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.

LPA1 lysophosphatidic acid receptors (LPA1Rs) are normally present on the surface of the cell. Our initial findings were that HMG-CoA reductase inhibitors (atorvastatin and mevastatin) induce the sequestration of the G protein-coupled LPA1R in recycling endosomes, most likely by inhibiting the recycling of tonically internalized receptors. Whereas, co-addition of geranylgeranylpyrophosphate (GGPP) or geranylgeraniol (GGOH) prevented atorvastatin-induced sequestration of LPA1Rs, the geranylgeranyltransferase-I inhibitor, GGTI-298, mimicked atorvastatin and induced LPA1R sequestration. This suggested that statin-induced endosomal sequestration was caused by defective protein prenylation. The likely targets of atorvastatin and GGTI-298 are the Rho family GTPases, RhoC and RhoA, since both inhibitors greatly reduced the abundance of these GTPases and since knockdown of endogenous RhoC or RhoA with small interfering RNAs (siRNAs) led to endosomal sequestration of LPA1R. Knockdown of RhoC was much more potent at inducing endosomal sequestration than knockdown of either RhoA or RhoB. In contrast, atorvastatin, GGTI-298, siRNA against RhoA, B, or C did not alter the internalization or recycling of transferrin receptors, indicating that recycling of transferrin receptors is distinct from LPA1Rs. Thus, these results, for the first time, implicate RhoA and RhoC in endocytic recycling of LPA1Rs and identify atorvastatin and GGTI-298 as novel inhibitors of this process. / Per the request of the author and advisor, and with the approval of the Graduate Education office, the following changes were made to this thesis: Replaced original page 1 with Errata Page 2. Replaced original pages 3-28 with Errata Pages 3 – 16. Replaced original pages 69-71 with Errata pages 17 – 19.

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.

The quaternary status of G protein-coupled receptors (GPCRs) is important, unknown and controversial. Estimates of size from numerous pharmacological, biochemical and biophysical studies range from monomers to octamers. Accounts of stability vary from constitutive oligomers to a spontaneous, ligand-regulated interconversion between monomers and dimers. In the present investigation, the oligomeric size of GPCRs in live Chinese hamster ovary (CHO) cells has been examined by two methods. Both are based on the efficiency of Förster resonance energy transfer (FRET) between fluorophore-tagged receptors, as determined from emission spectra via spectral deconvolution. In the first, the apparent FRET efficiency (Eapp) was measured for cells expressing eGFP- and eYFP-tagged M2 muscarinic receptors at different ratios of acceptor to donor. Eapp then was related to the pair-wise efficiency (Ep) according to a model that enumerates all pathways for the transfer of energy between single donors and acceptors within an oligomer of given size (n). Each value n returned a distinct and well-defined value of Ep. Fluorescence lifetime imaging provided an independent estimate of Ep that was in close agreement with the model-based value when n = 4, identifying the M2 receptor as a tetramer. In the second approach, the M1 and M2 muscarinic receptors and the β1 and β2 adrenergic receptors were tagged with GFP2 and eYFP, and the value of Eapp was estimated for each pixel in the image of a cell. The distributions of Eapp from 34–40 cells expressing each receptor were compared with those predicted for populations of dimers, trimers and tetramers, the latter configured as a square and a rhombus. In each case, the combined data were well described in terms of a rhombus. Distributions obtained for the M2 and β2 receptors were not affected by agonists or inverse agonists, nor was there evidence for appreciable numbers of dimers or larger oligomers. Taken together, the results suggest that GPCRs of Family 1 exist largely or wholly as constitutive tetramers.

The current epidemic of obesity has been linked to an increase in fat intake associated with the Western diet. Nutrient-induced stimulation of enteroendocrine cells in the small intestine leads to the release of hormones that contribute to satiety and the control of food intake. In particular, ingested fat, specifically in the form of free fatty acids, is potent activator of enteroendocrine cells in the proximal small intestine. However, the underlying signaling cascade that free fatty acids initiate in these enteroendocrine cells, which leads to secretion of satiety hormones, is not known. In general, my research is focused on identifying nutrient-responsive pathways in enteroendocrine cells involved with the release of satiety signals and using this information to begin to develop novel drug delivery strategies to reduce food intake. In general, my results revealed that activation of the fatty acid receptor GPR120 was ecessary for the linoleic acid-induced intracellular calcium rise, a necessary precursor for hormone release. Using patch clamp recording, I discovered that linoleic acid activated enteroendocrine cells by inducing membrane depolarization, a process requiring the calcium-activated, monovalent cation permeable channel TRPM5, which is activated downstream of GPR120. To validate the unexpected finding that TRPM5 was involved in fattyacid signaling, I performed experiments using bitter compounds, whose transduction pathway is known to involve TRPM5. Enteroendocrine cells express the bitter taste receptors and release cholecystokinin in response to bitter stimuli, suggesting the probable role of gut in initiation of protective behavior against ingestion of potentially harmful substances. Armed with the data on the specifics of the fatty acid transduction, I performed experiments using nanoparticles to determine their utility for delivering pharmaceuticals specifically to the enteroendocrine cells. I fabricated and characterized PLGA nanoparticles and performed intracellular uptake studies in order to optimally delivery payloads inside cells. Finally, I validated their use by using cell-based assays to determine the effects of internalized PLGA nanoparticles on ion channels and signaling pathways involved in CCK release. Taken together, this dissertation research has identified the signaling pathways (pharmacological targets) involved in fatty acid-mediated satiety hormone release and validated the potential therapeutic use of nanoparticle-mediated drug delivery for the eventual control of food intake.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20691号 / 工博第4388号 / 新制||工||1682(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 田中 宏明, 教授 高野 裕久, 教授 米田 稔 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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