Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

Regulator of G Protein Signaling 4 (RGS4) mediates motor defects in Parkinson's disease. Small molecule RGS4 inhibitors (e.g. CCG-50014) modify buried cysteine residues, but the structural and dynamic mechanisms underpinning specificity of inhibitors for RGS4 within the RGS family are poorly understood. We used NMR and other biophysical methods to examine ligand-induced structural changes and the dynamics of unliganded RGS4 and RGS8 that allow ligand binding. NMR and fluorescence spectroscopy data reveal details of the hidden, excited conformational state of RGS4 that exposes Cys148, one of the buried cysteines bound by inhibitors. We further show that specificity of RGS4 inhibitors is driven by differential accessibility of the target cysteine compared to its equivalent in RGS8. Cys148 is buried beneath the lid at the center the α4-α7 helix bundle, and this bundle is destabilized in RGS4 compared to RGS8. Notably, helix 6 is highly destabilized in RGS4 compared to RGS8 and is likely the key mediator of access to Cys148. Our findings provide key insight into the mechanism of allosteric RGS4 inhibition and show that dynamics drive inhibitory specificity among RGS proteins.

Background: RGS9-deficient mice show drug-induced dyskinesia but normal locomotor activity under unchallenged conditions. Results: Genes related to Ca2+ signaling and their functions were regulated in RGS9-deficient mice. Conclusion: Changes in Ca2+ signaling that compensate for RGS9 loss-of-function can explain the normal locomotor activity in RGS9-deficient mice under unchallenged conditions. Significance: Identified signaling components may represent novel targets in antidyskinetic therapy. The long splice variant of the regulator of G-protein signaling 9 (RGS9-2) is enriched in striatal medium spiny neurons and dampens dopamine D2 receptor signaling. Lack of RGS9-2 can promote while its overexpression prevents drug-induced dyskinesia. Other animal models of drug-induced dyskinesia rather pointed towards overactivity of dopamine receptor-mediated signaling. To evaluate changes in signaling pathways mRNA expression levels were determined and compared in wild-type and RGS9- deficient mice. Unexpectedly, expression levels of dopamine receptors were unchanged in RGS9-deficient mice, while several genes related to Ca2+ signaling and long-term depression were differentially expressed when compared to wild type animals. Detailed investigations at the protein level revealed hyperphosphorylation of DARPP32 at Thr34 and of ERK1/2 in striata of RGS9-deficient mice. Whole cell patch clamp recordings showed that spontaneous synaptic events are increased (frequency and size) in RGS9-deficient mice while long-term depression is reduced in acute brain slices. These changes are compatible with a Ca2+-induced potentiation of dopamine receptor signaling which may contribute to the drug-induced dyskinesia in RGS9-deficient mice.

In order to function, proteins must be folded into their native shape. While this can sometimes occur spontaneously, the process can be hindered by thermodynamic barriers, trapped intermediates, and aggregation prone hydrophobic interactions. Molecular chaperones are proteins that help client proteins or substrates overcome these barriers so that they can be folded properly. One such chaperone is the chaperonin CCT, a large MDa protein made up of 16 paralogous subunits that form a double ring structure. CCT encapsulates its substrates in a central cavity, where they are sequestered and folded, using ATP binding and hydrolysis to drive conformational changes in the CCT-substrate complex. CCT mediates the folding of many substrates involved in a variety of cellular process, including the cytoskeletal proteins actin and tubulin, and the G protein subunit Gabg, which signals downstream of GPCRs in a variety of cellular processes. We showed that CCT is responsible for folding the b-propeller containing proteins, mLST8 and Raptor, which are subunits of the mTOR complexes. The mTOR complexes (mTORC1 and mTORC2) are master regulators of cell growth and survival by controlling processes such as protein synthesis, energy metabolism, cell survival pathways and autophagy. CCT folds mLST8 and Raptor and help them assemble into the mTOR complexes. As a result, CCT is required for functional mTOR signaling. Furthermore, we solved a 4.0 Ǻ resolution structure of mLST8 bound to CCT. Surprisingly, mLST8 is found in the center of the folding cavity, in between the rings, despite previous evidence suggesting that substrates bind only in the apical domains. Given its role in folding and assembling the mTOR complexes, G proteins, and many other proteins involved in cell survival pathways, CCT has been implicated in cancer. CCT upregulation often correlates with a worse prognosis, likely because uncontrolled growth requires increased chaperone capacity. The peptide CT20P has been shown to have cytotoxic effects in cancer cells, likely through its binding to CCT. We characterized CT20P, showing that it binds to CCT and inhibits its substrate-folding functions in cells. We specifically showed that a GFP-CT20P fusion protein inhibited the assembly of two important signaling complexes Gbg and mTORC1. These results show that CT20P is a useful inhibitor for the study of CCT function.

Phosducin-like protein (PhLP) has been shown to interact with the cytosolic chaperonin containing TCP-1 (CCT), and the βγ subunit dimer of heterotrimeric G proteins (Gβγ). Here we provide details obtained from cryo-electron microscopic and biochemical studies on the structure of the complex between the cytosolic chaperonin CCT and PhLP. Binding of PhLP to CCT occurs through only one of the two chaperonin rings, making multiple contacts with CCT through both its N- and C-terminal domains. In addition, we show that PhLP acts as a co-chaperonin along with CCT in mediating the assembly of the G protein βγ subunit and that assembly is dependant upon the phosphorylation of PhLP by the protein kinase CK2. Variants of PhLP lacking the CK2 phosphorylation sites, or variants with an inability to bind Gβγ block the assembly process and inhibit G protein signaling. PhLP forms a complex with CCT and nascent Gβ prior to the release of Gβγ from the ternary complex and subsequent association with the Gγ subunit to form the Gβγ dimer. In order to understand the mechanism of Gβγ dimer assembly and the role of PhLP phosphorylation in the assembly process, we provide here a method for the purification of the PhLP·CCT·Gβ ternary complex of sufficient purity for structural studies.

Chemokines are important chemical signals that guide lymphocyte movement within the immune system and promote the organization and functions of germinal centers (GCs) in the secondary lymphoid tissues. Previous studies have shown that GC T cells exhibit high expression of chemokine receptor 4, CXCR4, but that these cells are unable to migrate to the ligand for this receptor, the chemokine CXCL12. This “migratory paralysis” to CXCL12 was found to be correlated with the expression of two Regulators of G-protein Signaling, RGS13 and RGS16 in the GC T cells. The objective of my research was to determine whether RGS13 and RGS16 expression were associated with CXCL12-mediated CD4+ T cell migration. Because human GC T cells are rare and vary from one individual to another, I utilized two human neoplastic CD4+ T cell lines (i.e. Hut78 and SupT1) to facilitate and standardize my research. I also confirmed my observations using primary CD4+ T cells. Hut78 cells behaved similarly to GC T cells interms of CXCL12-mediated migration and RGS13 and RGS16 expression, while SupT1 cells appeared similar to CD4+ T cells that resided outside of GCs. The effect of RGS13 and RGS16 expression in the various CD4+ T cells was examined by altering the natural levels of these genes using RNA-mediated silencing and/or gene overexpression analysis after which, I examined the ability of the cells to migrate to CXCL12. RNA-mediated silencing of RGS16-, but not RGS13-, expression in Hut78 T cells resulted in a doubling of the migration rate in response to CXCL12. Over-expression of RGS13 or RGS16 in SupT1 and primary CD4+ T cells resulted in migration that was decreased by fifty percent. Because GC T cells demonstrated decreased migration to CXCL12 signals that may help them leave the GC, I reasoned that these cells may have an increased opportunity over other CD4+ T cells to become infected by the Human Immunodeficiency Virus (HIV) trapped on Follicular Dendritic Cells in the GCs of infected subjects. Examination of GC T cells obtained from HIV-infected subjects indicated that these cells were more frequently infected by HIV than other CD4+ T cells thereby confirming my postulate. My research indicated that RGS13 and RGS16 were associated with CXCL12-mediated CD4+ T cell migration and suggests that these molecules may play an important role in HIV pathogenesis within the GC.

Many proteins cannot fold into their native state without the assistance of one or more molecular chaperones. Chaperonins are an essential class of chaperones that provide an isolated chamber for proteins to fold. CCT, a group II chaperonin found in eukaryotes assists in the folding of actins, tubulins, and many other cellular proteins. PhLP1 is a member of the phosducin protein family that assists CCT in the folding of Gβ and its subsequent assembly with Gγ. However, previous studies have not addressed the scope of PhLP1 and CCT-mediated Gβγ; assembly. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of all Gγ subunits that form dimers with Gβ2 and the assembly of Gγ2 with Gβ1-4, without affecting the specificity of the Gβγ interactions. These findings suggest that PhLP1 has a general role for the assembly of all Gβγ combinations. Although the role of PhLP1 as a co-chaperone for Gβγ assembly has been established, other possible functions for PhLP1 either as a co-chaperone or otherwise are yet to be investigated. A known tumor suppressor protein, PDCD5, was found to interact with PhLP1 in a co-immunoprecipitation proteomics screen. The data presented in Chapter 3 show that PDCD5 binds PhLP1 indirectly through a ternary complex with CCT. Our results signify that the apoptotic function of PDCD5 is cytosolic, is phosphorylation dependent, and most likely involves CCT. Moreover, structural analysis suggests that over-expressed PDCD5 blocks β-actin from entering the CCT folding cavity, suggesting a co-chaperone role for PDCD5 in inhibiting or enhancing folding of yet-to-be determined CCT substrates. Compared to PhLP1, the functions of other members of the phosducin family, PhLP2A, PhLP2B, and PhLP3, are poorly understood. They have no role in G-protein signaling, but appear to assist CCT in the folding of actin, tubulin and proteins involved in cell cycle progression. Chapter 4 investigates the possibility of PhLP2 and/or PhLP3 acting as co-chaperones in the folding and assembly of actins and tubulins. In addition, another mediator of cellular signaling, 14-3-3ε, was found to interact with PhLP2A in a phosphorylation dependent manner and relieve the inhibition of β-actin folding caused by PhLP2A over-expression.

A fundamental question in biology is how proteins, which are synthesized by the ribosome as a linear sequence of amino acids, fold into their native functional state. Many proteins require the assistance of molecular chaperones to maneuver through the folding process to protect them from aggregation and to help them reach their native state in the very concentrated protein environment of the cell. This study focuses on the roles of Phosducin-like Protein 1 (PhLP1) and Programmed Cell Death Protein 5 (PDCD5) as molecular co-chaperones of the Cytosolic Chaperonin Complex (CCT).Signaling in retinal photoreceptors is mediated by canonical G protein pathways. Previous in vitro studies have demonstrated that Gβ subunits rely on CCT and its co-chaperone PhLP1 to fold and assemble into Gβγ and RGS-Gβ5 heterodimers. The importance of PhLP1 in the assembly process was first demonstrated in vivo in a retinal rod photoreceptor-specific deletion of PhLP1. To test whether this mechanism applied to other cell types, we prepared a second mouse line that specifically disrupts the PhLP1 gene in cone photoreceptor cells and measured the effects on G-protein expression and cone visual signal transduction. In PhLP1 depleted cones, Gt2 and RGS9-Gβ5 levels were dramatically reduced, resulting a 60-fold decrease in cone sensitivity and a 50-fold increase in cone photoresponse recovery time. These results demonstrate a common mechanism of Gβγ and RGS9-Gβ5 assembly in rods and cones, underlining the significance of PhLP1/CCT-mediated folding in G protein signaling.PDCD5 has been proposed to act as a pro-apoptotic factor and tumor suppressor. However, the mechanisms underlying its apoptotic function are largely unknown. A proteomics search for PhLP1 binding partners revealed a robust interaction between PDCD5 and CCT. PDCD5 formed a complex with CCT and β-tubulin, a key CCT folding substrate, and specifically inhibited β-tubulin folding. Cryo-electron microscopy studies of the PDCD5-CCT complex suggested a possible mechanism of inhibition of β-tubulin folding. PDCD5 binds the apical domain of the CCTβ subunit, projecting above the folding cavity without entering it. Like PDCD5, β-tubulin also interacts with the CCTβ apical domain, but a second site is found at the sensor loop deep within the folding cavity. These orientations of PDCD5 and β-tubulin suggest that PDCD5 sterically interferes with β-tubulin binding to the CCTβ apical domain and inhibits β-tubulin folding. Given the importance of tubulins in cell division and proliferation, PDCD5 might exert its apoptotic function at least in part through inhibition of β-tubulin folding.

Yes / Background: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins. / This work was supported by the DFG and SFB670. TYR acknowledges support from the Professorinnen Program of the University of Cologne.