Natriuretic peptides form a family of structurally-related peptides known to regulate salt and water homeostasis and to cause vasodilation. Synthesis of atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides occurs mainly in the heart and brain and has been identified recently in the female reproductive tract. The expression of ANP and CNP, as well as their cognate guanylyl cyclase receptors (NPR-A and NPR-B, respectively), have been detected in the rat ovary. / We have shown previously that the expression of the natriuretic peptides and their receptors, in the rat ovary, is modulated by the estrous cycle. Since estrogen and the gonadotropins (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) are important regulators of follicular development and ovarian function we hypothesized that expression of the natriuretic peptide system is modulated by these hormones. In order to test this hypothesis, the expression of the natriuretic peptide system (peptide and receptor) was evaluated in ovarian cells (granulosa and residual tissue cells) obtained from immature female rats treated with either diethylstilbestrol (DES), an estrogen analogue, or equine chorionic gonadotropin (eCG), a gonadotropin which possesses both LH and FSH activity. (Abstract shortened by UMI.)

Regulators of G protein signaling (RGS) are proteins that negatively regulate G protein-coupled receptor signaling. Although a conserved core domain is necessary and sufficient for their GTPase accelerating protein (GAP) activity, many RGSs possess C- and N-terminal protein-binding motifs that augment GAP activity and participate in other cellular regulatory mechanisms. / Human RGS 1 functionally complements a Saccharomyces cerevisiae mutant lacking the RGS homologue Sst2p. We demonstrate that deletion of the N-terminus or RGS domain negatively affects this ability in Sst2p-deficient strains, whereas deletion of the C-terminal 10 residues of RGS 1 does not. Coexpression of the N-terminus and RGS domains restores complementation of Sst2p to that of wild type. The conservative replacement of sequential residues spanning the N-terminus of RGS1 causes little loss of function. These results suggest that the N-terminal and RGS domains of RGS I function in concert to effect signaling and that the C-terminal 10 residues of RGS I are not required for this activity. Further, residues present in the N-terminus are not highly conserved suggesting that overall structure, rather than individual residues or motifs, may be important for function.

The eIF2α kinases PERK and PKR are well characterized regulators of protein synthesis. When they are activated in response to various cellular stresses, they inhibit translation initiation by phosphorylating eIF2α on a key residue. Recently, PKR has been shown to regulate the expression of specific proteins independent of its ability to inhibit protein synthesis, and we wished to identify additional targets of this particular function. The cellular levels of cyclin D1, a cell cycle control protein that regulates progression through G1, decrease under conditions that activate various eIF2α kinases. We demonstrate that this is not due to the inhibition of cyclin D1 synthesis, but rather the induction of cyclin D1 degradation by the 26S proteasome. This degradation is ubiquitin-dependent, and although cyclin D1 synthesis is unaffected eIF2α phosphorylation is required. Our work provides evidence of a functional cross-talk between the eIF2α kinases and the degradation of specific proteins during periods of acute stress. PKR has also been shown to regulate the function of the transcription factors Stat1 and Stat3, which mediate the cellular response to a wide variety of extracellular signals. In order for these proteins to accumulate in the nucleus and drive gene transcription, they must first be phosphorylated on a conserved tyrosine residue. The transcriptional activity of Stat proteins is tightly regulated, and therefore the phosphorylation and subsequent dephosphorylation events are critically important. Herein we demonstrate that the tyrosine phosphorylation of Stat1 and Stat3 is compromised by the catalytic activity of PKR, and that this proceeds through the nuclear phosphatase TC-PTP. This decrease in tyrosine phosphorylation abrogates the transcriptional activity of Stat1 and Stat3 normally observed in response to IFN-γ and IL-6 stimulation, respectively. Phosphorylation of both TC-PTP and eIF2α by PKR is required for the inhibition of Stat1 / Les kinases PERK et PKR sont des régulateurs reconnus de la synthèse de protéines. En réponse à différents stress cellulaires, elles empêchent l’initiation de la traduction en phosphorylant un résidu-clé du module alpha de la protéine eIF2. Récemment, PKR a été montrée de régler l'expression des protéines spécifiques independent de sa capacité d'empêcher la synthèse de protéine, et nous avons souhaité identifier les cibles additionnelles de cette fonction particulière. Les niveaux cellulaires de cycline D1, une protéine de contrôle qui dirige la progression du cycle cellulaire à travers G1, diminuent lorsque PERK et PKR sont activés. Nous démontrons que ceci ne découle pas de l’inhibition de la synthèse de cycline D1, mais plutôt de l’induction de sa dégradation par le protéasome 26S. Cette dégradation dépend de l’ubiquitine et requiert la phosphorylation d’eIF2 au module alpha, bien que la synthèse de cycline D1 ne soit pas affectée. Nos travaux fournissent des preuves concluantes d’un échange fonctionnel entre les kinases eIF2α et la dégradation de protéines exprimées durant les périodes de stress aiguë.Il a été démontré que PKR régule aussi la fonction des facteurs de transcription Stat1 et Stat3, qui aiguillonnent la réponse cellulaire à une grande variété de signaux extracellulaires. La phosphorylation de ces protéines sur un résidu tyrosine mène à leur exportation et à leur accumulation dans le noyau cellulaire, d’où elles stimulent la transcription génique. L’activité transcriptionnelle des protéines Stat est strictement contrôlée et chaque événement de phosphorylation ou de déphosphorylation revêt une importance cruciale. Nous révélons que la phosphorylation d’un résidu tyrosine de Stat1 et Stat3 est compromise par l’activité catalytique de PKR et que la phosphatase nucléaire TC-PTP en est la responsable. Cette diminution des niveaux de phosphorylation du rési

The pathway of membrane insertion and assembly of a signal anchor sequence specific for the outer mitochondrial membrane has been investigated. Signal anchor protein insertion into the outer membrane, in vitro, was found to overlap with a general import pathway followed by the outer membrane protein, porin, as well as matrix-targeted proteins. However, signal anchor protein insertion did not require a postreceptor import step involved in porin insertion and matrix protein translocation. Also, in contrast to the membrane insertion of porin, signal anchor protein insertion did not require nucleoside triphosphates for transfer of bound precursor to the membrane assembled form. Following outer membrane integration, a hybrid protein containing the signal anchor sequence of yeast Tom70 was found to assemble into homodimers. Dimerization was mediated by the transmembrane domain. A sequence motif containing alanine residues clustered on one face of the predicted membrane-spanning $ alpha$-helix was important for dimerization, perhaps allowing favourable close packing of the transmembrane helices.

The neuron-specific K+/Cl- cotransporter 2 (KCC2) is one of the major cation Cl- cotransporter (CCC) proteins in the central nervous system that controls Cl- homeostasis via mediating Cl- extrusion. KCC2 has been established as an essential protein in promoting the maturation of synaptic inhibition during development and controlling neuronal excitibility in the adult central nervous system (CNS). Although various mechanisms are known to regulate KCC2 gene expression, accumulating evidence has shown KCC2 activity can be altered on much shorter time scales in a manner of 10-20 minutes. This suggested post-translational mechanisms, which take place much more rapidly than gene expression, may also be an important means by which KCC2 function can be regulated. However, what these post-translational mechanisms are and how they contribute to the control of KCC2 function remain elusive. In this thesis, I set out to characterize the molecular mechanisms governing the post-translational regulation of KCC2, including its endocytosis, quaternary assembly, and cell surface expression. Specifically, my results suggest: 1) Endogenous KCC2 interacts with the clathrin-mediated endocytosis (CME) machinery; 2) KCC2 is constitutively endocytosed via a CME-dependent mechanism; 3) A di-leucine motif, 657LL658, is essential for both KCC2 constitutive endocytosis and the binding of KCC2 to the CME adaptor protein AP-2 complex; 4) Two regions within KCC2 proximal and central carboxyl terminus respectively mediate KCC2 dimerization, and in particular mutating the 657LL658 residues to alanines completely abolishes KCC2 dimerization; 5) The 657LL658 sequence is highly conserved amongst closely related K+/Cl- cotransporter proteins, but absent from the more distant Na+/Cl- cotransporters controlling Cl- uptake, suggesting an evolutionarily conserved mechanism may regulate the constitutive endocytosis and dimerization of functionally homologous KCC members; and 6) KCC2 expression at the neur / Le co-transporteur d’ions K+ et Cl- (KCC2), spécifique aux neurones, est l’un des plus importants co-transporteurs de cations chlorure (CCC) du système nerveux central. Il contrôle l’homéostasie de la cellule en régulant l’extrusion d’ions chlorure. KCC2 est reconnu comme étant une protéine essentielle à la maturation de l’inhibition synaptique au cours du développement et au contrôle de l’excitabilité des neurones dans le système nerveux central de l’adulte. Bien que les mécanismes régulant l’expression de KCC2 soient connus, de nouvelles données suggèrent que l’activité de KCC2 peut aussi être modifiée sur de plus courtes périodes, de l’ordre de 10 à 20 minutes. Ceci suggère qu’un contrôle post-traductionnel, agissant beaucoup plus rapidement qu’un contrôle transcriptionnel, pourrait aussi être important dans la régulation de l’activité de KCC2. Toutefois, la nature de ces mécanismes post-traductionnels et la façon par laquelle ils contribuent à la fonction de KCC2 demeurent inconnus.Dans cette thèse, je vise à caractériser les mécanismes moléculaires qui gouvernent la régulation post-traductionnelle de KCC2, incluant son expression à la surface cellulaire, son endocytose et son assemblage quaternaire. Plus spécifiquement, mes résultats suggèrent que: 1) Le KCC2 endogène interagit avec la machinerie d’endocytose par clathrines (CME) ; 2) Le KCC2 est endocyté constitutivement par un mécanisme dépendant des clathrines; 3) Le motif di-leucine 657LL658 est essentiel non seulement à l’endocytose constitutive de KCC2, mais aussi à l’interaction entre KCC2 et le complexe protéique adaptateur AP-2; 4) Deux régions à l’intérieur de KCC2, l’une proximale et l’autre centrale à l’extrémité C-terminale, sont responsables de la dimérisation de KCC2 qui peut être abrogée par la substitution des leucine 657 et 658 par des alanines; 5) La séquence 657LL658 est très conservée par

The cadherins are a family of calcium-dependent cell adhesion molecules. The regulators of cadherin expression have not been identified. We have examined the ability of hormones to regulate the expression of cadherin subtypes present in the mouse testis, ovary and uterus. 17-$ beta$ estradiol caused a rapid and significant increase in N-cadherin mRNA levels in the Sertoli cells of the testis and granulosa cells of the ovary. This hormone was also capable of stimulating E-cadherin mRNA levels in the epithelial cells of the ovary and uterus. Progesterone was capable of increasing E-cadherin mRNA levels in the uterus but not in the ovary. These studies are the first demonstration that steroids are capable of regulating cadherin mRNA levels in vivo. Furthermore, this is the first demonstration of a physiological role for 17-$ beta$ estradiol in the testis. / Follicle stimulating hormone was capable of increasing N-cadherin mRNA levels in mouse testes and in cultured mouse Sertoli cells. The stimulatory effects of FSH on Sertoli cells were mimicked by cholera toxin and forskolin, suggesting the involvement of the cAMP signal transduction pathway. Furthermore, there was a synergistic effect between 17-$ beta$ estradiol and elevated levels of cAMP on N-cad mRNA levels in cultured Sertoli cells. Our observations indicate that steroids and follicle-stimulating hormone are key regulators of cadherin expression.

The distribution of glucocorticoid receptors (GR) in the rat testis was examined in vivo using (3H)-dexamethasone 21-mesylate (DM) which binds covalently to GR. After injection with $ sp3$H-DM, the testes of adrenalectomized, adult rats were processed for light microscope (LM) radioautographic (RAG) analysis. Control rats received simultaneously a 25-or 50- fold excess of cold dexamethasone. Quantitation of the label confirmed the presence of specific DM binding sites in both Leydig cells (86% of the label) and to a lesser degree, in the cellular layers of the seminiferous epithelium (14% of the label). These binding data were confirmed by LM immunocytochemistry. Interstitial macrophages were non-specifically labeled. In the Leydig cell, an electron microscope (EM) quantitative RAG analysis of 3H-DM binding sites showed that smooth endoplasmic reticulum (sER) and mitochondria were heavily labeled, with 53% and 31% of the total label, respectively. Cytosol (exclusive of all structures) and nucleus showed comparatively weak labeling, with 9% and 7% of the label, respectively. All other structures showed little or no labeling.

An immunocytochemical procedure for the human glucocorticoid receptor (GR) was developed herein in order to assess its level and subcellular distribution in a well-studied system of childhood acute lymphoblastic leukemia cells (CEM), in which sensitive and resistant subclones have been established, and in different tissue preparations (lymph node biopsies, bone marrow aspirates and peripheral blood) from patients with various forms of leukemia or lymphoma. In the absence of preincubation with steroid, the GR was localized predominantly in the cell cytoplasm and to a weaker extent in the nucleus of CEM cells. Following incubation with glucocorticoid, an increase in nuclear GR was observed in the steroid-sensitive CEM-C7 and C7E$ sb2$A cell lines but not in the resistant C7 subclones (4R4, 3R43 and ICR27). Incubation of human peripheral mononuclear cells with 10$ sp{-6}$M dexamethasone for 30 or 60 minutes in vitro resulted in a marked increase in nuclear GR immunoreactivity. Since nuclear translocation of GR is a critical step in glucocorticoid hormone action on target cells, assessment of the subcellular distribution of GR may provide functional tests for steroid responsiveness. Furthermore, the effect of glucocorticoids on the regulation of GR and its mRNA was studied by immunocytochemistry, RIA and in situ hybridization.

No description available.

Multidrug resistance (MDR) in cancer is a phenomenon that impairs efficient treatment that has now become a major hinderance to chemotherapy. ABC transporters such as the P-glycoprotein (P-gp or ABCB1) and the multidrug resistance-associated protein 1 (MRP1 or ABCC1) are known to actively efflux chemotherapeutic agents out of cells thereby inducing MDR. A promising approach in order to specifically target MDR cells is to take advantage of their increased sensitivity (collateral sensitivity) to certain compounds. The second chapter of this thesis aims to elucidate the mechanism of collateral sensitivity in MRP1 cells. Overall, the results show a hypersensitivity of these cells to glutathione (GSH) modulating agents. Such hypersensitivity in cells is caused by the presence of MRP1, which extrudes GSH out of the cell. Collateral sensitivity can be achieved by stimulating the extrusion with verapamil (Vrp), Apigenin (Api) or by inhibiting the GSH synthesis with butathione sulfoximine (BSO). Finally, GSH depletion leads to reactive oxygen species (ROS) accumulation, which likely triggers apoptosis. The third and fourth chapters aim toresolve the collateral sensitivity mechanism induced in P-gp expressing cells. The study of Vrp, progesterone (Pro) and deoxycorticosterone (DOC) induced collateral sensitivity lead to the conclusion that two requirements ar necessary for validating this phenomenon. The first requirement, common to the three compounds, is the presence of and active P-gp. This P-gp sees its ATPase activity stimulated and the electron transport chain higher demand generated leads to the formation of higher levels of ROS. It seems that these ROS alone are not sufficient and they need to potentiate something else. In the case of Vrp it seems that increase Ca2+ level would be the second requirement. For Pro and DOC, the presence of cholesterol at the membrane levels seems to be enough. This allows the / La résistance multiple à la chimiothérapie (MDR) dans le cancer est un phénomène qui nuit à l'efficacité du traitement et est devenue un important problème. Les transporteurs ABC comme la P-glycoprotéine (P-gp ou ABCB1) et la protéine associée à la résistance multiple 1 (MRP1 ou ABCC1) sont reconnue pour pomper de façon active les médicaments hors des cellules et de ce fait induisent la MDR. Une approche prometteuse visant à cibler spécifiquement ces cellules MDR est de prendre avantage de leur sensibilité accrue (sensibilité collatérale) face à certains composés.Le second chapitre de cette thèse visait à élucider le mécanisme de sensibilité collatérale chez les cellules exprimant MRP1. Ensemble les résultats montre une sensibilité accrue de ces cellules face aux agents modulant la GSH. Cette hypersensibilité est causée par la présence de MRP1 qui pompe la GSH hors de la cellule. La sensibilité collatérale a pue être obtenue en stimulant le pompage en utilisant la verapamil (Vrp) ou l'Apigenin (Api), ou en inhibant la synthèse de la GSH avec la buthionine sulfoximine (BSO). Finalement, la diminution de la GSH résulte en une accumulation d'espèce réactive d'oxygène (ROS) déclanchant l'apoptose.Les chapitres trois et quatre étaient dirigés vers la résolution du mécanisme de sensibilité collatérale induite chez les cellules exprimant la P-gp. L'étude de la sensibilité collatérale induite par la Vrp, la progestérone (Pro) et la désoxycorticostérone (DOC) a mené conclusion que deux pré-requis sont nécessaire pour l'obtention du phénomène. Le premier, commun pour les trois composés, est la présence d'une P-gp active. Cette P-gp voit sont activité ATPase être stimulé et l'élévation de la demande de la chaîne de transport d'électrons entraîne la formation de niveau élevés de ROS. Il semble que ces ROS seuls ne sont pas suffisant et ils on bes