Understanding the structure-function relationship of proteins such as G protein-coupled receptors (GPCRs) requires detailed information on their three-dimensional (3D) structure and dynamics. Recent progress in the structural elucidation of receptor-G protein or -arrestin complexes yields valuable starting points to studying their function and dynamics. I investigated the dynamic process of β2 adrenergic receptor-G protein recognition and binding using molecular dynamics (MD) simulations. MD simulation is a well-established technique to monitor the time-resolved motions of proteins and is used to investigate the dynamics of GPCRs. In order to start MD simulations, the structures that are often incompletely resolved have to be further completed. Finally, the results from computational analysis have to be subjected to experimental validation. To promote broad applicability of that workflow, I automated critical steps such as modeling of missing segments or interactive analysis and visualization of the results of MD simulations. Web-tools allow researchers besides different methodological expertise to apply unfamiliar techniques on their biological systems of interest without the need to download software or databases. To facilitate usage, I included visualization allowing intuitive understanding and analysis. We developed the NGL molecular web-viewer, which accesses hardware-accelerated graphics through WebGL. This and other recent developments, reviewed by Mwalongo and others, opened up new possibilities for web molecular graphics and render- ing techniques, superseding plugin- and Java-based viewers and making them comparable with desktop tools, such as PyMOL, VMD, or UCSF Chimera. Here, I implemented and applied tools for interactive modeling of missing segments into single-particle cryo-electron microscopy (cryo-EM) density maps, for interactive analysis of structure-functional dynamics, and more general to promote interdisciplinary research. To allow non-expert users access to bioinformatics methods and biophysical techniques, all tools were generated as web-services allowing interactive analysis and visualization.:Introduction 1 GPCRs as a prime example in structural and pharmaceutical biology 2 Modeling missing segments into cryo-EM density maps 3 Research goal I: Web service for modeling of missing segments into cryo-EM density maps 4 From static to dynamics to investigate GPCR function 5 The complexity of sharing MD simulations 6 Research goal II: Interactive visual sharing of MD simulations Manuscripts 1 FragFit: a web-application for interactive modeling of protein segments into cryo-EM density maps 2 MDsrv: viewing and sharing molecular dynamics simulations on the web. 3 Bringing Molecular Dynamics Simulation Data into View Summary 1 Modeling of missing fragments into cryo-EM density maps 2 InteractiveanalysisofMDsimulationsontheweb . . . . . . References Appendix A Additional Manuscripts 1 SL2: an interactive webtool for modeling of missing segments in proteins 2 GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors. 3 A fragment based method for modeling of protein segments into cryo-EM density maps 4 Structural Insights into the Process of GPCR-G Protein Complex Formation 5 Sharing Data from Molecular Simulations Appendix B Darstellung des eigenen Beitrags Selbständigkeitserklärung Curriculumvitae & Publikationen Danksagung

In the nervous system galanin primarily displays a modulatory role. The galaninergic system consists of a number of bioactive peptides with a highly plastic expression pattern and three different receptors. The lack of receptor subtype selective ligands and antibodies have severely hampered the charac-terization of this system. Therefore, most of the knowledge has been drawn from experiments with transgenic animals, which has given some major conclusions, despite the compensatory effects seen in several animal studies. Therefore, the production of subtype selective ligands is of great importance to delineate the galanin system and slowly experimental data from receptor subtype selective ligand trials is emerging. This thesis aims at studying galanin receptor-ligand interactions and to increase and improve the utilized tools in the galanin research field, espe-cially the development of novel galanin receptor subtype selective ligands. Paper I demonstrates the potential to N-terminally extend galanin ana-logues and the successful development of a galanin receptor 2 (GalR2) selec-tive ligand. In addition, a cell line stably expressing galanin receptor 3 (GalR3) was developed, to improve and simplify future evaluations of sub-type selective galanin ligands. Paper II measures the affinities of M617 and M871 to GalR3 and demon-strates that M871 preferentially binds GalR2. Furthermore, the relatively high affinity of M617 was evaluated by assessing the contribution in recep-tor interaction of individual amino acid residues in the C-terminal part of the M617. In conclusion, this thesis has provided a novel design strategy for galanin receptor ligands and increased the understanding of ligand interactions with the GalR3. Furthermore, M1145 has together with new analogues proven to be highly GalR2 specific, holding promises to future delineation of the galaninergic system as a therapeutic target.

Metabolite-sensing G protein-coupled receptors (msGPCRs) are GPCRs that are activated by metabolites originating from various sources. Several of the known msGPCRs are expressed on immune cells and adipocytes as well as the gut epithelium and metabolic tissues like the pancreas. Some of their known agonists are produced endogenously while others are of exogenous origin. Examples for agonists of exogenous origin are metabolites produced by (intestinal) bacteria. The expression profile and the nature of their agonists link msGPCRs to functions in the regulation of metabolic processes and immune cell responses. Both receptors investigated in my thesis project, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84, are msGPCRs highly expressed on cells of the innate immune system such as neutrophils, monocytes, and macrophages. HCA3 is a member of the HCA family consisting of three receptors, which show high sequence homology. Especially HCA2 and HCA3 only differ in a few positions resulting in an amino acid sequence differing in 17 amino acids and the extended C-terminus of HCA3. While HCA1 and HCA2 are present in the genome of all mammals, HCA3 is only present in higher primates such as chimpanzee, orangutan, and human. As a result, there is a lack of accessible animal models and the receptor is still insufficiently studied. 3 hydroxyoctanoic acid (3HO), 3 hydroxydecanoic acid (3HDec) and aromatic D-amino acids D-phenylalanine (D-Phe) and D-tryptophan (D-Trp) are previously reported agonists of HCA3 with 3HO being its endogenous agonist. Regarding its physiological function, it is known that the receptor is involved in a negative feedback loop in the regulation of lipolysis and fatty acid oxidation under prolonged fasting conditions in adipocytes. Further, it has been shown that aromatic D-amino acids induce chemotaxis in human neutrophils. GPR84 is a receptor for medium chain fatty acids (MCFAs) with a chain length of 9 to 14 carbon atoms (C9 - C14) and their hydroxylated derivatives. Thus, GPR84 and HCA3 share 3HDec as a common agonist. Further, both receptors couple to Gαi proteins resulting in the inhibition of adenylyl cyclase and subsequent decrease of intracellular cyclic AMP (cAMP) levels, and induce the phosphorylation and activation of extracellular signal regulated kinase1/2 (ERK1/2). As opposed to HCA3, GPR84 is present in the genome of most mammals and various studies have linked GPR84 to pro-inflammatory functions and processes like phagocytosis, chemotaxis and upregulation of pro-inflammatory cytokine release. Most of these studies on GPR84 were performed using surrogate agonists. Because HCA3 is still poorly understood, the aim of my project was to shed some light on its function by evolutionary and functional analyses of HCA3 orthologs. Moreover, detailed analyses of its signal transduction and components involved in receptor-mediated downstream signaling events were performed as part of the present dissertation. Since HCA3 and GPR84 share at least one agonist and are co-expressed in different types of immune cells, we studied signaling of both receptors simultaneously. Our functional analyses of human and great ape HCA3 orthologs using cAMP inhibition assays revealed the evolutionary conservation of the endogenous agonist 3HO. By further functionally analyzing the primate HCA3 orthologs, we found both aromatic D-amino acids, D-Phe and D-Trp, to activate human HCA3 with the highest potency. Although D-Phe and D-Trp were previously described to induce HCA3-mediated chemotaxis in neutrophils a link to where the two D-amino acids would originate from in sufficiently high concentrations in a physiological context was missing. After extensive review of literature, we found that some intestinal bacteria and bacteria used to ferment food and beverages produce and secrete D-amino acids. This led us to investigate whether other structurally related D-amino acid metabolites produced by bacteria also activate HCA3. These investigations resulted in the discovery of lactic acid bacteria (LAB)-derived metabolites as highly potent agonists of HCA3. We tested both the Phe-metabolites D-phenyllactic acid (D-PLA) and L-PLA as well as the racemic mixture of the Trp-metabolite indole-3-lactic acid (ILA). All three compounds specifically induced activation of HCA3, but we found D-PLA to be a 35-fold more potent agonist than the L-enantiomer. Further, D-PLA proved to be 10-fold more potent than 3HO and 240-fold more potent than D Phe. Since D-PLA is known to be present in LAB-fermented foods such as Sauerkraut, we investigated whether D-PLA is absorbed and enters the blood circulation after oral ingestion of 100 mg D-PLA or Sauerkraut (5-6 g per kg body weight), respectively. Both, ingestion of the pure compound and of Sauerkraut resulted in a significant increase of D-PLA in the plasma post-prandial resulting in concentrations sufficiently high to activate HCA3. To examine HCA3 signaling and the involved components in more detail, we used several inhibitors of internalization and signaling components. The goal was to examine whether there are differences in the activation of signaling pathways, recruitment of signaling components, internalization behavior and endocytic routes of HCA3 and GPR84 in response to 3HO vs 3HDec and decanoic acid (C10) vs 3HDec respectively. Initial analyses of the signaling kinetics of the two receptors, using dynamic mass redistribution (DMR) measurements, indicated differences between the two respective agonists for both HCA3 and GPR84. DMR measurements allow for a time-resolved recording of the activated signaling cascades, independent of the activated pathways. Additional use of pertussis toxin to inhibit Gαi proteins and dynasore to block dynamin-2 function revealed that signaling of both receptors induced by both respective agonists was completely dependent on G protein activation, but differentially dependent on dynamin-2 function suggesting differences in desensitization and internalization mechanisms. Using cAMP inhibition and ERK1/2 activation assays, we further investigated the role of internalization for HCA3 and GPR84 signal transduction. Interestingly, ERK1/2 activation downstream of both receptors was strongly reduced when internalization was inhibited, while cAMP inhibitory signaling of HCA3 induced by both 3HO and 3HDec and GPR84 signaling induced by C10 were significantly reduced by blocking dynamin-2 function but not internalization in general. 3HDec-induced cAMP inhibition downstream of GPR84 was completely insensitive to inhibition of both. Further experiments using dominant negative dynamin-2 mutants verified dependence of HCA3-mediated Gαi and ERK1/2 signaling on dynamin-2 function. Moreover, qualitative analysis of confocal images revealed that subcellular distribution of HCA3 but not GPR84 is altered when dynamin-2 function is impaired. Interestingly, analysis of β-arrestin 2 recruitment by a luminescence-based assay (DiscoverX PathHunter) and imaging of HEK293T cells expressing mRuby-tagged receptor and YFP-tagged β-arrestin 2, showed that only 3HO- but not 3HDec-induced activation of HCA3 leads to recruitment of β-arrestin 2 and subsequent co-localization in intracellular vesicles. GPR84 data suggests that the receptor does not interact with β-arrestin 2 at all. Finally, we also analyzed HCA3 signaling kinetics and β-arrestin 2 recruitment in response to the newly identified agonists D-PLA and L-PLA and the previously known agonist D-Phe. We found signaling kinetics of L-PLA and D-Phe to be similar to 3HDec-induced kinetics, while D-PLA appears to be similar to 3HO. This was further supported by the fact that D-PLA also induced β-arrestin 2 recruitment, while L-PLA and D-Phe did not. Taken together, these findings advanced our understanding of HCA3 and GPR84. The work provides evidence that HCA3 evolved as a signaling system to communicate uptake of fermented food (together with fermenting bacteria) to the immune system. Our data in combination with literature reporting positive, anti-inflammatory properties of D-PLA and LAB in general suggests that at least some of the described positive effects are mediated by HCA3. Furthermore, we showed for the first time biased signaling of these two receptors in response to their natural agonists. Our work increases the knowledge about specific signaling components involved in downstream signaling of the respective receptor in response to the different agonists, potentially linking e.g. activation of HCA3 by 3HO and D-PLA, but not 3HDec and D-Phe, to the inhibition of pro-inflammatory cytokine release through β-arrestin 2 dependent mechanisms. Moreover, 3HDec-induced signaling downstream of GPR84 is very different from that downstream of HCA3. This suggests that 3HDec also triggers different physiological responses in immune cells depending on its local concentration and the expression levels of the two receptors. However, these findings still need to be validated in cells of innate immunity like neutrophils and macrophages that endogenously express both receptors. At last, the physiological consequences such as increased ROS-production, pro-/anti-inflammatory cytokine release, migration, and phagocytosis need to be addressed in future studies to get a better understanding of the function as well as interplay of HCA3 and GPR84 in innate immunity and their suitability as drug targets.

Screening compounds with image-based analysis is an important part in the processof drug discovery. It is an efficient way to screen compounds as it gives moreinformation than for example HTS. High-content screening as it is also called, hasreally progressed in recent years, as the field of data science evolves, and with it sodoes the efficiency of how images can be processed into information. Anotherimportant part of the drug discovery field is the family of receptors GPCRs, a largefamily of over 800 different receptors in humans. The reason GPCRs are importantin drug discovery is because of the large number of drugs targeting them. In thisexperiment we wanted to use image-based analysis to challenge drugs orcompounds that were said to be specific and see if they actually are that specific, orif we can see indications of the drug also working somewhere else. While the drugswe tested did not appear to cause any morphological perturbations large enough todistinguish them from the control, some drugs appear to cluster differently. Thismight suggest that they affect multiple targets, but it needs to be followed up upon inorder to draw any substantial conclusions.

Les récepteurs couplés aux protéines G (RCPG) sont des protéines membranaires impliquées dans la communication entre cellules via des messagers circulants (hormones, neurotransmetteurs) ainsi que dans la perception de notre environnement (vision, odorat, goût). Ils sont essentiels à de nombreuses fonctions physiologiques vitales (cardiaques,respiratoires...) et comportementales (relations sociales et affectives) et sont donc une cible thérapeutique de choix pour la découverte de nouveaux médicaments.Au sein de l'équipe Canaux, de l’Institut de Biologie Structurale, un biocapteur original a été créé se basant sur la fusion de ces RCPG avec un canal ionique (Kir6.2) appelé Ion Channel-Coupled Receptor (ICCR). Les changements conformationnels du récepteur induit par son activité (fixation de ligand, activation des protéines G) sonttraduits par le canal ionique en courant électrique aisément détectable par des techniques électrophysiologiques. Cette nouvelle génération de biocapteurs permet d'étudier en temps réel l’activité des RCPG par des techniques électrophysiologiques très sensibles.Le travail de thèse s’est principalement focalisé sur l’étude du récepteur de l’ocytocine (OXTR) impliqué dans l’accouchement, l’allaitement et le lien social. La technologie ICCR a été utilisée pour trois des projets de cette thèse. Le premier avait pour but l’étude des mécanismes moléculaires de la dépendance au cholestérol du récepteur del’ocytocine, et a ainsi permis d’identifier un nouveau mécanisme de régulation allostérique entre le cholestérol et la fixation des ligands. Un second projet a porté sur l’utilisation de ce biocapteur pour identifier de nouveaux types de ligands, plus spécifiques de certaines voies intracellulaires, appelés ligands biaisés. Enfin, un troisième projet a mis en relief l’effet de certains composés environnementaux sur les RCPG et a permis de mettre en avant de nouveaux récepteurs ciblés par ce type de composés.Pour terminer, un projet parallèle s’est porté sur l'étude de la formation de pores par des protéines bactériennes dépendantes des RCPG. Il s’agit des « pore-forming toxins » (PFTs) de la famille des hémolysines gamma, produitespar un des pathogènes humains les plus virulents, Staphylococcus aureus. Certaines de ces toxines sont capables de sefixer sur des RCPG très spécifiques, les récepteurs aux chimiokines, et ont donc un rôle important dans les infections virales et dans certaines pathologies cancéreuses. Les travaux ont notamment permis d’obtenir des informations nouvelles sur le mécanisme d’insertion de ces pores dans la membrane. / G protein-coupled receptors (GPCRs) are membrane proteins involved in communication between cells via circulatingmessengers (hormones, neurotransmitters) as well as in the perception of the environment (vision, smell, taste). Theyare essential for many physiological functions (cardiac, respiratory...) and behavioral (social and emotional responses)and therefore represent interesting therapeutic targets.Within the Channels team, at the Institute of Structural Biology, an original biosensor was created, based onthe fusion of a GPCR to an ion channel (Kir6.2), called an Ion Channel-Coupled Receptor (ICCR). Conformationalchanges of the receptor induced by its activity (ligand binding, activation of G proteins) are directly transmitted to theion channel and allow the generation of an electrical signal easily detectable by electrophysiological techniques. Thesenew biosensors are powerful tools to study GPCR activity in real time.The main focus of the thesis was the study of the oxytocin receptor (OXTR), involved in childbirth,breastfeeding and social bonding. ICCR technology has been used for three projects during the thesis. The first aimedat studying the molecular mechanisms of cholesterol dependency of the oxytocin receptor and allowed theidentification of a new allosteric regulation mechanism between cholesterol and the ligand binding. A second projectfocused on the use of this biosensor to identify new types of ligands, selective to certain intracellular pathways, calledbiased ligands. Finally, a third project highlighted the effect of certain compounds, known as endocrine disruptors, onGPCRs. Endocrine disruptors are environmental pollutants which have potentially harmful effects on human health.Finally, a parallel project was dedicated to the study of pore formation by GPCR-dependent bacterial toxins.These proteins are called pore-forming toxins (PFTs), from the gamma hemolysin family and are produced by one ofthe most virulent human pathogens Staphylococcus aureus. Some of these toxins are able to bind very specifically tocertain GPCRs, members of the chemokine receptor family. They therefore play a vital role in numerous viralinfections and in some cancerous pathologies. New information concerning the mechanism of membrane insertion ofthese toxins during pore formation was discovered during this work.

In this work, the evolutionary biophysics approach is applied to the two of the largest protein superfamilies present in human genomes, namely P-loop fold nucleoside triphosphatases (P-loop NTPases) and G-protein coupled receptors (GPCRs). This approach combines comparative analysis of protein structures and sequences with molecular modeling techniques in order to reveal not only the conservation of particular residues among proteins within each superfamily but also their role in the fundamental mechanisms underlying common functions. The study of the hydrolysis activation mechanism in P-loop NTPases started with the molecular dynamics simulations of Mg-NTP complexes (Mg-ATP and Mg-GTP) in the presence of K+, NH4+, and Na+ ions. These simulations showed that in the presence of large cations (K+ and NH4+), the conformation of the phosphate chain of ATP and GTP is extended, with large distances between alpha- and gamma-phosphates. This conformation is similar to the shape of ATP and GTP molecules (or their analogs) in the crystal structures of various P-loop NTPases. To clarify the role of monovalent cations in P-loop NTPases, MD simulations were conducted for two cation-dependent GTPases: tRNA modification GTPase MnmE and translation factor EF-Tu. MD simulations of Mg-GTP/EF-Tu complex bound to the tRNA and ribosome fragment in the presence of K+ ions have shown consistent binding of a potassium ion from the solution between alpha- and gamma-phosphates (AG site), similar to the cation binding in MnmE and other cation-dependent P-loop GTPases. In both proteins, binding of K+ ion in the AG site led to the rotation of gamma-phosphate, making this group more eclipsed with alpha-phosphate. The new rotated position of gamma-phosphate was stabilized by a novel H-bond with the backbone nitrogen of the K-3 residue (relative to the ubiquitously conserved Lys) of the P-loop motif. The activation mechanism observed in MD simulations of MnmE and EF-Tu could be envisioned as basic for P-loop NTPases, as these cation-dependent proteins are among the most ancient members of the P-loop superfamily. This mechanism was used as a basis for extensive comparative analysis of representative proteins from all major classes of P-loop NTPases. Based on the established conservation and presence of the key features in active sites of P-loop NTPases, the chain of events where rotation of gamma-phosphate triggers the nucleophilic attack and gamma-phosphate cleavage has been proposed as the basic universal activation mechanism of NTP hydrolysis in P-loop NTPases. The second part of this work explores the activation of GPCRs as sodium-translocating receptors. Crystal structures of the novel Na-pumping microbial rhodopsin along with the recent avalanche of GPCR structures provided the basis for comparative structure analysis, focused on investigating the similarities in the Na-binding sites of the two superfamilies. Structure superposition of GPCRs and microbial rhodopsins (MRs) based on comparison of their Na-binding sites was used to produce structure-guided sequence alignments of the two superfamilies. The only residue universally conserved between the two superfamilies was Trp in the helix 6/F (Trp6.48 in GPCRs). In both families, the signaling mechanism directly involves this residue, which is likely to be an ancient feature inherited from the common ancestor of MRs and GPCRs – the Na-pumping light-activated rhodopsin. The similarity of GPCRs with light-activated sodium pumps endorses the suggestion that GPCRs may also function as Na+ ion translocators. A model of GPCR activation accompanied by translocation of Na+ was constructed to demonstrate how this mechanism can explain the voltage sensitivity of certain Class A GPCRs. Two modes of activation were modeled – one where Na+ ion is transported into the cytoplasm and the one where Na+ ion is expelled to the intracellular space. The two modes quantitatively describe the behavior of voltage-activated and voltage-suppressed GPCRs, respectively. Finally, further structure scrutiny and rotamer analysis provided a plausible pathway of Na+ transmembrane translocation through the helical bundle of GPCRs.

Neuropeptide und ihre Rezeptoren regulieren im menschlichen Körper essentielle Funktionen. Fehlfunktionen können zu schwerwiegenden Krankheiten führen, weswegen die Erforschung dieser Peptid-Rezeptor-Systeme von hohem Wert ist. Die Komplexität der Signalisierung erschwert die Forschung in Säugetiermodellen, weswegen auch Modelle von Invertebraten herangezogen werden können, wo viele homologe Neuropeptide zu finden sind. In dieser Arbeit wurde das ein Homolog zum Neuropeptid Y (NPY) im Rundwurm Caenorhabditis elegans (C. elegans) identifiziert und charakterisiert. Das NPY-System besteht aus 3 Peptiden und 4 Rezeptoren, welche in Säugetieren vor allem den Energiehaushalt regulieren, aber auch bei Stress, Depression und Angstzuständen eine Rolle spielen. In C. elegans wurden 41 dem NPY ähnliche Rezeptoren (NPR) und über 30 Neuropeptide identifiziert, welche auch Funktionen in der Nahrungssuche zeigen. In dieser Arbeit wurde gezeigt, dass das humane und C. elegans System pharmakologische und funktionale Gemeinsamkeiten aufweisen. Hier wurden der NPR-1 und NPR-11 als NPY-ähnlichste Rezeptoren identifiziert und erstmals ein NPY-ähnliches Peptid beschrieben, das FLP-34-1. Des Weiteren wurde eine bekannte Methode für Zellkulturexperimente in C. elegans etabliert, der Biolumineszenz-Energietransfer (BRET), welcher die Bindung von FLP-34-1 an NPR-11 in vivo zeigte. Zudem konnte mit dieser Methode eine Peptid-induzierte Internalisierung von NPR-11 Richtung Endosomen in vivo in Echtzeit gezeigt werden. Die Ergebnisse dieser Arbeit helfen die molekularen Mechanismen der Peptid-Rezeptor-Interaktionen besser zu verstehen und unterstützen damit auch die Forschung an höheren Tieren.

G-protein coupled receptor signaling (GPCR) is essential for regulating a large variety of hormonal, sensory and neuronal processes in eukaryotic cells. Because the regulation of these physiological responses is critical, GPCR signaling pathways are carefully controlled at different levels within the cascade. Phosducin-like protein 1 (PhLP1) can bind the G protein βγ dimer and participate in GPCR signaling. Recent evidence has supported the concept that PhLP1 can serve as a co-chaperone of the eukaryotic cytosolic chaperonin complex CCT/TRiC to mediate G βγ assembly. Although a general mechanism of PhLP1-mediated G βγ assembly has been postulated, many of the details about this process are still missing. Structural analysis of key complexes that are important intermediates in the G βγ assembly process can generate snapshots that provide molecular details of the mechanism beyond current understanding. We have isolated two important intermediates in the assembly process, the Gβ1-CCT and PhLP1-Gβ1-CCT complexes assembled in vivo in insect cells, and have determined their structures by cryo-electron microscopy (cryo-EM). Structural analysis reveals that Gβ1, representing the WD40 repeat proteins which are a major class of CCT substrates, interacts specifically with the apical domain of CCTβ. Gβ1 binding experiments with several chimeric CCT subunits confirm a strong interaction of Gβ1 with CCTβ and map Gβ1 binding to α-Helix 9 and the loop between β-strands 6 and 7. These regions are part of a hydrophobic surface of the CCTβ apical domain facing the chaperonin cavity. Docking the Gβ molecule into the two 3D reconstructions (Gβ1-CCT and PhLP1-Gβ1-CCT) reveals that upon PhLP1 binding to Gβ1-CCT, the quasi-folded Gβ molecule is constricted to a more native state and shifted to an angle that can lead to the release of folded Gβ1 from CCT. Moreover, mutagenesis of the CCTβ subunit suggests that PhLP1 can interact with the tip of the apical domain of CCTβ subunit at residue S260, which is a downstream phosphorylation target site of RSK and S6K kinases from the Ras-MAPK and mTOR pathways. These results reveal a novel mechanism of PhLP1-mediated Gβ folding and its release from CCT. The next important step in testing the PhLP1-mediated Gβγ assembly hypothesis is to investigate the function of PhLP1 in vivo. We have prepared a rod-specific PhLP1 conditional knockout mouse in which the physiological consequences of the loss of PhLP1 functions have been characterized. The loss of PhLP1 has led to profound consequences on the ability of these rods to detect light as a result of a significant reduction in the expression of transducin (Gt) subunits. Expression of other G protein subunits as well as Gβ5-RGS9-1 complexes was also greatly decreased, yet all of this occurs without resulting in rapid degeneration of the photoreceptor cells. These results show for the first time the essential nature of PhLP1 for Gβγ and Gβ5-RGS dimer assembly in vivo, confirming results from cell culture and structural studies.

The sensation of mechanical signals is vital for all animals. For this task Drosophila larvae are equipped with chordotonal organs. These are specialized mechanosensory organs which are composed of multicellular subunits. In this study I show how metabotropic signaling by the adhesion GCPR CIRL interacts with part of the ionotropic pathways during mechanosensation in sensory neurons of the pentascolopidial chordotonal organ (lch5). CIRL modulates cAMP levels in sensory neurons and thereby shapes the receptor potential response to mechanical stimuli. Here, CIRL forms a functional interaction with the TRP channel NOMPC in which nompC is epistatic to Cirl. Furthermore, the evidence presented suggest the presence of another target of CIRL and the involvement of a further signaling pathway besides cAMP modulation. In the second part of the study, I describe a method to express the anion-selective channelrhodopsin GtACR1 in individual of the five neurons of the lch5. For this I used the MARCM approach which generates genetic mosaics during the development of the neurons of interest. Thereby a specific subset of cells deriving from a common precursor expresses the desired protein GtACR1.

Hormonally-triggered regulatory hierarchies play a major role in organismal development. Disruption of a single member of such a hierarchy can lead to irregular development and disease. Therefore, knowledge of the members involved and the mechanisms controlling signaling through such pathways is of great importance in understanding how resulting developmental defects occur. Type II transmembrane serine proteases (TTSPs) make up a family of cell surface-associated proteases that play important roles in the development and homeostasis of a number of mammalian tissues. Aberrant expression of TTSPs is linked to several human disorders, including deafness, heart and respiratory disease and cancer. However, the mechanism by which these proteases function remains unknown. The ecdysone-responsive Stubble TTSP of Drosophila serves as a good model in which to study the functional mechanism of the TTSP family. The Stubble protease interacts with the intracellular Rho1 (RhoA) pathway to control epithelial development in imaginal discs. The Rho1 signaling pathway regulates cellular behavior via control of gene expression and actin cytoskeletal dynamics. However, the mechanism by which the Stubble protease interacts with the Rho1 pathway to control epithelial development, in particular leg imaginal disc morphogenesis, has yet to be elucidated. The Stubble protein consists of several conserved domains. One approach to a better understanding of the mechanism of action of Stubble in regulating Rho1 signaling is to define which of the conserved domains within the protease are required for proper function. Sequence analysis of twelve recessive Stubble mutant alleles has revealed that the proteolytic domain is essential for proper function. Alleles containing mutations which disrupt regions of the protease domain necessary for protease activation or substrate binding, as well as those with deletions or truncations that remove some portion of the proteolytic domain, result in defective epithelial development in vivo. In contrast, mutations in other regions of the Stubble protein, including the disulfide-knotted and cytoplasmic domains, were not observed. Another important step for defining the connection between Stubble and Rho1 signaling is to identify a Stubble target that acts as an upstream regulator of the Rho1 pathway. We performed a genetic screen in which 97 of the 147 Drosophila non-olfactory and non-gustatory G-protein-coupled receptors (GPCRs), a family of proteins that has been shown to be protease-activated and to activate Rho1 signaling, were tested for interactions with a mutant allele of Stubble. We found 4 genomic regions uncovering a total of 7 GPCRs that interact genetically when in heterozygous combination with a Stubble mutant. Further analysis of these genes is necessary to determine if any of these GPCRs is targeted by Stubble during activation of the Rho1 pathway.