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.

5 Abstract The thesis is focused on the role of regulator of G-protein signaling NtRGS1 in control of growth and cell proliferation of tobacco cell line BY-2. The protein NtRGS1 is an important candidate for being plant G-protein coupled receptor. Heterotrimeric G-proteins are involved in key signaling mechanisms in eukaryotic cells. Basic principles of this type of signaling are well conserved between plants and animals and related higher taxa. Outstanding difference of plant G-protein system is altered enzymatic activity of Gα subunit of the G-protein heterotrimer. These alterations correlate with chimeric structure and function of investigated NtRGS1 protein. The interaction of Gα and NtRGS1 is absolutely essential for running of heterotrimeric G-protein signaling in plants. Truncated versions of NtRGS1 fused to GFP were crated in the aim of protein characterization. The truncated proteins were investigated in respect of analysis of the role of NtRGS1 domains in protein targeting. Dynamic changes in NtRGS1 and selected truncated versions induced by experimental application of nutrition, especially sugars were described. Expression if Gα and NtRGS1 were investigated simultaneously. Influence of modulation of Gα and NtRGS1 expression on growth parameters of tobacco cell line BY-2 were described. Key words:...

G protein-coupled receptors (GPCRs) are heptahelical, transmembrane proteins that mediate a plethora of physiological functions by binding ligands and releasing G proteins that interact with downstream effectors. GPCRs signal as monomers, complexes of the same receptor subtype (homomers), or complexes of different receptor subtypes (heteromers). Recently, heteromeric GPCR complexes have become attractive targets for drug development since they exhibit distinct signaling and cell-specific localization from their homomeric counterparts. Yet, the effect of heteromerization on the pharmacology of many GPCR homomers remains unknown. Therefore, we have undertaken the task to examine the effect of heteromerization on Gs signaling through the adenosine 2A receptor (A2AR) and Gi signaling through the dopamine type 2 receptor (D2R) since the A2AR-D2R heteromer is an emerging therapeutic target for Parkinson’s disease (PD). We examined the effect of heteromerization on A2AR and D2R homomeric signaling using electrophysiology and the Xenopus laevis oocyte heterologous expression system. G protein-coupled inwardly rectifying potassium channels (GIRKs) were used as reporters for Gi signaling because activation leads to direct Gbeta-gamma (Gβγ)-mediated stimulation of the GIRK current. We also coupled GIRK channels to Gs signaling by overexpressing Gαs and signaling throughGαsβγ. Our electrophysiological assay is innovative because it allows us to optimize the conditions of heteromerization and directly observe GPCR signaling at the G protein level. Our data demonstrate that heteromer formation alone decreases dopamine-elicited Gi signaling through the D2R and CGS-21680-elicited Gs signaling through the A2AR. Furthermore, this reciprocal antagonism was predominately due to changes in efficacy versus potency. We also examined crosstalk observing that applying agonists or antagonists to the adjacent receptor further modulate this inhibition with the combination of agonists and antagonists relieving inhibition. Mutating the A2AR-D2R heteromer interface abrogated all of the aforementioned ligand-induced effects on G protein signaling through the A2AR-D2R heteromer. We are currently aiming to validate our results from the oocyte experiments with an in vivo model. Our data further elucidate the effect of various ligands on G protein signaling through the A2AR- D2R heteromer, which may facilitate future studies that examine A2AR-D2R heteromer signaling.

Thesis advisor: Charles S. Hoffman / Glucose detection in yeast occurs via a cAMP signaling pathway that is similar to that of other signaling pathways in humans. The presence of glucose in the environment ultimately represses, as a result of cAMP signaling, the transcription of the gene fbp1. Adenylate cyclase is known to convert ATP to cAMP, and is thus a central protein in the propagation of the signal. Mutant forms of the adenylate cyclase gene (git2) have been found by the inability for the organism to repress fbp1 transcription in the presence of glucose. In this study, two questions were under investigation. The first was focused on the ability of the mutations to affect the dimerization of the catalytic domain. The second investigated multiple protein-protein interactions in the leucine rich-repeat (LRR) domain of adenylate cyclase. Both domains contain mutations that confer an activation defect, and they are thus are thought to have a relationship. / Thesis (BS) — Boston College, 2005. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Transmembrane signal transduction by ligand-activated G protein-coupled receptors (GPCRs) controls virtually every aspect of mammalian physiology, and this receptor class is the target of 40-50% of currently marketed pharmaceuticals. In addition to the clinical use of direct GPCR agonists and antagonists, it is now believed that GPCR effectors and regulators may also be viable drug targets with improved therapeutic efficacy and specificity. The prototypic role of Regulator of G protein Signaling (RGS) proteins is inhibition of G protein signaling through acceleration of GTP hydrolysis by GΑ, which promotes re-association of GΑ and GΒΓ subunits with the receptor at the cell membrane. In this way, RGS proteins determine the magnitude and duration of the cellular response to GPCR stimulation. Though RGS protein biochemistry has been well elucidated in vitro, the physiological functions of each RGS family member remain largely unexplored. RGS6 belongs to the R7 subfamily of RGS proteins originally identified in brain. Our acquisition of an RGS6-/- mouse allowed us to survey RGS6 expression in all tissues of the body revealing the greatest expression of RGS6 in brain. Despite robust neural RGS6 expression, little is known regarding functional roles of RGS6 in the brain and spinal cord. In addition, we identified several novel, higher molecular weight RGS6 immunoreactive bands specifically present in the nervous system. The plan of this thesis work was multifaceted. We sought to elucidate novel GPCR signaling cascades modulated by RGS6 in brain while simultaneously characterizing the expression patterns and identity of the novel RGS6 species specifically detected in the nervous system. Considering the large diversity of RGS6 isoforms present in brain, the abundance of potential RGS6 binding partners, and the possibility of discovering new mechanisms involved in RGS6 regulation, elucidation of the novel RGS6 molecular species is of paramount importance. Utilizing RGS6-/- mice we identified RGS6 as a critical modulator of two GPCRs in brain. First, by inhibiting the serotonin receptor 1A (5-HT1AR)-adenylyl cyclase (AC) axis, RGS6 functions to promote anxiety- and depression-related behaviors in mice. As a result, RGS6-/- mice exhibit a robust anxiolytic and antidepressant phenotype remarkably similar to that of animals treated chronically with therapeutic doses of selective serotonin reuptake inhibitors (SSRIs). RGS6 also inhibits GABAB receptor (GABABR)-G protein- activated inwardly rectifying potassium (GIRK) channel current in cerebellar granule cells, and loss of RGS6 results in cerebellar ataxia and gait abnormalities reversible by GABABR blockade. Furthermore, evaluation of voluntary alcohol drinking behaviors in WT versus RGS6-/- mice revealed a striking reduction in alcohol intake resulting from RGS6 loss in both acute and chronic alcohol consumption paradigms due, at least in part, to potentiation of GABABR signaling. Thus, RGS6 inhibitors have potential clinical utility in the treatment of mood disorders and alcoholism. We have shown that one novel RGS6 immunoreactive band expressed in the brain and spinal cord is a phospho-protein sensitive to Λ phosphatase-mediated dephosphorylation. Further, new information acquired from PCR amplification of RGS6 mRNA species from human brain cDNA libraries has necessitated substantial revisions to the RGS6 splicing scheme devised by the Fisher laboratory in 2003. To the 36 isoforms generated from two alternate transcription start sites (RGS6L vs. RGS6), the inclusion or exclusion of exons 14 and 17, and variable splicing to one of 7 different 3' terminal exons, we have added the possible insertion of three novel internal exons (A1, A2, A3), a retained intron, and two new 3' terminal exons. As a result, the number of RGS6 mRNAs present in brain could be as many as 248 unique species, an astonishing diversity unprecedented in the RGS protein family.

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.:Introduction; Materials and methods; Results; Discussion