Seven transmembrane (7-TM) G protein coupled receptors (GPCR) constitute the largest family of integral membrane proteins in eukaryotes with more than 1000 members and encoding more than 2% of the human genome. These proteins play a key role in the transmission and transduction of cellular signals responding to hormones, neurotransmitters, light and other agonists, regulating basic biological processes. Their natural abundance together with their localization in the cell membrane makes them suitable targets for therapeutic intervention. Consequently, GPCR are proteins with enormous pharmacologic interest, representing the targets of about 50% of the currently marketed drugs. The current limitations in the experimental techniques necessary for microscopic studies of the membrane as well as membrane proteins emerged the use of computational methods and specifically molecular dynamics simulations. The lead motif of this thesis is the study of GPCR by means of this technique, with the ultimate goal of developing a methodology that can be generalized to the study of most 7-TM as well as other membrane proteins. Since the bovine rhodopsin was the only protein of the GPCR family with a known threedimensional structure at an atomic level until very recently, most of the effort is centered in the study of this receptor as a model of GPCR.The scope of this thesis is twofold. On the one hand it addresses the study of the simulation conditions, including the procedure as well as the sampling box to get optimal results, and on the other, the biological implications of the structural and dynamical behavior observed in the simulations. Specifically, regarding the methodological aspects of the work, the bovine rhodopsin has been studied using different treatments of long-range electrostatic interactions and sampling conditions, as well as the effect of sampling the protein embedded in different one-component lipid bilayers. The binding of ions to lipid bilayers in the absence of the protein has also been investigated. Regarding the biological consequences of the analysis of the MD trajectories, it has been carefully addressed the binding site of retinal and its implications in the process of isomerization after photon uptake, the alteration a group of residues constituting the so-called electrostatic lock between helices TM3 and TM6 in rhodopsin putatively used as common activation mechanism of GPCR, and the structural effects caused by the dimerization based on a recent semi-empirical model. Finally, the specific binding of ions to bacteriorhodopsin has also been studied. The main conclusion of this thesis is provide support to molecular dynamics as technique capable to provide structural and dynamical informational about membranes and membrane proteins, not currently accessible from experimental methods). Moreover, the use of an explicit lipidic environment is crucial for the study the membrane protein dynamics as well as for the protein-protein and lipidprotein interactions.

<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

Lysophosphatidic acid (LPA) is a mitogenic lipid that enhances cell growth, proliferation and motility through binding and activation of at least four receptors, LPA1/Edg2, LPA2/Edg4, LPA3/Edg7, and PPAR and #947;. Here, we show that LPA stimulation inhibits the cell cycle regulator and tumor suppressor, p53. Ten M LPA reduced the cellular levels of total p53 and p53 phosphorylated at serine 15 by approximately 50% in A549 cells and this effect was sustained for at least 6 h. This resulted in a corresponding decrease in p53-mediated transcription. Transient-transfection of the Edg-family LPA receptors, LPA1-3 in HepG2 cells, which do not respond to LPA, also showed this inhibitory response. The response was specific to LPA receptors since neither Gi-coupled M2 muscarinic acetylcholine receptors, nor a mutant LPA1 receptor (LPA1 R124A), which is unable to bind LPA, inhibited p53 activity. Both transient-transfection of the LPA-degrading lipid phosphate phosphatase-1 (LPP-1), or exogenous addition of phospholipase B, which decreases exogenous lysophosphatidate, reversed the LPA receptor-induced decrease in p53-mediated transcription. Although pertussis toxin did not prevent the inhibition of p53, a mutant LPA1 receptor (LPA1 and #8710;361), which lacks the C-terminal PDZ-binding domain, failed to inhibit p53 function. This establishes LPA-mediated inhibition of p53 function requires an interaction with PDZ-containing proteins. These data establish a novel role for LPA-mediated receptor activation in diminishing p53 activity; which, in addition to LPAs well-characterized effects on growth-promoting signaling pathways, is likely to contribute to the survival and proliferation of cancer cells. Of the Edg-family LPA receptors, the LPA1 receptor is the most widely expressed. In the next study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. LPA treatment induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removing agonist following incubation resulted in LPA1 recycling back to the surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). Finally, our results indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.

<p>The increasing number of biological databases today requires that users are able to search more efficiently among as well as in individual databases. One of the most widespread problems is redundancy, i.e. the problem of duplicated information in sets of data. This thesis aims at implementing an algorithm that distinguishes from other related attempts by using the genomic positions of sequences, instead of similarity based sequence comparisons, when making a sequence data set non-redundant. In an automatic updating procedure the algorithm drastically increases the possibility to update and to maintain the topicality of a non-redundant database. The procedure creates a biologically sound non-redundant data set with accuracy comparable to other algorithms focusing on making data sets non-redundant</p>

Ph.D.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 114-126.

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.

Somatostatin (SST) is a peptide hormone that was originally identified in the hypothalamus and subsequently found throughout the central nervous system and in various peripheral organs. Generally classified as an inhibitory factor, SST is secreted by endocrine, neuronal and immune cells and acts to regulate cell secretion, neurotransmission and cell proliferation. There are five receptor-subtypes known to engage SST, termed SSTR1-5, all belonging to the superfamily of G-protein coupled receptors (GPCRs). Within the past few years, there has been a prepondef8:llce of evidence to suggest the importance of GPCR dimerization in receptor-biogenesis, regulation and pharmacology. It has been previously reported in our laboratory, that human (h) SSTR5 homo- and heterodimerizes with hSSTR1 in an agonist-regulated manner. However, it was unclear as to the contribution of each subtype in the formation of the hSSTR1/hSSTR5 heterodimer, the possible molecular determinants involved and the effects of heterodimerization on the pharmacology of the receptors. Furthermore, the dimerization properties of other hSSTRs including their heterodimerization remain undetermined. Here, we demonstrate that agonist binding to hSSTR5 and not hSSTR1 modulates the formation of the heterodimer, with particular emphasis on its carboxyl-terminal tail in specifying the interaction. We also determined the mechanics of the hSSTR2 homodimer, unlike the previous hSSTRs investigated, forms constitutive dimers that dissociate into monomers following activation with agonist. This feature is important for receptor trafficking, as preventing their dissociation impairs agonist-mediated endocytosis. Lastly, we investigated the heterodimerization of hSSTR2 and hSSTR5, an interaction that, like the hSSTR1/hSSTR5 heterodimer, is subtype-specific, requiring selective-activation of hSSTR2 and not hSSTR5. The heterodimer exhibited enhanced signalling characteristics including, prolonged activation of MAP kinases and an increase in the induction of the cyclin-dependent kinase inhibitor p27Kip1. These enhanced properties of the heterodimer conferred an extended growth inhibitory response. Dimerization of GPCRs, with particular emphasis on heterodimers, generates novel receptors with unique properties distinct from those of the individual receptor monomers/homodimers. An understanding on the mechanisms involved in GPCR dimerization could provide a rationale in future drug design.