Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 140-150).

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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.

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Gene prediction in newly sequenced genomes is a known challenging. Although sophisticated comparative pipelines are available, computationally derived gene models are often less than perfect. This is particularly true when multiple very similar paralogs are present. The issue is aggravated further when genomes are assembled only at a preliminary draft level to contigs or short scaffolds rather than to chromosomes. However, these genomes deliver valuable information for studying gene families. High accuracy models of protein-coding genes are needed in particular for phylogenetics and for the analysis of gene family histories. In this dissertation, I established a tool, the ExonMatchSolver-pipeline (EMS-pipeline), that can assist the assembly of genes distributed across multiple fragments (e.g. contigs). The tool in particular tackles the problem of identifying those coding exon groups that belong to the same paralogous genes in a fragmented genome assembly. The EMS-pipeline accommodates a homology search step with a protein input set consisting of several highly similar paralogs as query. The core of the pipeline uses an Integer Linear Programming Implementation to solve the paralog-to-contig assignment problem. An extension to the initial implementation estimates the number of paralogs encoded in the target genome and can handle several paralogs that are situated on the same genomic fragment. The EMS-pipeline was successfully applied to simulated data, several showcase examples and to deuterostome genomes in a large scale study on the evolution of the arrestin protein family. Especially at high genome fragmentation levels, the tool outperformed a naive assignment method. Arrestins are key signaling transducers that bind to activated and phosphorylated G protein-coupled receptors and can mediate their endocytosis into the cell. The refined annotations of arrestins resulting from the application of the EMS-pipeline are more complete and accurate in comparison to a conventional database search strategy. With the applied strategy it was possible to map the duplication- and deletion history of arrestin paralogs including tandem duplications, pseudogenizations and the formation of retrogenes in detail. My results support the emergence of the four arrestin paralogs from a visual and a non-visual proto-arrestin. Surprisingly, the visual ARR3 was lost in the mammalian clades afrotherians and xenarthrans. Segmental duplications in specific clades and the 3R-WGD in the teleost stem lineage, on the other hand, must have given rise to new paralogs that show signatures of diversification in functional elements important for receptor binding and phosphate sensing. The four vertebrate orthology groups show an interesting pattern of divergence of three endocytosis motifs: the minor and major clathrin binding site and the adapter protein-2 (AP-2) binding motif. Identification of such signatures, of residues that determine specificity between paralogs and are positively selected after duplication was made possible by high quality alignments obtained by genome inquiries, dense species sampling and consideration of fragmented loci from poorly assembled genomes in the framework of the EMS-pipeline, that was established in this dissertation.:1 Introduction 2 1.1 Basics and definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1.1 What is a gene? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1.2 What is a tree in phylogenetics? . . . . . . . . . . . . . . . . . . 3 1.1.3 What are paralogs and orthologs? . . . . . . . . . . . . . . . . . 4 1.1.4 Central dogma in molecular biology: From DNA to protein . . 5 1.2 Gene duplications as evolutionary playground . . . . . . . . . . . . . . 12 1.2.1 Mechanisms of gene duplication . . . . . . . . . . . . . . . . . . 13 1.2.2 Evolutionary fate of duplicated genes . . . . . . . . . . . . . . . 14 1.3 Identification and annotation of protein homologs . . . . . . . . . . . . 15 1.3.1 Challenges of existing resources . . . . . . . . . . . . . . . . . . 16 1.3.2 Similarity search approaches without consideration of the gene structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.3.3 Gene structure aware gene annotation approaches . . . . . . . . 19 1.3.4 Graph-based inference of orthology relationships . . . . . . . . 21 1.3.5 Chance and challenge of fragmented assemblies . . . . . . . . . 21 1.4 Applied phylogenetic methods . . . . . . . . . . . . . . . . . . . . . . . 22 1.4.1 Phylogenetic inference in a nutshell . . . . . . . . . . . . . . . . 23 1.4.2 Inference of natural selection in inter-species data sets . . . . . 29 1.4.3 Detection of specificity determining positions . . . . . . . . . . 32 1.5 Multi-talents in cell signaling: The cytosolic arrestin proteins . . . . . . 34 1.5.1 Functions of arrestins in cell signaling . . . . . . . . . . . . . . . 34 1.5.2 Arrestin activation by GPCR binding . . . . . . . . . . . . . . . 36 1.5.3 Functions of arrestins in cellular trafficking . . . . . . . . . . . . 37 1.5.4 Evolution of arrestins . . . . . . . . . . . . . . . . . . . . . . . . 39 2 The ExonMatchSolver-pipeline 42 2.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.1 Pipeline overview . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.2 Exon assembly as an assignment problem . . . . . . . . . . . . . 43 2.2.3 Solving the Paralog-to-Contig Assignment Problem . . . . . . . 46 2.2.4 Post-processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.2.5 Implementation and usage . . . . . . . . . . . . . . . . . . . . . 48 2.2.6 Performance assessment by simulations . . . . . . . . . . . . . . 50 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2.3.1 Performance on simulated data . . . . . . . . . . . . . . . . . . . 50 2.3.2 Performance on real data - Two Showcase Examples . . . . . . . 51 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 3 Evolution of the arrestin protein family in deuterostomes 61 3.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.2.1 Database scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.2.2 Detailed gene annotation . . . . . . . . . . . . . . . . . . . . . . 63 3.2.3 Data resources used in the current study . . . . . . . . . . . . . 64 3.2.4 Alignment and building of phylogenetic trees . . . . . . . . . . 64 3.2.5 Identification of specificity determining positions . . . . . . . . 65 3.2.6 Testing for natural selection . . . . . . . . . . . . . . . . . . . . . 66 3.2.7 Assessement of conservation . . . . . . . . . . . . . . . . . . . . 66 3.2.8 Parsimonious reconstruction of exon gain and loss events . . . 67 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 3.3.1 Evolution of the arrestin fold family based on database inquiries 67 3.3.2 The refined arrestin annotations are more complete than database entries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 3.3.3 Arrestin paralog gain and loss patterns based on the refined annotations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 3.3.4 Evolution of arrestin functional elements . . . . . . . . . . . . . 88 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 3.4.1 Limitation of arrestin database annotations . . . . . . . . . . . . 96 3.4.2 Arrestins in early vertebrate evolution . . . . . . . . . . . . . . . 98 3.4.3 Sub- and neofunctionalization as consequence of the 3R-WGD . 102 3.4.4 Independent arrestin duplications in deuterostomes . . . . . . . 104 3.4.5 Loss of arrestin paralogs in different vertebrate orders . . . . . 106 3.4.6 Previously unknown interaction partners and isoforms . . . . . 108 4 Improvements on the ExonMatchSolver-pipeline 110 4.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 4.2.1 Estimation of the paralog number . . . . . . . . . . . . . . . . . 111 4.2.2 Subdivision of gene loci on the same contig . . . . . . . . . . . . 113 4.2.3 Implementation details . . . . . . . . . . . . . . . . . . . . . . . 113 4.2.4 Assessment of the ExonMatchSolver-pipeline Version 2 . . . 115 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 5 Conclusion and Outlook 119 A Additional figures 123 B Additional tables 134 C CV 152 Bibliography 156

Cardiac dysfunction is correlated with detrimental prognosis of sepsis and contributes to a high risk of mortality. After an initial hyperinflammatory reaction, most patients enter a protracted state of immunosuppression (late sepsis) that alters both innate and adaptive immunity. The changes of cardiac function in late sepsis are not yet known. MicroRNA-155 (miR-155) is previously found to play important roles in both regulations of immune activation and cardiac function. In this study, C57BL/6 mice were operated to develop into early and late sepsis phases, and miR-155 mimic was injected through the tail vein 48 h after cecal ligation and puncture (CLP). The effect of miR-155 on CLP-induced cardiac dysfunction was explored in late sepsis. We found that increased expression of miR-155 in the myocardium protected against cardiac dysfunction in late sepsis evidenced by attenuating sepsis-reduced cardiac output and enhancing left ventricular systolic function. We also observed that miR-155 markedly reduced the infiltration of macrophages and neutrophils into the myocardium and attenuated the inflammatory response via suppression of JNK signaling pathway. Moreover, overexpression of β-arrestin 2 (Arrb2) exacerbated the mice mortality and immunosuppression in late sepsis. Furthermore, transfection of miR-155 mimic reduced Arrb2 expression, and then restored immunocompetence and improved survival in late septic mice. We conclude that increased miR-155 expression through systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling and Arrb2 mediated immunosuppression.

Sepsis is an exaggerated systemic inflammatory response to persistent bacteria infection with high morbidity and mortality rate clinically. β-arrestin 2 modulates cell survival and cell death in different systems. However, the effect of β-arrestin 2 on sepsis-induced cardiac dysfunction is not yet known. Here, we show that β-arrestin 2 overexpression significantly enhances animal survival following cecal ligation and puncture (CLP)-induced sepsis. Importantly, overexpression of β-arrestin 2 in mice prevents CLP-induced cardiac dysfunction. Also, β-arrestin 2 overexpression dramatically attenuates CLP-induced myocardial gp130 and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CLP. Therefore, β-arrestin 2 prevents CLP-induced cardiac dysfunction through gp130 and p38. These results suggest that modulation of β-arrestin 2 might provide a novel therapeutic approach to prevent cardiac dysfunction in patients with sepsis.

Our previous studies have shown that β-arrestin 2 plays an anti-apoptotic effect. However, the mechanisms by which β-arrestin contribute to anti-apoptotic role remain unclear. In this study, we show that a deficiency of either β-arrestin 1 or β-arrestin 2 significantly increases serum deprivation (SD)-induced percentage of apoptotic cells. β-arrestin 2 deficient-induced apoptosis was inhibited by transfection with β-arrestin 2 full-length plasmid, revealing that SD-induced apoptosis is dependent on β-arrestin 2. Furthermore, in the absence of either β-arrestin 1 or β-arrestin 2 significantly enhances SD-induced the level of pro-apoptotic proteins, including cleaved caspase-3, extracellular-signal regulated kinase 1/2 (ERK1/2) and p38, members of mitogen-activated protein kinases (MAPKs). In addition, a deficiency of either β-arrestin 1 or β-arrestin 2 inhibits phosphorylation of Akt. The SD-induced changes in cleaved caspase-3, ERK1/2 and p38 MAPKs, Akt, and apoptotic cell numbers could be blocked by double knockout of β-arrestin 1/2. Our study thus demonstrates that β-arrestin inhibits cell apoptosis through pro-apoptotic ERK1/2 and p38 MAPKs and anti-apoptotic Akt signaling pathways.

Objective: Stress, either physical or psychological, can modulate immune function. However, the mechanisms associated with stress-induced immune suppression remain to be elucidated. β-Arrestin 2 serves as adaptor, scaffold, and/or signal transducer. The role of β-arrestin 2 in stress-induced immune suppression is not known yet. Methods/Results: Here, we demonstrate that β-arrestin 2 deficiency in mice increases the sensitivity to the chronic stress-induced reduction in the number of splenocytes. Interestingly, the stress-induced suppression of T helper-type (Th) 1 cytokines and the increased production of Th2 cytokines were greatly enhanced in β-arrestin 2-deficient mice compared with wild-type mice. Moreover, inhibition of PI3K in β-arrestin 2-deficient mice exerts an additive effect on the stress-induced reduction in the number of splenocytes. Conclusion: Our study demonstrates that a deficiency in β-arrestin 2 augments stress-induced immune suppression.

Background: Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that β-arrestin 2 plays a critical role in cell apoptosis. The role of β-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established. Scope of Review: EC cells HEC1B and Ishikawa were transfected with either β-arrestin 2 RNA interfering (RNAi) plasmid or β-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of β-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally β-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3β. Overexpression of β-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3β. Taken together, our studies demonstrate for the first time that β-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells. Major Conclusions: Resveratrol primes EC cells to undergo apoptosis by modulating β-arrestin 2 mediated Akt/GSK3β signaling pathways. General significance: These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by β-arrestin 2 and may provide insights into a potential clinical relevance in EC.