<p>Chemokine receptors are members of the seven transmembrane receptor (7TMR) superfamily and are regulated by the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system. CCL19 and CCL21 are endogenous agonists for the chemokine receptor CCR7. They are known to be equipotent in promoting Gi/o mediated calcium mobilization, chemotaxis and inhibition of adenylyl cyclase activity. Here we test the hypothesis that these ligands are biased agonists that differentially activate the G-protein coupled Receptor Kinase (GRK)/ b-arrestin system.</p><p>In order to test whether these ligands have distinct activity, murine T lymphocytes were used to compare the effects of CCL19 and CCL21 activation of CCR7 at endogenous expression levels. While each ligand stimulates similar chemotactic responses, we also find that CCR7 ligands lead to differential signaling. For instance, CCL19 is markedly more efficacious than CCL21 for the activation of ERK and JNK, but not AKT in these cells. Furthermore, ERK activation and chemotaxis are maintained as separate pathways, also distinguishable by their dependency upon PKC and PI3 kinase, respectively. Thus, CCL19 and CCL21 stimulate equal activation of PI3 kinase, AKT, and chemotaxis, but are in fact biased agonists leading to differential activation of MAP kinase in murine T lymphocytes. </p><p>To determine the mechanism of CCR7 ligand bias, we used HEK-293 cells expressing CCR7 to compare the proximate signaling events following CCL19 and CCL21 activation. We found striking differences in the activation of the GRK/ b-arrestin system. CCL19 leads to robust CCR7 phosphorylation and b-arrestin2 recruitment catalyzed by both GRK3 and GRK6 while CCL21 activates GRK6 alone. This differential GRK activation leads to distinct functional consequences. Only CCL19 leads to the recruitment of b-arrestin2-GFP into endocytic vesicles and classical receptor desensitization. In contrast, each agonist is fully capable of signaling to MAP kinase through b-arrestin2 in a GRK6 dependent fashion. </p><p>Therefore, CCR7 and its ligands represent a natural example of ligand bias whose mechanism involves differential GRK isoform utilization by CCL19 and CCL21 despite similar G-protein signaling. This study suggests that the GRK signatures of 7TMRs can determine the function of discrete pools of b-arrestin and thus guide its cellular effects.</p> / Dissertation

Skeletal muscle is a major organ comprising 30-40% of the human body mass. The coordination of processes resulting in mature muscle requires many genes, and their loss can result in debilitating muscle disorders. Of the strategies being developed to cure muscle diseases, enhancement of the natural process of muscle cell fusion in existing or introduced myogenic cells has great therapeutic potential. In this work, we determined whether a drug that stimulates proliferation and fusion of myoblasts could alleviate murine Duchenne muscular dystrophy. We also studied the necessity of a gene that is upregulated in early fusing human myoblast cultures and its role in muscle disease development.

Das Rett Syndrom (RTT) gehört zu den tiefgreifenden Entwicklungsstörungen des Gehirns von dem fast ausschließlich Mädchen betroffen sind (ICD-10, F84.10). Ursächlich für die Pathogenese sind Mutationen im X-chromsomalen MECP2-Gen, welches für den Transkriptionsfaktor Methyl-CpG binding protein 2 (MeCP2) kodiert. Unterschiedliche neurologische Symptome treten zwischen 6 und 18 Monaten nach der Geburt auf, wobei schwere Rhythmussstörungen der Atmung für ein Viertel plötzlicher Todesfälle bei Rett-Patientinnen verantwortlich gemacht werden. Der neuronale Atmungsrhythmus bei Säugern wird in verschieden Regionen des ponto-medullären Hirnstammes generiert, wobei der Prä-Bötzinger Komplex als essentiell für die Rhythmogenese der Atmung angesehen wird. Mittels Genexpressionsstudien in der Ventralen Respiratorischen Gruppe (VRG), die den Prä-Bötzinger-Komplex einschließt, zeigte sich eine massiv erhöhte, pathologische Expression des Serotoninrezeptor 5B sowohl auf mRNA-, als auch auf Proteinebene bei MeCP2-defizienten Mäusen zum postnatalen Entwicklungstag P40. Der Serotoninrezeptor 5B (5-HTR5B) gehört zur Klasse der G-Protein-gekoppelten Rezeptoren. Durch detaillierte Analysen des 5-HTR5B-Proteins konnte eine natürliche Trunkierung des Rezeptors nachgewiesen werden. Des Weiteren wurde eine ungewöhnliche intrazelluläre Lokalisierung in Membranen von vesikulären und tubulären Kompartimenten beobachtet. Trotz dieser ungewöhnlichen Eigenschaften besitzt der Rezeptor weiterhin die Fähigkeit, das inhibitorische G-Protein Gαi3 konstitutiv zu aktivieren und somit den Anstieg von cAMP zu verhindern. Durch genetisches Ausschalten des 5-HTR5B Proteins (knockout) konnte gezeigt werden, dass die durch 5-HTR5B-verminderte cAMP-Konzentration in der VRG ursächlich für den gestörten Atmungsrhythmus MeCP2-defizienter Mäuse ist. Die sich aus diesen Ergebnissen ableitende pharmakologische Strategie, die cAMP Konzentration zu erhöhen, führte zu einem deutlich verbesserten Atmungsrhythmus. Die Ergebnisse dieser Arbeit implizieren neue Therapieansätze zur Behandlung der Atmungs-störungen von Rett-Patienten.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

In insects, ecdysis is an important feature of growth and development and is tightly controlled by a variety of neuropeptides. In holometabolous insects, crustacean cardioactive peptide (CCAP) is one of many factors that regulate ecdysis behaviours; however, not much is known about the control of ecdysis in hemimetabolous insects. In this thesis, the CCAP-signaling pathway is shown to be essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus. The cDNA sequence of the CCAP gene has been cloned from the R. prolixus central nervous system (CNS) and the functional role of CCAP as a neuromodulator/neurotransmitter demonstrated. Specifically, the expression of RhoprCCAP in CNS neurons producing extensive CCAP-like immunoreactive processes within the neuropile indicates that CCAP plays central roles in coordination of other neurons. RhoprCCAP also acts as a neurohomone/neuromodulator released peripherally to coordinate many tissues. Thus, CCAP-like immunoreactive processes are found in neurohemal sites and also on peripheral tissues. The RhoprCCAP receptor (RhoprCCAPR) has been cloned and shown to be a G-protein coupled receptor (GPCR). RhoprCCAPR expression is observed in the CNS and certain peripheral tissues of R. prolixus. Also, CCAP stimulates hindgut contractions and increases the heartbeat rate in a dose-dependent manner. The involvement of CCAP in R. prolixus ecdysis has been investigated. Up-regulation of the RhoprCCAP transcript in the CNS and the RhoprCCAP receptor (RhoprCCAPR) transcript in the CNS and specific peripheral tissues was observed immediately prior to ecdysis. Also, decreasing staining intensity of CCAP-like immunoreactivity in neurons immediately following ecdysis indicates the release of CCAP during ecdysis. The critical importance of the CCAP-signalling pathway was further demonstrated by knockdown of the RhoprCCAP and RhoprCCAPR transcripts utilizing double stranded RNA interference. Insects with these transcripts knocked down have high mortality (up to 84%), typically at the expected time of ecdysis, or have ecdysis extremely delayed. Taken together, this thesis demonstrates that RhoprCCAP plays a crucial role in regulating ecdysis behaviours in R. prolixus, and clearly shows the conserved nature of the CCAP-signaling pathway in ecdysis for both holometabolous and hemimetabolous insects.

Transforming growth factor beta (TGFbeta) and Angiotensin II (AngII) signaling occurs through two distinct receptor superfamilies, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Through diametric actions, TGFbeta and AngII regulate various biological responses, including cell proliferation and migration. Previously, we identified the G protein-coupled receptor kinase 2 (GRK2), which acts through a negative feedback loop mechanism to terminate Smad signaling. To investigate the impact of TGFbeta-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). We show that activation of the TGFbeta signaling cascade results in increased GRK2 expression levels, consequently inhibiting AngII-induced ERK phosphorylation and antagonizing AngII-induced VSMC proliferation and migration. The inhibitory effect of TGFbeta on AngII signaling occurs at the MEK-ERK interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFbeta signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation. GRK2 is a key factor in mediating this crosstalk.

The evolution of the blood feeding adaptation has required precise coordination of multiple physiological processes in the insect, such as reproduction, behavior, digestion and diuresis. These processes are under careful synchronous hormonal control. For rapid excretion, multiple diuretic hormones are known. Although originally described based on their ability to stimulate hindgut contractions, the Aedes kinins have been shown to stimulate fluid secretion in female mosquitoes of Aedes aegypti. Aedes kinins are leucokinin-like neuropeptides released from neurosecretory cells in the brain and abdominal ganglia. They act by binding to the Aedes kinin receptor, a G proteincoupled receptor (GPCR). The Aedes kinin receptor has been cloned, sequenced, functionally characterized, and immunolocalized to stellate cells in the Malpighian tubules of Ae. aegypti. In addition to their myotropic and diuretic roles, leucokinin-like peptides and/or their receptors have been also been discovered in the nervous, digestive, and reproductive systems of other arthropod species. Therefore, the Aedes kinins have the potential to function in several simultaneous physiological processes that are stimulated by blood feeding. This thesis aims to understand better their role in the whole mosquito by investigating the Aedes kinin receptor's global expression as well as its in vivo contribution to post-prandial diuresis. Presence of the Aedes kinin receptor was investigated in the head, posterior midgut (stomach), hindgut, ovaries, and Malpighian tubules of both non blood-fed and blood-fed females by western blot using anti-receptor antibodies. The receptor was then immunolocalized in the posterior midgut and rectum. Finally, RNAi was employed to knock down kinin receptor expression, followed by measurement of in vivo urine excretion post blood feeding in a precision humidity chamber. Transcript and protein knockdown were confirmed by qPCR and immunohistochemistry, respectively. Results indicate widespread expression of the Aedes kinin receptor protein in organs novel for hematophagous insects and demonstrate the receptor's fundamental role in rapid diuresis. These findings strongly point to the Aedes kinins as integrative signaling molecules that could coordinate multiple physiological systems. The Aedes kinins could therefore have contributed to the success of the blood feeding adapation in mosquitoes.

Animal chemosensation involves several families of G protein-coupled receptors (GPCRs) and, though some of these families are well characterized in vertebrates and nematode worms, receptors have not been identified for most metazoan lineages. In this dissertation, I use a combination of bioinformatics approaches to identify candidate chemosensory receptors in three invertebrates that occupy key positions in the metazoan phylogeny. In the sea urchin Strongylocentrotus purpuratus, I uncovered 192 candidate chemosensory receptors many of which are expressed in sensory structures including pedicellariae and tube feet. In the cephalochordate Branchiostoma floridae, my survey uncovered 50 full-length and 11 partial odorant receptors (OR). No ORs were identified in the urochordate Ciona intestinalis. By exposing conserved amino acid motifs and testing the ability of those motifs to discriminate between ORs and non-OR GPCRs, I identified three OR-specific amino acid motifs that are common in cephalochordate, fish and mammalian ORs and are found in less than 1% of non-ORs from the rhodopsin-like GPCR family. To further investigate the antiquity of vertebrate ORs, I used the OR-specific motifs as probes to search for orthologs among the protein predictions from 12 invertebrates. My search uncovered a novel group of genes in the cnidarian Nematostella vectensis. Phylogenetic analysis that included representatives from the major subgroups of rhodopsin-like GPCRs showed that the cnidarian genes, the cephalochordate and vertebrate ORs, and a subset of genes S. purpuratus from my initial survey, form a monophyletic clade. The taxonomic distribution of these genes indicates that the formation of this clade began at least 700 million years ago, prior to the divergence of cnidarians and bilaterians. Furthermore, my phylogenetic analyses show that three of the four major subgroups of rhodopsin-like GPCRs existed in the ancestor of cnidarians and bilaterians. The utility of the new genes I describe here is that they can be used to identify candidate olfactory cells and organs in cnidarians, echinoderms and cephalochordates that can be tested for function. These genes also provide the raw material for surveys of other metazoans as their genomes become available. My sequence level comparison between chordates, echinoderms and cnidarians exposed several conserved amino acid positions that may be useful for understanding receptor mediated signal transduction. ORs and other rhodopsin-like GPCRs have roles in cell migration, axon guidance and neurite growth; therefore duplication and divergence in the rhodopsin-like gene family may have played a key role in the evolution of cell type diversity (including the emergence of complex nervous systems) and in the evolution of metazoan body plan diversity. / Graduate

Ste2p and Ste3p are well-characterized yeast pheromone G-protein Coupled Receptors (GPCR) those are involved in the signaling of mating responses that lead to cell fusion. Their signaling–associated interactions with G-protein/MAPK signal transduction machinery are well established, homologous to those in mammalian systems, and serve as a simplified model system in GPCR research. While the arrestin- mediated biased signaling mechanism of mammalian GPCR has not been discovered for the pheromone receptors, a recent demonstration of α-arrestins being involved in the internalization of the pheromone GPCR, Ste2p was reported. The present study was designed to reevaluate and extend the alternate functionality for pheromone receptors and to determine the role of yeast arrestins in the yeast mating. Specific residues in the TM6 of Ste2p exhibiting strong mating and constitutive MAPK signaling were combined and investigated in terms of their effect on MAPK signal transduction leading to cell cycle arrest as well as their impact on downstream mating projection formation and zygote formation events. Our findings indicate that Ste2p possess as specific residues that govern its relative bias for mediating MAPK signaling or mating events. Relative dose response experiments accounting for systemic and observation bias for these mutations yielded evidence of mutational-derived functional biases for Ste2p and further validated the alternate pheromone dependent functionalities for Ste2p. Further, arrestin knockout and knock-in studies showed that Art1 (Ldb19) is selectively involved in the regulation of zygote formation but not MAPK signal transduction following the binding of ligand to Ste2p receptors. In addition, ligand stimulated selective localization of Art1 (Ldb19) to the mating projection, implicating it in the regulation of downstream mating functionalities. Overall, while leaving the full mechanism of alternate/biased Ste2p signaling to be elucidated, these results highlight the possibility of continued relevance of the yeast pheromone-mating pathway as a simplified model for GPCR research in the context of arrestin-mediated biased GPCR signaling.

UDP-sugars were identified as extracellular signaling molecules, assigning a new function to these compounds in addition to their well-defined role in intracellular substrate metabolism and storage. Previously regarded as an orphan receptor, the G protein-coupled receptor (GPCR) P2Y14 (GPR105) was found to bind extracellular UDP and UDP-sugars. Little is known about the physiological functions of this GPCR. To study its physiological role a gene-deficient (KO) mouse strain expressing the bacterial LacZ reporter gene was used to monitor the physiological expression pattern of P2Y14. P2Y14 is mainly expressed in pancreas and salivary glands and in subpopulations of smooth muscle cells of the gastrointestinal tract, bronchioles, blood vessels and uterus. Among other phenotypical differences KO mice showed a significantly impaired glucose tolerance following oral and intraperitoneal glucose application. An unchanged insulin tolerance points towards an altered pancreatic islet function. Transcriptome analysis of pancreatic islets showed that P2Y14 deficiency significantly changed expression of components involved in insulin secretion. Insulin secretion tests revealed a reduced insulin release from P2Y14-deficient islets highlighting P2Y14 as a previously unappreciated modulator of proper insulin secretion.