Upon sperm entry, the vertebrate egg undergoes a series of cell divisions that create a number of smaller cells without increasing the embryonic mass. This induces an elevation of intracellular calcium transient that is conserved across species. In zebrafish, fertilization occurs through an opening in the chorion, the micropyle and in Xenopus it can occur anywhere in the animal hemisphere. Wnt signaling activation is required during dorsal-ventral axis specification and it needs to be suppressed during the regionalization of the brain. Axin is a negative regulator of Wnt signaling and contains an RGS (Regulator of G Protein Signaling) domain. RGS domains are typical of RGS proteins, which are involved in a distinct signaling pathway, G-protein signaling. RGS proteins exert a negative effect of G-protein signaling by accelerating the GTPase activity (GAP) of the Gα subunit, thus turning off the signaling. Axin contains an RGS domain, however, it is not clear whether Axin is directly involved in G-protein signaling. We will also present a work performed using another negative regulator of the Wnt signaling network called naked cuticle (Nkd). Nkd has been shown to modulate β-catenin dependent and independent Wnt signaling. In chapter 2, we will show that the Axin-RGS like function is dispensable during the formation of the dorsal-ventral axis. We manipulated this protein by creating a point mutation in a critical aminoacid within the Axin-RGS domain, known to be detrimental for the GAP function of RGS proteins, Axin1Q162A. Maternal depletion of Axin1 in Xenopus oocytes causes hyperactivation of Wnt signaling and results in dorsalization. Axin1Q162A is able to suppress the dorsalization of maternally depleted embryo and restore normal dorsa-ventral axis formation. In chapter 3, we will describe the role of Axin during the patterning of the vertebrate brain. We show that the point mutant is not able to restore normal brain development in zebrafish embryos after Axin knockdown. We hypothesize that Axin-RGS like function is necessary during the patterning of the vertebrate brain that occurs after zygotic transcription has been initiated. Moreover, we show that Axin-RGS like activity may be dispensable during this stage of development. Finally, we demonstrate that Axin1Q162A localization differs from the wildtype Axin1 and Axin1 but not Axin1Q162A is localized to the plasma membrane upon Gα overexpression in zebrafish embryos. Embryonic organ laterality is preceded by molecular and physiological asymmetries. In chapter 4 we describe the role of another Wnt antagonis, Nkd cuticle, during left-right patterning. Prior to organogenesis, a group of cells called Dorsal Forerunner Cells, (DFCs), migrate ahead of the dorsal blastoderm during gastrulation to form the Kupffer's vesicle (KV). This vesicle will trigger a signaling cascade that will culminate with left-right determination. We show data that support the requirement of Nkd in organ laterality and convergence and extension movements using zebrafish and Xenopus laevis.

The process of vertebrate development requires communication among many cells of the embryo in order to define the body axis (front/back, head/tail or left/right). The Wnt signaling network plays a key role in a vast array of cellular processes including body axis patterning and cell polarity. One arm of the Wnt signaling network, the non-canonical Wnt pathway, mediates intracellular Ca2+ release via activation of heterotrimeric G proteins. Regulator of G protein Signaling (RGS) proteins can accelerate inactivation of G proteins by acting as G protein GAPs and are uniquely situated to control the amplitude of a Wnt signal. I hypothesize that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this modulation is essential for developmental patterning events. To this end, this thesis is focused on the effects G protein regulation has on intracellular Ca2+ release dynamics and developmental patterning events. I combine cellular, molecular and genetic analyses with high resolution Ca2+ imaging to provide new understanding of the role of RGS proteins on Wnt mediated Ca2+ release dynamics and developmental patterning events. In chapter 2, I describe endogenous Ca2+ dynamics from the very first cell divisions through early somitogenesis in zebrafish embryos. I find that each phase of zebrafish development has a distinct pattern of Ca2+ release, highlighting the complexity of Ca2+ ion and cellular physiology. In Chapter 3, I identify rgs3 as potential modulator of Ca2+ dynamics and Chapter 4 expands upon these observations by providing data supporting that Rgs3 function is necessary for appropriate frequency and amplitude of Ca2+ release during somitogenesis and that Rgs3 functions downstream of Wnt5 activity. My results provide new evidence that a member of the RGS protein family is essential for modulating the non-canonical Wnt network to assure normal tissue patterning during development. In Chapter 5, I report the identification and characterization of Rgs3b, a paralogue to Rgs3, in zebrafish. I describe results indicating that Rgs3b is poised to interact with Wnt11 indicating that individual RGS genes may have unique roles in modulating Wnt/Ca2+ signaling in different tissues or different stages. In conclusion, this thesis provides data supporting that individual RGS proteins are critical in modulating the frequency and amplitude of Wnt/Ca2+ signaling in different tissues and at different developmental stages and this is a substantial breakthrough in understanding how RGS proteins function to fine-tune known signaling pathways

Atrial arrhythmias are very common clinically relevant conditions that are strongly associated with aging and parasympathetic tone. Additionally, ATP-sensitive K+ (KATP) channel activation has been reported to facilitate the development of re-entrant atrial arrhythmias. Since KATP channels are direct effectors of Gαi/o and RGS4 is an inhibitor of Gαi/o-signaling, we here investigate whether KATP channel activity is increased under decreased RGS4 activity in a manner that enhances susceptibility to AF. We show that loss of RGS4 facilitates the induction of atrial arrhythmias under parasympathetic challenge both in whole animals and isolated atrial tissues. Furthermore, using both genetic disruption (Kir6.2 ablation) and pharmacologic blockade (tolbutamide), we show that loss of functional KATP channels decreases the incidence of pacing-induced re-entry and prolongs repolarization in RGS4-deficient atria. Our findings are consistent with the conclusion that enhanced KATP channel activity may contribute to pacing-induced re-entrant rotors in the RGS4-deficient mouse model.

Atrial arrhythmias are very common clinically relevant conditions that are strongly associated with aging and parasympathetic tone. Additionally, ATP-sensitive K+ (KATP) channel activation has been reported to facilitate the development of re-entrant atrial arrhythmias. Since KATP channels are direct effectors of Gαi/o and RGS4 is an inhibitor of Gαi/o-signaling, we here investigate whether KATP channel activity is increased under decreased RGS4 activity in a manner that enhances susceptibility to AF. We show that loss of RGS4 facilitates the induction of atrial arrhythmias under parasympathetic challenge both in whole animals and isolated atrial tissues. Furthermore, using both genetic disruption (Kir6.2 ablation) and pharmacologic blockade (tolbutamide), we show that loss of functional KATP channels decreases the incidence of pacing-induced re-entry and prolongs repolarization in RGS4-deficient atria. Our findings are consistent with the conclusion that enhanced KATP channel activity may contribute to pacing-induced re-entrant rotors in the RGS4-deficient mouse model.

Regulators of G protein signaling (RGSs) are negative regulators of G protein coupled receptors (GPCRs). Our lab has demonstrated that yeast Saccharomyces cerevisiae is a useful system to study RGS and G protein signaling. Mammalian RGSs can be expressed in yeast and favored to interact with mammalian GPCRs as well. / Based on the observation that human RGS1 causes yeast cell growth arrest, I therefore used RGS1 expressing yeast cells to screen a mouse T cell cDNA library in order to find potential interacting proteins. From the screen, I identified a mouse sphingomyelin synthase 1 (SMS1) cDNA. By using a series of different apoptotic stimuli, such as hydrogen peroxide, osmotic stress, exogenous ceramide and its precursors, high temperature etc., SMS1 expression was found to suppress cell growth arrest and prevent viability decline, indicating that SMS1 represents an anti-apoptotic protein that functions by decreasing the intracellular level of pro-apoptotic ceramide. / Gene analysis further indicated that the SMS1 gene consists of 16 exons spread over a 256kb portion of mouse chromosome 19. It is alternatively spliced to produce 4 different transcripts (SMS1alpha1, SMS1alpha2, SMS1beta and SMS1gamma) and encode 3 different proteins (SMS1alpha, SMS1beta and SMS1gamma). Notably, I found that SMS1beta protein does not interfere with SMS1alpha anti-apoptotic function, although both of these two proteins contain the protein-protein interaction domain, sterile alpha motif (SAM), at their N-terminus. / I also carried out a study to examine GPCR-RGS interactions using the yeast expression system. Our lab had noticed that there was an extra RGS5 related protein that was detected by western blot analysis in the protein extracts prepared from yeast and HEK293 cells expressing RGS5. The size of the band was approximately 2 times the molecular weight of RGS5, indicating the possibility that RGS5 forms a dimer. To further examine this hypothesis, I, therefore, performed a series of experiments, included yeast 2 hybrid assays, to demonstrate that RGS5 does interact with itself. This is the first report that RGS can form a dimer. The implications for this finding are discussed in detail.

No description available.

Les protéines RGS (Regulator of G-protein Signaling) sont des inhibiteurs des voies de signalisation des protéines G. RGS4 atténue l’activité de protéines G dans plusieurs tissus tel que la diminution de son activité peut accroître la sévérité de la bradycardie, cardiomyopathies liées au diabète, l’invasion de cellule cancéreuse du sein, résistance à l’insuline et intolérance au glucose. RGS4 a été localisé à la membrane plasmique ainsi que dans des compartiments intracellulaires, cependant, son mode de trafique intracellulaire reste méconnu. En utilisant des outils de microscopie confocale sur cellules vivantes et méthode de détection d’activité des voies de signalisation conditionnée par les protéines G, nous avons caractérisé l’importance de deux sites de palmitoylation, ces deux sites : Cys2 et Cys12 montrent des intérêts complémentaires dans le trafic de RGS4 vers la membrane cellulaire. Dans un axe linéaire, nous avons identifié DHHC3 et 7, deux enzymes de palmitoylation participant au trafique intracellulaire de RGS4 et donc à la maximalisation de son activité inhibitrice des voies de signalisation contrôlées par Galphaq. Enfin des marqueurs de membranes endosomales, les protéines rab ont permis de caractériser les voies de trafic intracellulaire empruntée par RGS4, par exemple RGS4 est internalisé de la membrane plasmique par Rab5 et recyclé à la membrane cellulaire par Rab11. L’activation ou inhibition de Rab5 et 11 ont permis d’observer des changements d’activité de RGS4. Ces travaux confèrent une base de données pour des études ultérieures visant à développer des stratégies thérapeutiques à accroître les fonctionnalités de RGS4. / RGS proteins (Regulator of G-protein Signaling) are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues such that loss of its function may lead to bradycardia, diabetic cardiomyopathy, breast cancer cell invasion, insulin resistance and glucose intolerance. RGS4 has been localized to both plasma membrane and intracellular pools, however, the nature of its intracellular trafficking remains to be elucidated. G-protein inhibition requires the presence of RGS4 at the plasma membrane. In this work, we characterized the complementary roles of two putative palmitoylation sites on RGS4 to target intracellular compartments and plasma membrane. We identified palmitoylation on Cys2 and 12 respectively important for RGS4 endosomal targeting and plasma membrane localization, when mutations were introduced to the palmitoylation sites, RGS4 capability of inhibiting Gq-mediated signaling was impaired. As a continuum we identified two palmitoylating enzymes, DHHC3 and 7 as modulator of RGS4 localization and function. Knock downs of DHHC3 and 7 impaired RGS4 endosomal and plasma membrane targeting and capability of inhibiting M1-muscarinic receptor signaling. Finally we used live cell confocal microscopy to define RGS4 intracellular trafficking routes. Specifically Rab5 mediated RGS4 trafficking from the plasma membrane to intracellular compartments while Rab11 mediated RGS4 trafficking to the plasma membrane. Activation and inhibition of Rab5 and 11 routes impaired RGS4 capability of inhibiting M1-muscarinic receptor signaling pathway. These novel findings provide a strong rationale for future studies aimed at developing new strategies to increase the function of RGS4.

Regulators of G protein signaling (RGS) are a diverse group of proteins, which play a fundamental role in modulation of G protein coupled receptor signal transduction. RGS proteins are primarily known as GTPase activating proteins (GAPs) for Gá subunits. In addition to the RGS domain, which is responsible for GAP activity, most RGS proteins also contain other structural motifs. The R7 family of RGS proteins for example, which consists of RGS-6, 7, 9 and 11 gene products, also contains DEP, DHEX and GGL domains. All R7 RGS proteins are obligatory binding partners with G protein beta subunit, G beta sub five, which binds to the GGL domain. In my dissertation work, I provide insights into significance of the multi-domain architecture of G beta sub five-RGS7. I have identified a novel intramolecular interaction within the G beta sub five-RGS7 complex; between the DEP domain of RGS7 and G beta sub five subunit. My experimental evidence supports the idea that G beta sub five-RGS7 can exist in at least two hypothetical conformations: "closed" where the DEP domain and G beta sub five subunit are bound to each other, and "open" where DEP and G beta sub five are not interacting, and as a result both these proteins can associate with other binding partners. My results indicate that in its "open" conformation, G beta sub five-RGS7 can selectively inhibit calcium mobilization elicited by stimulated muscarinic acetylcholine receptor type 3 (M3R). This inhibition is mediated by direct interaction between the third intracellular loop of M3R and the DEP domain of RGS7. In addition to the effect on M3R signaling, I observed that the G beta sub five-RGS7 complex redistributes from the cytosol to endocytic vesicles in an M3R-specific manner. These results identify a novel molecular mechanism that can impart receptor-subtype selectivity on signal transduction via G protein-coupled receptors. Lastly, I have identified a small group of compounds that inhibits the DEP-G beta sub five interaction. These compounds may serve as starting points for design of G beta sub five-RGS7 modulators in the future.

No description available.

Univerzita Karlova v Praze Přírodovědecká fakulta Studijní program: Fyzikální chemie Mgr. Lenka Řežábková Studium úlohy proteinů 14-3-3 v regulaci G-proteinové signalizace Role of the 14-3-3 proteins in the regulation of G-protein signaling Disertační práce Školitel: doc. RNDr. Tomáš Obšil, Ph.D. Konzultanti: doc. RNDr. Petr Heřman, CSc. doc. RNDr. Jaroslav Večeř, CSc. Praha, 2012 Abstract The 14-3-3 family of phosphoserine/phosphothreonine-binding proteins dynamically regulates the activity of their binding partners in various signaling pathways that control diverse physiological and pathological processes such as signal transduction, metabolic pathways, cell cycle and apoptosis. More than 300 different cellular proteins from diverse eukaryotic organisms have been described as binding partners for the 14-3-3 proteins. During my Ph.D., I was particularly interested in the role of 14-3-3 proteins in the regulation of G protein signaling pathway. The 14-3-3 proteins affect the G protein signaling via the interaction with negative regulators of G protein cascade - the RGS proteins and phosducin. I employed both biochemical and biophysical approaches to understand how the activity and function of RGS3/14-3-3 and phosducin/14-3-3 complexes are regulated. I solved the low-resolution solution structure of...