1,720,986 research outputs found
Signaling by internalized G-protein-coupled receptors
No full textG-protein-coupled receptors (GPCRs) are cell surface receptors and are generally assumed to signal to second messengers such as cyclic AMP (cAMP) exclusively from the plasma membrane. However, recent studies indicate that GPCRs can continue signaling to cAMP after internalization together with their agonists. Signaling from inside the cell is persistent and appears to trigger specific downstream effects. Here, we will review these recent data, which form the basis for a novel concept of intracellular GPCR signaling and suggest new and intriguing
scenarios for the functions of GPCRs in the endocytic compartment. We propose that current models of GPCR signaling should be revised to accommodate the ability of receptors to change their signaling properties depending on their subcellular localization
Technology insight : modern methods to monitor protein-protein interactions reveal functional TSH receptor oligomerization
No full textThe formation of supramolecular structures (dimers or oligomers) is emerging as an important aspect of G-protein-coupled receptor (GPCR) biology. In some cases, GPCR oligomerization is a prerequisite for membrane targeting or function; in others, the relevance of the phenomenon is presently unknown. Although supramolecular structures of GPCRs were initially documented by classical biochemical techniques such as coimmunoprecipitation, many recent advances in the field of GPCR oligomerization have been prompted by the introduction of two new biophysical assays based on Förster's resonance energy transfer-fluorescence resonance energy transfer and bioluminescence resonance energy transfer. These modern techniques allow the study of protein-protein interaction in intact cells, and can be used to monitor monomer association and dissociation in vivo. Recently, oligomerization has also been reported in the case of the TSH receptor (TSHR). This review will focus on the previously unsuspected implications that oligomerization has in TSHR physiology and pathology. It is now clear that TSHR oligomerization is constitutive, occurs early during post-translational processing, and may be involved in membrane targeting and activation by the hormone or by stimulating antibodies. Oligomerization between inactive mutants and wild-type TSHR provides a molecular explanation for the dominant forms of TSH resistance
Direct visualisation of cyclic AMP levels in three dimensional cultures of thyroid follicles
No full textCyclic AMP (cAMP) is the principal intracellular mediator of TSH effects in the thyroid, inducing both thyroid hormone production and cell proliferation. This notion derives from a large series of evidences, mainly obtained by biochemical approaches on cell lines or primary thyrocytes. However, given the limited resolution in time and space of the techniques employed so far, little is know about the spatial localization and temporal dynamics of cAMP signaling in thyroid cells. Moreover, it is well known that the functional unit of the thyroid is a multicellular structure, i.e. the thyroid follicle. Therefore, a deeper understanding of TSH/cAMP cascade and of thyrocyte biology in general will probably require an in vivo approach, either on isolated thyroid follicles or on whole animals. The aim of this study was to develop a method for real-time monitoring of cAMP levels in living thyroid follicles. To this purpose we established a protocol for three dimensional culture of thyroid follicles, which results in good morphology and allows microscopic visualization. To monitor intracellular cAMP levels, we employed a Fluorescence Resonance Energy Transfer based sensor (Epac1-cAMPs), the fluorescence of which is inversely proportional to cAMP levels. Thyroid follicles obtained from a transgenic mouse expressing Epac1-cAMPs were then isolated and cultured as mentioned above. By this approach we were able to evaluate the kinetics of cAMP accumulation and clearance after TSH stimuli of different intensity and duration. The kinetics observed after a short application of TSH were unexpectedly fast, with t1/2 of ~1 and 3 min. for cAMP accumulation and degradation, respectively. These data are consistent with an unpredicted speed of TSH association and dissociation from its receptor. The newly developed method paves the way to a series of in vivo studies aimed at further elucidating the spatio-temporal organization of TSH/cAMP signaling cascade
Effects of 8-Cl-cAMP on growth and apoptotic process in poorly differentiated thyroid cencer cell lines
No full textTools that are highly effective in the treatment of differentiated thyroid cancer (DTC) loose their therapeutic potentials in poorly differentiated tumors. The synthetic analog 8-Cl-cAMP has been known to have an antiproliferative effect in a variety of cancer cells and is tested as antineoplastic agent in clinical trials. The signaling mechanisms that govern the 8-Cl-cAMP-induced growth inhibition are still uncertain and data in thyroid neoplasia are lacking. Therefore, we tested the effects of 8-Cl-cAMP on the growth and apoptotic process in anaplastic (ARO), papillary (NPA) and follicular (WRO) thyroid carcinoma cell lines. Our proliferation data show that growth of ARO, NPA and WRO was inhibited by more than 50% after the treatment with 8-Cl-cAMP in a time- and dose-dependent manners. To test whether apoptosis occurs in 8-Cl-cAMP treated cells, we analyzed cell cycle, DNA fragmentation and caspase activity. We found induction of apoptosis in all the cell lines with different sensitivities. Since MAPKs are involved in the regulation of proliferation and apoptosis, we investigated modification of ERKs, that are preferentially activated in response to mitogens, and p-38 MAPKs, that are activated in response to cell stresses. Following the treatment with 8-Cl-cAMP, no modification of ERK phosphorylation was found while a marked and progressive induction of p38-MAPK phosporylation was seen in all the cell lines. We also evaluated the Akt phosphorylation, as a marker of the PI3K proliferative pathway that as been implicated in thyroid cell proliferation, and we found poor modifications of Akt phosphorylation state after 8-Cl-cAMP treatment.
In conclusion, 8-Cl-cAMP has a potent inhibitory effect on WRO, NPA and ARO cell growth which is accompanied by a pro-apoptotic effect via p38-MAPK. Therefore, 8-Cl-cAMP has a potential to be tested in vivo as a therapeutic agent for poorly DTC
Syndromes of hormone resistance in the hypothalamic-pituitary-thyrod axis
No full textForty years have elapsed since the first description of a syndrome of resistance in the hypothalamic-pituitary-thyroid axis, i.e., resistance to thyroid hormone action. In the last two decades many other types of resistance have been discovered, including resistance to the action of thyrotropin-releasing hormone (TRH), of thyroid-stimulating hormone (TSH), and of thyroid hormones (THs); the latter can be due not only to thyroid hormone receptor defects but also to alteration in genes encoding TH-specific transporters or components involved in metabolic pathways of THs. Moreover, alteration in genes encoding for second messengers may cause forms of resistance other than those due to receptor mutations, the most important one being that of an inactivating mutation in the G-protein α-subunit leading to TSH resistance in the setting of pseudohypoparathyroidism type 1a. Recognition of these rare thyroid disorders is of great importance not only for informed genetic counselling but also for avoiding diagnostic mistakes that may lead to incorrect and potentially dangerous treatments
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
PKA II mediates cAMP-dependent phosphorylation of CREB and activation of gene transcription in differentiated rat thyroid cells
No full textThyroid stimulating hormone (TSH) is the key modulator of thyroid cell function. Binding of TSH to its receptor (TSHR) results in an increase of cAMP intracellular concentration, which activates protein kinase A (PKA) to phosphorylate CREB and other substrates. Eventually, this leads to gene transcription modification associated with both proliferation and differentiation. Two major types of PKA (PKA I and II) exist in mammal cells, but their specific role in TSH signaling is poorly understudy so far. Therefore, the aim of this study was to dissect PKA I/II effects on gene transcription in thyroid cells. In order to selectively activate PKA I or II, pairs of cAMP analogs with different affinities for sites A and B of PKA regulatory subunits were used. The effect of PKA I or II activation on the transcription of early genes, genes involved in cell replication and genes associated with thyroid differentiated function was evaluated in FRTL5 cells by quantitative real-time PCR. The results indicate that PKA II, but not PKA I, stimulation can mimic the effect of TSH treatment on these genes. In addition, pre-treatment with a PKA II inhibitor was able to abolish the effect of TSH. Since CREB is the major transcriptional mediator of cAMP signal, we also evaluated the effect of PKA I or II activation on CREB phosphorylation. Also in this case, only PKA II activation lead to efficient CREB phosphorylation, and TSH-dependent activation was abolished by pre-incubation with the PKA II inhibitor. Finally, silencing of PKA regulatory subunit 2B (Prkar2b) by RNA interference resulted in down-regulation of proliferative genes. These results indicate that PKA II is the principal mediator of cAMP transcriptional effects in thyroid cells. Further studies are needed to evaluate the role of PKA I, which could play a role in post-transcriptional modifications rather than directly affecting gene transcription
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Somatostatin Receptor Type 2 (SSTR2) Internalization and Intracellular Trafficking in Pituitary GH-Secreting Adenomas: Role of Scaffold Proteins and Implications for Pharmacological Resistance
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