Aktivierte G-Protein gekoppelte Rezeptoren aktivieren heterotrimere GProteine, in dem sie den Austausch von GDP zu GTP am G-Protein katalysieren. Theoretische Untersuchungen mittels eines vereinfachten kinetischen Modells des Gi/o-Protein Zyklus legen nahe, dass nicht nur GDP-,sondern auch GTP-gebundene Gi/o-Proteine mit aktivierten α2A-adrenergen Rezeptoren (α2A-AR) interagieren können. Demgemäß sollten aktivierte Gi/o-Proteine mit aktivierten α2A-AR vermehrt interagieren, wenn mehr α2A-AR aktiviert werden als für eine maximale G-Protein Aktivierung nötig sind. Dies sollte zu einer paradoxen Deaktivierung von Gi/o-Proteinen und deren Effektorproteinen, z.B. dem G-Protein gekoppelten, einwärtsgleichrichtenden Kaliumkanal (GIRK-Kanal) führen. Mittels FRET lässt sich in lebenden und in permeabilisierten Zellen unter Kontrolle der intrazellulären Nukleotide die Aktivierung von α2A-AR, die Interaktion von Gi/o-Proteinen mit α2A-AR und die Aktivierung von Gi/o-Proteinen bestimmen. Die Arbeit zeigt auf mehreren Ebenen, dass Go-Proteine mit aktivierten α2A-AR interagieren und im nukleotidfreiem Zustand sequestriert werden können: (I) Go-Proteine,irreversibel durch GTPγS aktiviert werden abhängig von der Rezeptor Aktivierung in Abwesenheit von Nukleotiden deaktiviert, (II) Go-Proteine interagieren in Gegenwart niedriger Nukleotidkonzentrationen in wesentlich größer Fraktion mit aktivierten α2A-AR als in Gegenwart hoher Nukleotidkonzentrationen, (III) Go Proteine können in Gegenwart niedriger GTP und GTPγS-Konzentrationen bei Aktivierung des α2A-AR inaktiviert werden. Die Arbeit zeigt exemplarisch an der Signalkaskade des α2A-AR und Go, dass der G-Protein Zyklus in lebenden Zellen reversibel ist, woraus eine Deaktivierung aktivierter G-Proteine und aktivierter G-Protein Effektoren resultieren kann. Dies erklärt paradoxe Befunde zur Deaktivierung von GIRK-Kanälen in Myozyten durch A1-Rezeptoren. / G protein coupled receptors activate heterotrimeric G proteins by catalyzing the exchange of GDP with GTP at the Gα subunit. Kinetic modelling of the Gi/o protein cycle suggests, that both GDP- and GTP-bound Gi/o proteins interact with activated α2A-adrenergic receptors (α2A-AR). Consequently, upon activating more α2A-AR then required for maximal Gi/o protein activation, the interaction of activated Gi/o proteins with activated α2A-AR will become incresingly prominent and ultimately lead to a paradoxic deactivation of Gi/o proteins and their effectors such as G protein coupled inwardly rectifying potassium channels. Using means of FRET allows the detection of the receptor activation, receptor/G protein interaction and G protein activation in single living cells and in single permeabilized cells while controlling the intracellular nucleotide composition.Data suggest, that activated Go proteins may be sequestrated at activated α2A-AR in their nucleotide-free state: (I) Go proteins irreversibly activated by GTPγS become inactivated upon receptor stimulation in the absence of nucleotides, (II) Go proteins interact with activated α2A-AR to a large extent in the presence of low concentrations of nucleotide, (III) Go proteins may be inactivated upon activation of α2A-AR in the presence of low concentrations of GTP or GTPγS. Taken together, the data demonstrate the reversibility of the G protein cycle in living cells for the paradigm α2A-AR/Go pathway. The data thereby explain the paradoxic inactivation of G protein coupled inwardly rectifying potassium channels in myocytes upon activation of adenosine A1 receptors.

1 Summary The first part of this thesis is preoccupied with the identification of protein alterations in the membrane fraction of HEK293-E2M11 cells after prolonged TRH treatment. The isolated membrane fraction enriched in plasma membranes contained markedly increased the amount of Na,K-ATPase, TRH receptor and G-proteins compared to the postnuclear supernatant. By using 2D electrophoresis and mass spectrometry, the levels of 42 proteins were identified to be altered in samples of PM- enriched fractions from TRH-treated (16 h; 10 μM) cells. Out of these proteins only ezrin and stomatin-like 2 are known to be localized in the plasma membrane. Five proteins (mitofilin, MTHSP75, prohibitin, stomatin like-2, peroxiredoxin III) whose levels were increased after the prolonged TRH treatment represent proteins localized in mitochondria. All of them are important for proper structure and function of mitochondria. The ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax was markedly higher in cells treated with TRH than in control untreated cells. Hence, it can be concluded that prolonged TRH treatment may significantly affect mitochondrial membrane and function of mitochondria. The second part of this thesis deals with the identification of molecular protein complexes of TRH-R and/or Gq/11 protein. The presumed...

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

Latrophilin, alternatively named calcium-independent receptor of α-latrotoxin (CIRL), resembles a prototype of the adhesion class G-protein coupled receptors (GPCRs). Initially identified as a high-affinity receptor for α-latrotoxin, a component of the black widow spider, latrophilins are now associated with various distinct functions, such as synaptic exocytosis, tissue polarity and fertility (Tobaben et al., 2002; Langenhan et al., 2009; Promel et al., 2012). Despite these exploratory efforts the precise subcellular localisation as well as the endogenous ligand of CIRL still remains elusive. In this work genetic experiments, imaging approaches and behavioural studies have been used to unravel the localisation and physiological function of the latrophilin homolog dCirl in Drosophila melanogaster. Containing only one latrophilin homolog together with its genetic accessibility and well-established transgenic approaches, Drosophila seemed an ideally suited model organism. The present study showed that dCirl is widely expressed in the larval central nervous system including moto- and sensory neurons. Further, this work revealed that removal of the latrophilin homolog does not greatly affect synaptic transmission but it seems that aspects of the postsynaptic structural layout are controlled by dCIRL in the fruit fly. Additionally, dCirl expression at the transcriptional level was confirmed in larval and adult chordotonal organs, specialised mechanosensors implicated in proprioception (Eberl, 1999). Expression of dCIRL at the protein level could not yet been confirmed in moto- and sensory neurons likely due to low endogenous expression. However, behavioural studies using dCirl knockout mutant larvae indicated a putative mechanosensory function of dCIRL regarding touch sensitivity and locomotion behaviour. The second part of this thesis presents a strategy to examine interactions between several presynaptic proteins in living cells. The attempt described in this work is based on the discovery that GFP when split into two non-fluorescent fragments can form a fluorescent complex. The association of the fragments can be facilitated by fusing them to two proteins that interact with each other. Therefore, the split GFP method enables direct visualization of synaptic protein interactions in living cells. In initial experiments I could show that full length reporter protein fusions with n-Synaptobrevin (n-Syb), Synaptotagmin (Syt) and Syntaxin (Syx) allow expression in Drosophila and confirmed that fusion to either end of each synaptic protein did not impair expression or influence the viability of transgenic flies. Further, transgenes containing protein fusions of Syx, Syt, and n-Syb with split GFP fragments were established in previous studies (Gehring, 2010). The present work characterises the interaction of these protein fusions during different stages of synaptic vesicle turnover at active zones such as synaptic vesicle docking at the presynaptic membrane and vesicle fusion. These results suggest that the spGFP assay seems only partly suitable for resolving fast and transient protein-protein interactions at larval Drosophila active zones in vivo. / Latrophilin, auch als Calcium-unabhängiger Rezeptor für α-Latrotoxin (CIRL) bezeichnet, repräsentiert einen Prototyp der Adhäsions G-Protein gekoppelten Rezeptorklasse. Ursprünglich als hoch-affiner Rezeptor für α-Latrotoxin entdeckt, werden Latrophiline heute mit zahlreichen verschiedenen Funktionen, wie synaptischer Exozytose, Gewebepolarität und Fertilität assoziiert (Tobaben et al., 2002; Langenhan et al., 2009; Promel et al., 2012). Trotz dieser Fortschritte sind die genaue subzelluläre Lokalisation sowie der endogene Ligand noch weitgehend unbekannt. Diese Studie verwendet genetische Ansätze, bildgebende Verfahren und Verhaltensstudien, um die Lokalisation und physiologische Funktion des Latrophilinhomologs dCirl in Drosophila melanogaster aufzuklären. Die Tatsache, dass Drosophila nur ein einziges Latrophilin Homolog besitzt, zusammen mit den genetischen Möglichkeiten und den sehr gut etablierten transgenen Methoden, machen die Fruchtfliege zu einem idealen Modellorganismus. Die erhobenen Daten belegen, dass dCirl verstärkt im larvalen Nervensystem, einschließlich motorischer und sensorischer Neurone, exprimiert wird. Weiterhin konnte gezeigt werden, dass in dCirl Knockout-Mutanten die basale synaptische Transmission unverändert ist, vermutlich aber Teile der postsynaptischen Struktur durch dCIRL in der Fruchtfliege kontrolliert werden. Zusätzlich konnte nachgewiesen werden, dass dCirl auf Transkriptionsebene in den larvalen und adulten Chordotonalorganen exprimiert wird, spezifische Mechanosensoren, die an der Propriozeption beteiligt sind (Eberl, 1999). Die Expression von dCIRL auf Proteinebene in motorischen und sensorischen Neuronen konnte aufgrund niedriger endogener Expressionslevel noch nicht verifiziert werden. Allerdings deuten Verhaltensstudien, die Berührungsempfindlichkeit und Lokomotion untersuchen, auf eine mögliche mechanosensorische Funktion von dCIRL in den Larven von Drosophila hin. Der zweite Teil dieser Arbeit zeigt eine Strategie auf, die es ermöglicht, das Zusammenspiel verschiedener präsynaptischer Proteine in vivo zu untersuchen. Die hier beschriebene Methode basiert auf der Entdeckung, dass sich zwei nicht-fluoreszierende Fragmente des grün leuchtenden Proteins (GFP), zu einem fluoreszierenden Komplex zusammenlagern können. Diese geteilten GFP-Fragmente (split-GFPs) werden mit zwei unterschiedlichen Proteinen fusioniert, die miteinander interagieren. Die split-GFP Methode ermöglicht so eine direkte Visualisierung von Protein-Protein-Interaktionen in lebenden Zellen. In ersten Experimenten konnte ich zeigen, dass Synaptobrevin (n-Syb), Synaptotagmin (Syt) und Syntaxin (Syx), die mit vollständigen Fluorophoren markiert wurden, für die Expression in Drosophila geeignet sind und bestätigen, dass sowohl die N-terminale als auch die C-terminale Proteinfusion möglich ist. Zudem konnte durch diese Versuche die Überlebensfähigkeit der transgenen Fliegen überprüft werden. In vorangegangenen Studien wurden Transgene hergestellt, die Proteinfusionen von n-Syb, Syt und Syx mit split-GFP Fragmenten enthalten (Gehring, 2010). Die vorliegende Arbeit charakterisiert die Wechselwirkung dieser Proteinfusionen während unterschiedlicher Stufen der synaptischen Vesikelfreisetzung an der aktiven Zone, wie beispielsweise dem Vesikel-docking an der präsynaptischen Membran und der Vesikelfusion. Die Ergebnisse dieser Studie deuten darauf hin, dass die split-GFP Technik nur bedingt geeignet ist um schnelle und transiente Protein-Protein Interaktionen an der larvalen aktiven Zone von Drosophila in vivo darzustellen.

In dieser Arbeit wurden Einzelmolekültechniken zur Untersuchung von G-Protein-gekoppelten Rezeptoren (GPCR) und G-Proteinen in der Zellmembran lebender Zellen etabliert und angewendet. GPCR stellen die größte Familie membrangebundener Rezeptoren dar und leiten Signale über heterotrimere G-Proteine in das Zellinnere weiter. Auch wenn jüngst sowohl inaktive, als auch aktive Konformationen von GPCR und G-Proteinen mittels Röntgenstrukturanalyse aufgelöst werden konnten, sind die Dynamiken ihrer Aktivierung und Deaktivierung bisher nur bruchstückhaft bekannt. In der Vergangenheit wurden die Schritte der Signalkaskade, beginnend mit der Bindung des Rezeptorliganden bis hin zur Bildung von sekundären Botenstoffen, erfolgreich mit Fluoreszenz-Resonanz-Energie-Transfer-Techniken aufgeklärt. Diesen experimentell bestimmten Aktivierungszeiten stehen Daten aus Modellierungsstudien gegenüber, die sehr viel schnellere Konformationsänderungen vorhersagen, welche bereits in Studien mittels Kernspinresonanzspektroskopie nachgewiesen werden konnten. Folglich ist anzunehmen, dass die Zeitdomäne, innerhalb der die Aktivierung der GPCR stattfindet, sehr breit gefächert ist. Ein Ziel der vorliegenden Arbeit war es, diese mehrere Größenordnungen umfassenden Zeitskalen der GPCR-Aktivierung, welche in der Literatur beschrieben werden, mittels bildgebender Einzelmolekülverfolgung (SPT) und Fluoreszenz-Korrelations-Spektroskopie (FCS) zu untersuchen. Beide Verfahren liefern durch Einzelmolekülspuren oder Korrelationskurven eine Art Fingerabdruck des dynamischen Verhaltens des untersuchten Systems, was jeweils mit Vor- und Nachteilen verbunden ist. Die Stärke der Techniken zeigte sich bei dem vorliegenden Projekt vor allem in ihrer Kombination: Die klassische FCS bietet die Möglichkeit, Dynamiken über einen weiten Zeitraum von Mikrosekunden bis Sekunden auszuwerten, allerdings nur innerhalb eines kleinen, optisch definierten Detektionsvolumens. Die bildgebende Einzelmolekülverfolgung liefert hingegen ein großes Sichtfeld und ermöglicht somit die parallele Analyse vieler Einzelmolekülereignisse über die Zelle verteilt, jedoch auf Kosten der Zeitauflösung. Durch die Anwendung von SPT und FCS konnte in dieser Arbeit ein Zeitbereich der Rezeptor- (und G-Protein-) Dynamiken von Mikrosekunden bis Sekunden gefunden und diskutiert werden. Um die selektive Anregung der Plasmamembran zu gewährleisten, wurde die Interne Totalreflexionsfluoreszenzanregung verwendet. Diese eignet sich ideal als Grundlage für die spätere Analyse mittels SPT und FCS, welche komplementär nutzbar sind und mit dem gleichen zellulären Assay und unter Verwendung der gleichen Fluoreszenzmarker betrieben werden können. Die Studie am Beispiel der α2A- und β2-adrenergen Rezeptoren sowie des Gαi1-Proteins demonstrierte das enorme Potential dieser Einzelmolekültechniken für die Untersuchung von GPCR und skizziert die Komplexität deren Dynamik, wie sie auch durch neueste Modellierungsstudien vorhergesagt wird. / In this work single molecule techniques for the investigation of G-protein coupled-receptors (GPCR) and G-proteins in living cells were established and applied. GPCR constitute the largest family of membrane bound receptors and transduce extracellular stimuli via heterotrimeric G-proteins. Very recently inactive as well as active conformations of GPCR and G-proteins have been deduced from X-ray crystallography. Nevertheless, only little is known about the dynamics of receptor activation and deactivation. Techniques based on Fluorescence Resonance Energy Transfer (FRET) have allowed for the study of the GPCR signaling cascade from ligand binding up to second messenger generation. However, the reported activation times based on such FRET investigations seem to contradict data from molecular modelling studies which predict much faster conformational changes and are supported by recent Nuclear Magnetic Resonance (NMR) spectroscopy data. Thus, the timescale of GPCR activation remains actively debated, and is quite likely widely spread. One objective of this work was to experimentally probe this orders-of-magnitude broad time scale for GPCR activation reported in the literature using Single Particle Tracking (SPT) and Fluorescence Correlation Spectroscopy (FCS). By directly probing the dynamic behavior via both single particle traces and correlation curves, the advantages and disadvantages offered by each technique can be compensated. In combination SPT and FCS pool forces: Classical FCS allows evaluating dynamics within a broad time domain from the microsecond to the second range. The compromised small ‘field of observation’ comes with the advantage of high temporal resolution. By contrast, SPT allows parallel analysis of many single receptor or G-protein events distributed over at least the dimension of a single cell, however with lower temporal resolution. In this study the application of FCS and SPT allowed for the detection of receptor (and G-protein) dynamics. Selective illumination of the plasma membrane was achieved by using Total Internal Reflection Fluorescence. Data was subsequently analyzed by SPT as well as FCS, both methods working with the same cellular assay as well as fluorescent probes. Exemplarily investigating the α2A- and β2-adrenergic receptor as well as the Gαi1-protein, uncovers a wide timescale of receptor dynamics from microseconds to seconds, closing the gap between times that already could have been solved with other fluorescent techniques such as FRET. This study reveals the enormous potential of single molecule methods for the investigation of GPCR and delineates the complexity of GPCR dynamics as recently predicted by molecular modelling.

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.

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.

The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.

The hypothalamic peptide arginine vasopressin (AVP) is an important regulator of adrenocorticotropin (ACTH) release from the anterior pituitary. AVP stimulates ACTH secretion from corticotroph cells by activating the pituitary vasopressin receptor (V1b-R), a member of the G protein-coupled receptor (GPCR) family. In vitro, repeated stimulus of anterior pituitary cells with AVP results in rapid desensitisation. The aim of this research was to develop methods needed to use RNA interference (RNAi) to investigate the role of G protein-coupled receptor kinase 5 (GRK5) in this desensitisation process. This required the development of a model system using human embryonic kidney (HEK) 293 cells transfected with the pituitary vasopressin receptor, V1b-R. AVP binding to the V1bR activates the phosphoinositide signalling pathway, leading to production of inositol phosphates (IPs), which can be measured following radiolabelling of cells with myo-[³H]inositol. Stimulation of V1b-R-transfected cells for 15 min with AVP (100nM) increased IP production to 235.5 ± 23.4 % (n=3, p<0.02) of that seen in un-stimulated control cells. Following a 5 minute pre-treatment with 5nM VP, the IP response to stimulation with 100nM VP for 15 min was reduced to 62.8 ± 9.1 % (n=4, p<0.02) of that seen in control cells that were not pre-treated. These data indicate that AVP-desensitisation can be induced and measured in V1bR-transfected HEK293 cells following a brief pre-treatment with a physiological concentration of AVP. This model system will enable RNAi to be used to investigate the role of GRK5 in AVP-desensitisation. When using RNAi, it is essential to establish that the effects observed are the result of small interfering RNA (siRNA) specific degradation of the target mRNA. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the expression of GRK5 at the mRNA level in HEK293 cells. Human GRK5 mRNA was amplified using qRT-PCR with GRK5 specific primers, providing confirmation that GRK5 is expressed endogenously in HEK293 cells. GRK5 expression studies were carried out to evaluate whether the qRT-PCR methods developed would be suitable to measure knockdown of GRK5 mRNA using RNAi. These experiments were also designed to assess the impact of HEK293 cell culture methods on expression of GRK5. Expression of GRK5 did not vary with passage number (2-26 passages). The GRK5 expression in HEK293 cells that were maintained in culture for 5 days (grown to a confluence of approximately 100%) was 7.4 ± 0.9 fold greater (n=2, p<0.05) than for cells cultured for 3 days (grown to a confluence of approximately 65%). These data indicate that GRK5 expression is affected by HEK293 culture conditions. Furthermore, the results demonstrated that a significant difference in GRK5 expression could be measured in HEK293 cells using qRT-PCR. Therefore the results reported in this thesis provide the basis for future studies utilising RNAi to investigate mechanisms underlying V1b-R desensitisation.