data, together with those coming from a review of familial Pendred cases leaving in regions either with low or sufficient iodide supply, support the idea that the expression of thyroid phenotype in Pendred syndrome is more powerfully influenced by individual factors than by dietary iodide.

Thyroid gland development and function in the zebrafish model.

Thyroid development has been intensively studied in the mouse, where it closely recapitulates the human situation. Despite the lack of a compact thyroid gland, the zebrafish thyroid tissue originates from the pharyngeal endoderm and the main genes involved in its patterning and early development are conserved between zebrafish and mammals. In recent years, the zebrafish has become a powerful model not only for the developmental biology studies, but also for large-scale genetic analyses and drug screenings, mostly thanks to the ease with which its embryos can be manipulated and to its translucent body, which allows in vivo imaging. In this review we will provide an overview of the current knowledge of thyroid gland origin and differentiation in the zebrafish. Moreover, we will consider the action of thyroid hormones and some aspects related to endocrine disruptors.

Styrene oxide-induced 2'-deoxycytidine adducts: implications for the mutagenicity of styrene oxide.

The reaction between 2'-deoxycytidine and styrene 7,8-oxide (SO) resulted in alkylation at the 3-position and at the O(2)-position through the alpha- and beta-carbons of the epoxide but at the N(4)-position only through the alpha-carbon. The 3-alkylated adducts were found to deaminate to the corresponding 2'-deoxyuridine adducts (37 degrees C, pH 7.4) with half-lives of 6 min and 2.4 h for the alpha- and beta-isomers, respectively. The N(4)-alkylated products were stable at neutral pH. The O(2)-alkylated products were unstable being prone to depyrimidation and to isomerisation between alpha- and beta-isomers. In SO-treated double-stranded DNA, enzymatic hydrolysis allowed the identification of the beta3-deoxyuridine and alphaN(4)-deoxycytidine adducts (1.9 and 0.5% of total alkylation, respectively), in addition to the previously identified DNA-adducts. The 3-substituted uracil may have implications for the mutagenicity of SO.