Persistent cAMP-Signals Triggered by Internalized G-Protein–Coupled Receptors

Real-time monitoring of G-protein-coupled receptor (GPCR) signaling in native cells suggests that the receptor for thyroid stimulating hormone remains active after internalization, challenging the current model for GPCR signaling

Involvement of β-catenin and unusual behavior of CBP and p300 in glucocorticosteroid signaling in Schwann cells

In the nervous system, glucocorticosteroid hormones play a major role during development and adult life. Myelin-forming cells are among the targets of glucocorticosteroids, which have been shown to promote myelination both in the central and peripheral nervous system. Glucocorticosteroid-stimulated gene transcription is mediated by the glucocorticosteroid receptor (GR) that recruits coactivators of the p160 family, forming a docking platform for secondary coactivators, such as cAMP-response element binding protein (CREB)-binding protein (CBP) or its close homologue, p300. Here, we investigated the role of CBP and p300 in mouse Schwann cells (MSC80). We show that, although the CBP/p300 binding domain of steroid receptor coactivator-1 is crucial for GR transactivation, neither CBP nor p300 enhanced GR transcriptional activation, as shown by overexpression and small interfering RNA (siRNA) knocking-down experiments. Unexpectedly, overexpression of p300, considered as a coactivator of the GR, resulted in inhibition of GR transcriptional activity. Studies with p300 deletion mutants demonstrated that p300-dependent repression is related to its acetyltransferase activity. Functional and pull-down assays showed that β-catenin may be the coactivator replacing CBP in the GR transcriptional complex. Our results suggest the formation of a GR-coactivator complex within Schwann cells, indicating that glucocorticosteroids may act by means of unusual partners in the nervous system, and we show a repressive effect of p300 on nuclear receptors

GH/STAT5 signaling during the growth period in livers of mice overexpressing GH

GH/STAT5 signaling is desensitized in the liver in adult transgenic mice overexpressing GH; however, these animals present greater body size. To assess whether the STAT5 pathway is active during the growth period in the liver in these animals, and how signaling modulators participate in this process, growing transgenic mice and normal siblings were evaluated. STAT5 does not respond to an acute GH-stimulus, but displays higher basal phosphorylation in the livers of growing GH-overexpressing mice. GH receptor and the positive modulators glucocorticoid receptor and HNF1 display greater abundance in transgenic animals, supporting the activity of STAT5. The negative modulators cytokine-induced suppressor and PTP1B are increased in GH-overexpressing mice. The suppressors SOCS2 and SOCS3 exhibit higher mRNA levels in transgenic mice but lower protein content, indicating that they are being actively degraded. Therefore, STAT5 signaling is increased in the liver in GH-transgenic mice during the growth period, with a balance between positive and negative effectors resulting in accelerated but controlled growth.Fil: Martinez, Carolina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Piazza, Verónica Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Díaz, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Boparal, Raavnet K.. Southern Illinois University; Estados UnidosFil: Arum, Oge. Southern Illinois University; Estados UnidosFil: Ramirez, Maria Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Gonzalez, Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Becu, Damasia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Bartke, Andrzej. Southern Illinois University; Estados UnidosFil: Turyn, Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Miquet, Johanna Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; ArgentinaFil: Sotelo, Ana Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentin

Thyrotropin receptor trafficking relies on the hScrib–βPIX–GIT1–ARF6 pathway

G protein-coupled receptors are regulated by ligand stimulation, endocytosis, degradation of recycling to the cell surface. Little information is available on the molecular mechanisms underlying G protein-coupled receptors recycling. We have investigated recycling of the G protein-coupled thyroid stimulating hormone receptor (TSHR) and found that it relies on hScrib, a membrane-associated PDZ protein. hScrib directly binds to TSHR, inhibits basal receptor endocytosis and promotes recycling, and thus TSHR signalling, at the cell membrane. We previously demonstrated that hScrib is associated with a βPIX–GIT1 complex comprised of a guanine nucleotide exchange factor and a GTPase-activating protein for ADP ribosylation factors that is involved in vesicle trafficking. We used dominant-negative constructs and small interfering RNA to show that TSHR recycling is regulated by the interaction between hScrib and βPIX, and by the activity of GIT1. In addition, ARF6, a major target for GIT1, is activated during TSH stimulation of HEK293 and FRTL-5 thyroid cells, and plays a key role in TSHR recycling. Thus, we have uncovered an hScrib–βPIX–GIT1–ARF6 pathway devoted to TSHR trafficking and function

The endocytic protein GRAF1 is directed to cell-matrix adhesion sites and regulates cell spreading

The rho GTPase-activating protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1) remodels membranes into tubulovesicular clathrin-independent carriers (CLICs) mediating lipid-anchored receptor endocytosis. However, the cell biological functions of this highly prevalent endocytic pathway are unclear. In this article, we present biochemical and cell biological evidence that GRAF1 interacted with a network of endocytic and adhesion proteins and was found enriched at podosome-like adhesions and src-induced podosomes. We further demonstrate that these sites comprise microdomains of highly ordered lipid enriched in GRAF1 endocytic cargo. GRAF1 activity was upregulated in spreading cells and uptake via CLICs was concentrated at the leading edge of migrating cells. Depletion of GRAF1, which inhibits CLIC generation, resulted in profound defects in cell spreading and migration. We propose that GRAF1 remodels membrane microdomains at adhesion sites into endocytic carriers, facilitating membrane turnover during cell morphological changes

The polarity protein Scrib mediates epidermal development and exerts a tumor suppressive function during skin carcinogenesis

Background: The establishment and maintenance of polarity is vital for embryonic development and loss of polarity is a frequent characteristic of epithelial cancers, however the underlying molecular mechanisms remain unclear. Here, we identify a novel role for the polarity protein Scrib as a mediator of epidermal permeability barrier acquisition, skeletal morphogenesis, and as a potent tumor suppressor in cutaneous carcinogenesis. Methods: To explore the role of Scrib during epidermal development, we compared the permeability of toluidine blue dye in wild-type, Scrib heterozygous and Scrib KO embryonic epidermis at E16.5, E17.5 and E18.5. Mouse embryos were stained with alcian blue and alizarin red for skeletal analysis. To establish whether Scrib plays a tumor suppressive role during skin tumorigenesis and/or progression, we evaluated an autochthonous mouse model of skin carcinogenesis in the context of Scrib loss. We utilised Cre-LoxP technology to conditionally deplete Scrib in adult epidermis, since Scrib KO embryos are neonatal lethal. Results: We establish that Scrib perturbs keratinocyte maturation during embryonic development, causing impaired epidermal barrier formation, and that Scrib is required for skeletal morphogenesis in mice. Analysis of conditional transgenic mice deficient for Scrib specifically within the epidermis revealed no skin pathologies, indicating that Scrib is dispensable for normal adult epidermal homeostasis. Nevertheless, bi-allelic loss of Scrib significantly enhanced tumor multiplicity and progression in an autochthonous model of epidermal carcinogenesis in vivo, demonstrating Scrib is an epidermal tumor suppressor. Mechanistically, we show that apoptosis is the critical effector of Scrib tumor suppressor activity during skin carcinogenesis and provide new insight into the function of polarity proteins during DNA damage repair. Conclusions: For the first time, we provide genetic evidence of a unique link between skin carcinogenesis and loss of the epithelial polarity regulator Scrib, emphasizing that Scrib exerts a wide-spread tumor suppressive function in epithelia