Gene therapy is a promising treatment option for cancer. Ideally, a therapeutic gene is delivered specifically into tumor cells sparing the neighboring normal cells. For this purpose gene delivery vectors are designed that can recognize structures, which are exclusively expressed on tumor cells (i.e. the tumor-associated antigens -TAA-). Retroviral vectors are commonly used for gene therapy by modifying the envelope protein responsible for the recognition of the target cell. The Vesicular Stomatitis Virus G protein (VSV-G) is a well-liked choice for pseudotyping the retroviral vectors since it confers on the viral particle stability to allow concentration to high titers necessary for the clinical applications. However, the main drawback of VSV-G, the ubiquitously expressed receptor and thus the broad target range, hinders the use of this protein for targeted gene therapy. In this thesis, we aimed to modify the VSV-G for targeted gene therapy against Prostate Stem Cell Antigen (PSCA) -expressing tumors. Therefore we followed two approaches. The first approach comprised of the fusion of a single-chain antibody fragment against PSCA to the N-terminus of VSV-G. In the second approach the VSV-G was modified by insertion of a small epitope. We could demonstrate that two positions in the N-terminal region of VSV-G protein permit insertion of a ten amino acid long epitope. These mutant VSV-G proteins were successfully assembled into retroviral particles. We demonstrated that the mutant retroviral particles can be used for targeting to PSCA-positive cells using nanobeads. The nanobeads were chemically coupled to antibodies against the epitope in the VSV-G protein and PSCA on the tumor cell. These bispecific nanobeads allowed the recruitment of mutant retroviral particles to the PSCApositive cells. Our results point out the potential of these mutant retroviral particles in targeted gene delivery. Further studies will be necessary to assess the efficiency of in vivo targeted gene therapy using these mutant retroviral particles.

No description available.

In order for G protein signaling to occur, the G protein heterotrimer must be assembled from its nascent polypeptides. The most difficult step in this process is the formation of the Gβγ dimer from the free subunits since both are unstable in the absence of the other. Recent studies have shown that phosducin-like protein (PhLP1) works as a co-chaperone with the cytosolic chaperonin complex (CCT) to fold Gβ and mediate its interaction with Gγ. However, these studies did not address questions concerning the scope of PhLP1 and CCT-mediated Gβγ assembly, which are important questions given that there are four Gβs that form various dimers with 12 Gγs and a 5th Gβ that dimerizes with the four regulator of G protein signaling (RGS) proteins of the R7 family. The data presented in Chapter 2 shows that PhLP1 plays a vital role in the assembly of Gγ2 with all four Gβ1-4 subunits and in the assembly of Gβ2 with all twelve Gγ subunits, without affecting the specificity of the Gβγ interactions. The results of Chapter 3 show that Gβ5-RGS7 assembly is dependent on CCT and PhLP1, but the apparent mechanism is different from that of Gβγ. PhLP1 seems to stabilize the interaction of Gβ5 with CCT until Gβ5 is folded, after which it is released to allow Gβ5 to interact with RGS7. These findings point to a general role for PhLP1 in the assembly of all Gβγ combinations, and suggest a CCT-dependent mechanism for Gβ5-RGS7 assembly that utilizes the co-chaperone activity of PhLP1 in a unique way. Chapter 4 discusses PhLP2, a recently discovered essential protein, and member of the Pdc family that does not play a role in G protein signaling. Several studies have indicated that PhLP2 acts as a co-chaperone with CCT in the folding of actin, tubulin, and several cell cycle and pro-apoptotic proteins. In a proteomics screen for PhLP2A interacting partners, α-tubulin, 14-3-3, elongation factor 1α, and ribosomal protein L3 were found. Further proteomics studies indicated that PhLP2A is a phosphoprotein that is phosphorylated by CK2 at threonines 47 and 52.

No description available.

<p>The neuromodulator dopamine signals through the dopamine D2 receptor (D2R) to modulate central nervous system functions through diverse signal transduction pathways. D2R is a prominent target for drug treatments in disorders where dopamine function is aberrant, such as schizophrenia. D2R signals through distinct G protein and β-arrestin pathways and drugs that are functionally selective for these pathways could have improved therapeutic potential. How D2R signals through the two pathways is still not well defined, and efforts to elucidate these pathways have been hampered by the lack of adequate tools for assessing the contribution of each pathway independently. To address this, Evolutionary Trace was used to produce D2R mutants with strongly biased interactions for either G protein or β-arrestin. Additionally, various permutations of these mutants were used to identify critical determinants of D2R functional selectivity. D2R interactions with the two major downstream signal transducers were effectively dissociated and G protein signaling accounts for D2R canonical MAP kinase signaling cascade activation. Nevertheless, when expressed in mice, the β-arrestin biased D2R caused a significant potentiation of amphetamine-induced locomotion, while the G protein biased D2R had minimal effects. The mutant receptors generated here provide a new molecular tool set that enable a better definition of the individual roles of G protein and β-arrestin signaling in D2R pharmacology, neurobiology and associated pathologies.</p> / Dissertation

Oocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2. / text

Lysophosphatidic acid (LPA) belongs to a class of extracellular lipid signaling molecules. In the vasculature, LPA may regulate platelet activation and modulate endothelial and smooth muscle cell function. LPA has therefore been proposed as a mediator of cardiovascular disease. The bulk of circulating LPA is produced from plasma lysophosphatidylcholine (LPC) by autotaxin (ATX), a secreted lysophospholipase D (lysoPLD). Early studies suggest that some of the production of circulating LPA is platelet-dependent. ATX possesses an N-terminal somatomedin B-like domain suggesting the hypothesis that ATX interacts with platelet integrins which may localize ATX to substrate in the membrane and/or alter the catalytic activity of ATX. Using static adhesion and soluble binding assays we found that ATX does indeed bind to platelets and cultured mammalian cells in an integrin-dependent manner which is blocked by integrin function-blocking peptides and antibodies. This binding increases both the activity of ATX and localization of its product, LPA, to the platelet/cell membrane. LPA is generally stimulatory to human platelets although platelets from a small population of donors are refractory to LPA stimulation. Likewise LPA is inhibitory to murine platelets. We previously found that LPA receptor pan-antagonists reduce agonist-induced platelet activation, and partial stimulation of LPA5 specifically increases platelet activation in humans. Since both LPA5 and LPA4 are present at significant levels in human platelets, we hypothesized that LPA4 is responsible for an inhibitory pathway and LPA5 is responsible for an inhibitory pathway. We used mice deficient in LPA4 to test this model. Isolated platelet function tests revealed no major difference between lpa4-/- mice compared with WT mice although lpa4-/- mice were more prone to FeCl3-induced thrombosis. Paradoxically, chimeric mice reconstituted with lpa4-/- deficient bone marrow derived cells were protected from thrombosis. These discrepancies may be explained by involvement of endothelial cells and the relative scarcity of LPA receptors in murine platelets compared with human platelets. Taken together, these results demonstrate two critical regulators of LPA signaling and open up new avenues to further our understanding of atherothrombosis.

G protein-coupled receptors (GPCRs) form a link between the cell and their environment when signaling pathways are activated upon ligand binding. However, the ligands and functions for many GPCRs remain to be determined. G protein-coupled receptor 50 (GPR50) is one such orphan, and its exact role is yet unknown. There is however emerging functional and genetic evidence suggesting a function for GPR50 in psychiatric illness and lipid metabolism. It was hypothesised that investigating GPR50’s protein-protein interactions would lead to a greater understanding of the role of GPR50 in normal brain functioning and in mental illness. Putative protein interactors were initially isolated by a yeast two-hybid study and were further tested here. To address GPR50’s links to mental illness, the GPR50∆502-505 deletion variant associated with mood disorders was also investigated. To test this hypothesis I sought to confirm some of the key yeast two-hybrid interactions. Using co-immunoprecipitation and immunocytochemistry the interaction of GPR50 with reticulon family members Nogo-A, Nogo-C and RTN3, and with cell-cell adhesion molecule CDH8 and lipid-associated protein ABCA2 were validated. In order to identify the location of interactions, subcellular fractionation of mouse brain and rt-PCR and immunohistochemistry in developing and adult mouse brain were performed. GPR50 and several interactors were found to be enriched at the synapse by subcellular fractionation of whole adult brain, and at embryonic day 18 (E18) and 5 weeks by rt-PCR. Colocalisation of GPR50 and interactors was found in the amygdala, hypothalamus, cortex and specific brain stem nuclei by immunohistochemistry. The discovery of GPR50 expression in noradrenergic, serotonergic and dopaminergic nuclei in the adult brain stem suggests a further role for GPR50 in neurotransmitter signaling and stress. To investigate the function of GPR50 two assays were performed that measure processes which are known to be affected by Nogo and RTN3: The first assay was a neurite outgrowth assay in Neuroscreen-1 cells, a PC12 cell clone. A significant increase in neurite length was detected after transient overexpression of GPR50 and this effect was increased in the GPR50∆502-505/T532A variant. Additionally GPR50-overexpression resulted in an increase in filopodia formation suggesting a role in actin dynamics. As a second functional assay in vitro BACE1 activity assays were performed in HEK293 cells. GPR50 but not GPR50∆502-505/T532A overexpression resulted in a significant increase in BACE1 activity. Lastly a final series of pilot experiments were performed to gain insight into the secondary structure of the C-terminal domain and the effects of the polymorphisms on structure. The 35kDa GPR50 C-terminal domain was purified and Circular Dichroism studies indicated a predominantly unstructured protein with increased a- helical content in the GPR50∆502-505 variant. The results in this thesis indicate a role for GPR50 in neuronal development and synaptic functioning. The results also strengthen an association with major mental illness, with links to several disease mechanisms.

An anti-G"11 antibody was used to label neuronal cilia throughout the rat central nervous system. Immunoreactive cilia were observed in every examined region of the rat CNS, but not in monkey or mouse tissue. Antibodies to G"q and G"q/11 failed to label cilia. Immunoreactive cilia were observed as early as postnatal day 0 in spinal tissue, and postnatal day 3 in hypothalamic tissue. There was a statistically significant negative correlation between a region's mean cilium length and that region's distance to the nearest ventricle; regions nearest ventricles were those with the longest cilia. This correlation suggests neuronal cilia may function as chemosensors, detecting substances as they move out from the cerebrospinal fluid and into the extracellular space of the brain.

Les récepteurs couplés aux protéines G (RCPG) sont la famille de récepteurs membranaires la plus représentée chez les vertébrés, et la plus grande cible thérapeutique chez l'Homme. L'évolution du paradigme initial qui énonçait une stœchiométrie récepteur : protéine G : effecteur de 1 :1 :1 sera présentée sur le modèle des récepteurs aux chimiokines CXCR4 et CCR2. Grâce à la technique de transfert d'énergie par bioluminescence (BRET), les travaux réalisés durant cette thèse montrent (1) que c'est par un couplage alternatif de CXCR4 à Gα13 au lieu de la voie classique Gαi que les cellules de cancer du sein migrent pour former des métastases, (2) que la désensibilisation de CXCR4 implique le recrutement d'une combinaison définie de protéines (GRK et arrestines) permettant l'arrêt sélectif des multiples voies engagées en réponse à l'agoniste, et (3) que le protomère CXCR4 a un rôle déterminant dans l'engagement de la protéine Gαi et le recrutement de la β-arrestine par l'hétéro-oligomère CXCR4/CCR2 lorsque CCR2 est activé. Dans cette dernière et principale étude, les résultats montrent également que le dimère CCR2 peut s' assembler au dimère CXCR4 pour former un tétramère, et que l'activation de CCR2 influence la conformation du dimère CXCR4. Les phénomènes de coopérativité et d'activation asymétrique déjà rapportés pour cet hétérodimère pourraient donc impliquer l'interaction de quatre protomères. En conclusion les travaux effectués durant cette thèse démontrent une régulation supplémentaire de l'activité des récepteurs chimiokines au niveau de leur structure quaternaire, de leur signalisation, et de l'arrêt de cette signalisation. / G protein coupled receptors (GPCR) are the most represented cell surface receptors among vertebrates, and the major therapeutic target in humans. The initial paradigm stating a 1 :1 :1 stoichiometry for receptor :G protein :effector has evolved to a more complex model, as illustrated here with the example of the chemokine receptors CXCR4 and CCR2. Bioluminescence resonance energy transfer (BRET) was used to demonstrate that (1) CXCR4 is able to couple Gα13 instead of Gαi to promote breast cancer metastasis, (2) the multiple pathways engaged by stimulation of CXCR4 are selectively desensitized by the specific recruitment of a defined combination of proteins (GRKs and arrestins) and (3) the CXCR4 protomer plays a crucial role during Gαi engagement and β-arrestin recruitment by the CXCR4/CCR2 heterodimer upon CCR2 activation. In this last and main study, the results shown also demonstrate that CCR2 dimers could assemble with CX CR4 dimers into hetero-tetramers, and that CCR2 activation leads to a conformational change in the CXCR4 dimer. Former results showing cooperativity and asymmetric activation of a simple CXCR4/CCR2 heterodimer could then be applied to a tetramer. To conclude, the work done during this thesis demonstrates a more sophisticated regulation of chemokine receptors than previously suspected at 3 different levels: quaternary structure of the protomers, G protein signalling, and signalling termination