Among 18 family members in the myosin superfamily, myosin IX is unique by possessing a GTPase activating protein (GAP) for Rho. It is also attention-grabbing since it is a single-headed processive motor, as well as a minus-end directed motor. Although many biochemical properties have been revealed, its physiological function is largely unknown. As an initial step to address this question, I attempted to find the binding partner of myosin IXb using the yeast two-hybrid screen. Through the screen using the tail domain of myosin IXb as bait I found BIG1, a guanine nucleotide exchange factor (GEF) for ADP-ribosylation factor (Arfl), as a potential binding partner for myosin IXb. The interaction between myosin IXb and BIG1 was demonstrated by co-immunoprecipitation of endogenous myosin IXb and BIG1 with anti-BIG1 antibodies in normal rat kidney (NRK) cells. Using the isolated proteins, it was demonstrated that myosin IXb and BIG1 directly bind to each other. Various truncation mutants of the myosin IXb tail domain were produced and it was revealed that the binding region of myosin IXb to BIG1 is the zinc finger/GAP domain. Interestingly, the GAP activity of myosin IXb was significantly inhibited by addition of BIG1 with IC50 of 0.06 μM. The RhoA binding to myosin IXb was inhibited by the addition of BIG1 with a concentration similar to that which inhibit the GAP activity. Likewise, RhoA inhibited the BIG1 binding of myosin IXb. These results suggest that BIG1 and RhoA compete with each other for the binding to myosin IXb, thus resulting in the inhibition of the GAP activity by BIG1. The present study identified BIG1, the ArfGEF, as a new binding partner for myosin IXb, which inhibited the GAP activity of myosin IXb. Together, the results imply that the RhoGAP activity of myosin IXb is down-regulated by BIG1 at the Golgi, where myosin IXb could be involved in the regulation of actin cytoskeleton through the Rho-signaling pathway.

Modulation of Ca2+ channels is an important mechanism for regulation of synaptic strength. However, it is clear that some Ca2+ current types are insensitive to inhibitory modulation mediated by heterotrimeric G proteins (G protein inhibition), and among currents which are sensitive to G protein inhibition, there is great variation in the magnitude of Ca2+ current inhibition between cells of different origin. For the experiments in this dissertation, I utilized recently cloned Ca2+ channels to determine the minimal combination of Ca2+ channel subunits which would confer G protein sensitivity to the recombinant channels. In addition, I examined the role Ca2+ channel auxiliary subunits play in regulation of Ca2+ channel sensitivity to inhibitory G proteins, and whether channels which were sensitive to G protein inhibition were regulated equivalently by the auxiliary subunits. Finally, I investigated possible mechanisms by which these auxiliary subunits modulate G protein-mediated inhibition of Ca2+ channels. I found that α1A and α1B Ca2+ currents, when expressed in Xenopus oocytes, were sensitive to modulation by G proteins in the absence of any Ca2+ channel auxiliary subunits, while α1C currents were not modulated under the same conditions. I conclude from this data that Ca2+ channel α1 subunits are differentially sensitive to G protein modulation, and the α1 subunit of the class A and B Ca2+ channels is sufficient for G protein modulation. I also tested the ability of Ca2+ channel auxiliary subunits to modulate the magnitude of G protein-mediated inhibition Ca2+ currents. I found that the Ca2+ channel α2 subunit had no effect on the magnitude of G protein inhibition of α1A and α1B currents. However, the Ca2+ channel β3 subunit eliminated tonic G protein inhibition and sharply reduced the magnitude of muscarinic M2 receptor induced G protein inhibition of both α1A and α1B currents. I found, however, that while the magnitude of α1A and α1B current inhibition was equivalent in the absence of auxiliary subunits, the magnitude of inhibition was greater for the α1B channel after co-expression of the Ca2+ channel β3 subunit. These results indicate that the Ca2+ channel β3 subunit reduces the sensitivity of α1A and α1B Ca2+ channels to voltage-dependent G protein modulation, and does so to a greater extent for α1A channels when compared to α1B Ca2+ channels. I found that M2 receptor induced inhibition of α1B currents was more voltage-dependent after expression of the Ca2+ channel β3 subunit. Additionally, the rate relief of G protein inhibition dramatically increased after co-expression of the Ca2+ channel β3 subunit. I also co-expressed G protein subunits, and determined that inhibition of both α1B and α1Bβ3 currents was mediated by the G protein βγ subunit. Furthermore, the rate of voltage-dependent relief of G protein βγ subunit induced inhibition increased after co-expression of the Ca2+ channel β3 subunit, similar to the increased rate of relief of the M2 receptor induced G protein inhibition. These data, along with data which demonstrates that G protein inhibition results from the binding of the G protein βγ subunit to the Ca2+ channel (De Waard et al., 1997), indicate that the Ca2+ channel β3 subunit subunit reduces the magnitude of G protein inhibition of α1B Ca2+ currents by increasing the rate of dissociation of the G protein βγ subunit, such that moderate depolarizations used to activate the channel also relieve a large portion of the G protein inhibition.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 199-250.

Yeung, Barry Ho Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 137-154). / Abstracts in English and Chinese. / Abstract --- p.i / 論文摘要 --- p.iv / Acknowledgements --- p.vii / Publications based on work in this thesis. --- p.ix / List of abbreviations --- p.x / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Dorsal root ganglion cells --- p.1 / Chapter 1.1.1 --- Primary sensory neurons --- p.1 / Chapter 1.1.2 --- Non-neuronal cells --- p.3 / Chapter 1.1.2.1 --- Satellite glial cells --- p.3 / Chapter 1.1.2.2 --- Schwann cells --- p.6 / Chapter 1.2 --- Peripheral sensitization --- p.8 / Chapter 1.3 --- Neuron-glia interactions --- p.9 / Chapter 1.4 --- Aim of Thesis --- p.11 / Chapter Chapter 2 --- "Materials, media, buffers and solutions" --- p.13 / Chapter 2.1 --- Materials --- p.13 / Chapter 2.2 --- "Culture media, buffer and solutions" --- p.19 / Chapter 2.2.1 --- Culture media --- p.19 / Chapter 2.2.2 --- General culture buffers and culture plate coating reagents --- p.19 / Chapter 2.3 --- Antibodies used for identifying DRG cells --- p.23 / Chapter 2.3.1 --- Primary antibodies --- p.23 / Chapter 2.3.2 --- Secondary antibodies --- p.23 / Chapter Chapter 3 --- Methods --- p.24 / Chapter 3.1 --- Preparation of DRG cell cultures --- p.24 / Chapter 3.2 --- Preparation of neuron-enriched and glial cell cultures --- p.25 / Chapter 3.3 --- Immunocytochemistry --- p.26 / Chapter 3.4 --- Immunohistochemistry --- p.27 / Chapter 3.4 --- Determination of [3H]cAMP production in DRG cells --- p.28 / Chapter 3.4.1 --- Principle of assay --- p.28 / Chapter 3.4.2 --- Loading DRG cells with [3H]adenine --- p.28 / Chapter 3.4.3 --- Column preparation --- p.28 / Chapter 3.4.4 --- Measurement of [3H]cAMP production in DRG cells --- p.29 / Chapter 3.4.5 --- Data analysis --- p.30 / Chapter Chapter 4 --- Identification of DRG cells in dissociated cultures --- p.31 / Chapter 4.1 --- Introduction --- p.31 / Chapter 4.2 --- Aim of study --- p.34 / Chapter 4.3 --- Results --- p.35 / Chapter 4.3.1 --- Identification of DRG cells in isolated cultures --- p.35 / Chapter 4.3.2 --- Activation and proliferation of glial cells in isolated cell cultures --- p.36 / Chapter 4.3.3 --- Identification of glial cells in cultures --- p.38 / Chapter 4.3.4 --- Modification of staining methods --- p.40 / Chapter 4.3.5 --- Immunohistochemistry to identify DRG cells in DRG slices --- p.42 / Chapter 4.3.6 --- Comparison of antibody staining in whole DRG and isolated DRG cells --- p.44 / Chapter 4.4 --- Discussion --- p.44 / Chapter 4.5 --- Summary --- p.53 / Chapter Chapter 5 --- Characterization of GPCRs in isolated DRG cultures --- p.69 / Chapter 5.1 --- Introduction --- p.69 / Chapter 5.1.1 --- G-protein coupled receptors --- p.69 / Chapter 5.1.2 --- Pharmacological characterization of prostanoid receptors on DRG cells --- p.73 / Chapter 5.1.3 --- Gs- and Gi/o-coupled GPCRs in DRG cells --- p.75 / Chapter 5.1.3.1 --- Gs-coupled GPCR: β-adrenoceptors --- p.76 / Chapter 5.1.3.2 --- Gs-coupled GPCR: CGRP receptors --- p.79 / Chapter 5.1.3.3 --- Gi/o-coupled GPCR: α2-adrenoceptors --- p.82 / Chapter 5.1.3.4 --- Gi/o-coupled GPCR: Cannabinoid receptors --- p.85 / Chapter 5.1.3.5 --- Gi/o-coupled GPCR: 5-HT1Areceptors --- p.88 / Chapter 5.1.3.6 --- Gi/o-coupled GPCR: opioid and opioid-receptor-like 1 receptors --- p.90 / Chapter 5.2 --- Aims of study --- p.93 / Chapter 5.3 --- Results --- p.94 / Chapter 5.3.1 --- Characterization of prostanoid receptors in isolated DRG cells --- p.94 / Chapter 5.3.2 --- Characterization of CGRP receptors in isolated DRG cells --- p.96 / Chapter 5.3.3 --- Investigation of the effect of CGRP8.37 on CGRP responses --- p.97 / Chapter 5.3.4 --- Characterization of β1-adrenoceptors in isolated DRG cells --- p.97 / Chapter 5.3.5 --- Characterization of β2-adrenoceptors in isolated DRG cells --- p.98 / Chapter 5.3.6 --- Identification of β-adrenoceptor subtype mediating isoprenaline-stimulated responses.. --- p.99 / Chapter 5.3.7 --- Characterization of α2-adrenceptors in isolated DRG cells --- p.100 / Chapter 5.3.8 --- Characterization of cannabinoid 1 receptors in isolated DRG cells ... --- p.100 / Chapter 5.3.9 --- Characterization of cannabinoid 2 receptors in isolated DRG cells --- p.101 / Chapter 5.3.10 --- Characterization of 5-HT1A receptors in isolated DRG cells --- p.101 / Chapter 5.3.11 --- Characterization of μ-opioid receptors in isolated DRG cells --- p.102 / Chapter 5.3.12 --- Characterization of opioid-receptor-like 1 receptors in isolated DRG cells --- p.102 / Chapter 5.3.13 --- Effect of nerve growth factor on DRG cells --- p.103 / Chapter 5.4 --- Discussion --- p.106 / Chapter 5.5 --- Summary --- p.114 / Chapter Chapter 6 --- Conclusion and further studies --- p.134 / References --- p.137

We are interested in understanding the molecular mechanisms that govern synaptic growth and plasticity. Recent evidence from several laboratories suggests that small GTPases play an important role in the promotion of neurite outgrowth; however, their role in the control of synaptic growth and functional plasticity is not well understood. The goal of this thesis is to investigate the role of small GTPases (including Rac, Rho and Cdc42) in the regulation of synaptic growth in vivo, using the Drosophila larval neuromuscular junction (NMJ) synapses as a model system. Our results show that presynaptic overexpression of Rac, but not of Rho or Cdc42, positively regulates both synaptic structure and function. Genetic loss of Rac leads to embryonic lethality, making it impossible to assess the full loss-of-function phenotype using conventional mutants. To circumvent this, we use the MARCM (Mosaic Analysis with a Repressible Cell Marker) technique to generate single motor neuron clones devoid of all genetic copies of Rac. Our data suggest that Rac activity is crucial for normal synaptic development. In support of this conclusion, we demonstrate that genetic removal of trio, a guanine nucleotide exchange factor (GEF) that is known to activate Rac, leads to a drastic reduction in the number of synaptic boutons. In addition, genetic removal of one copy of trio is sufficient to suppress the gain-of-function phenotype of Rac. Moreover, we demonstrate that partial removal of the fragile X mental retardation gene (dfmr1), a known suppressor of Rac, enhances the gain-of-function phenotype of Rac. Taken together, our findings support a model in which Rac signaling positively regulates synaptic growth and function at the Drosophila larval NMJ.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2008" Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [115]-133).

Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 104 pages. Includes vita. Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 180-194.

Orientadores: Nilson Ivo Tonin Zanchin, Beatriz Gomes Guimaraes / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T09:39:45Z (GMT). No. of bitstreams: 1 Scapin_SandraMaraNaressi_D.pdf: 11335048 bytes, checksum: 153f9eea9142fb7f3cb17de59a608da6 (MD5) Previous issue date: 2007 / Resumo: As GTPases da família Rab regulam o transporte intracelular de vesículas em eucariotos. Cada Rab atua em uma via de transporte específica e seu mecanismo de ação se dá através da realização de um ciclo de ligação e hidrólise de GTP. Neste trabalho, foi determinada a estrutura cristalográfica das formas inativa (ligada a GDP) e ativa (ligada a GppNHp) da GTPase Rab11b, um membro da subfamília Rab11 que está envolvida na reciclagem de proteínas dos endossomos para a membrana plasmática, no tráfego de vesículas da rede trans-Golgi para a membrana plasmática e na fagocitose. Os resultados foram confrontados com os dados estruturais da Rab11a descritos anteriormente. A Rab11b inativa cristalizou como um monômero, o que gera conflitos a respeito da formação de dímeros funcionais pela Rab11a. A Rab11b e a Rab11a ativas divergiram em relação à posição e à interação da serina 20, que é importante na hidrólise de GTP, mas apresentaram taxas hidrolíticas semelhantes in vitro. Visando uma investigação mais ampla da família Rab, a GTPase Rab21 também foi cristalizada, mas os cristais difrataram até 2.90 Å de resolução. Ensaios de desnaturação térmica revelaram que a Rab21 é estruturalmente mais instável do que a Rab11, talvez pela presença de cisteínas que estão susceptíveis à oxidação, contribuindo para a agregação e precipitação da proteína. A Rab11 é bastante estável, e possivelmente forma estruturas do tipo beta-amilóide em altas temperaturas. Este trabalho envolveu também o estudo funcional da interação entre a proteína TIP41 humana (TIPRL) e o fator de transcrição MafB. A TIPRL é uma proteína conservada que foi identificada como uma ativadora de MAP quinases enquanto sua homóloga em levedura foi caracterizada como um antagonista da via de sinalização da quinase TOR que regula o crescimento celular. A MafB está envolvida no controle transcricional em diversos processos de desenvolvimento, mas seus reguladores ainda não estão bem estabelecidos. A interação direta entre a TIPRL e a MafB inteira, ou seu domínio bZIP isolado, foi confirmada através de ensaios de ligação in vitro. As proteínas co-localizaram no núcleo de células HEK293 e nossos resultados preliminares mostram que a TIPRL inibe a atividade transcricional da MafB in vivo, embora apenas interfira na ligação in vitro do domínio bZIP da MafB ao seu DNA-alvo mediante a estabilização do complexo TIPRL-bZIP. A TIPRL pode, portanto, constituir um novo regulador da atividade de MafB / Abstract: GTPases of the Rab family are responsible for the intracellular transport of vesicles. Each family member acts on a specific transport pathway and their function is regulated by GTP binding and hydrolysis, cycling between inactive (GDP-bound) and active (GTP-bound) forms. In this work, we describe the crystal structure of inactive and active forms of the GTPase Rab11b, a member of the Rab11 subfamily which is involved in recycling of proteins from endosomes to the plasma membrane, in polarized transport in epithelial cells, in the transport of molecules of the trans-Golgi network to the plasma membrane and in phagocytosis. The Rab11b structure showed several differences from the Rab11a isoform previously described. Inactive Rab11b crystallized as a monomer, contradicting the hypothesis about functional dimers formed by Rab11a. Active Rab11b differ from Rab11a relative to the position of the serine 20 sidechain, which is involved in GTP hydrolysis, although both GTPases show similar GTP hydrolysis rates in vitro. In order to obtain structural information on Rab GTPases, Rab21 was also crystallized, but the crystals diffracted to a relatively low resolution (2.90 Å). Rab21 is a cysteine rich protein, showing a higher instability relative to Rab11b. Thermal unfolding followed by circular dicroism confirmed this hypothesis. Both Rab11b and Rab11a show a relatively high thermal stability and circular dicroism analysis indicate that they undergo conversion to structures rich in beta-strands upon thermal denaturation. This work includes also studies on the function of TIPRL in regard to its interaction with the transcription factor MafB. TIPRL is a conserved human protein identified as an activator of MAP kinases whereas its yeast counterpart Tip41 functions as an antagonist of the TOR kinase pathway. MafB is a large member of the Maf family of bZIP transcription factors controlling developmental processes in vertebrates. Regulation of MafB is critical, for example, during erythroid differentiation. A direct interaction between TIPRL and full length MafB and the bZIP domain of MafB was confirmed by in vitro interaction assays. TIPRL is localized throughout the whole cell and overlaps with MafB in the nucleus of HEK293 cells. Preliminary assays showed that TIPRL inhibits transcriptional activation mediated by MafB in HEK293 cells, although MafB shows a higher binding affinity to its target DNA relative to TIPRL in vitro. This evidence indicates that TIPRL may control MafB activity in vivo / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular