36 results on '"Lanahan AA"'
Search Results
2. Transcriptional profiling in coronary artery disease: indications for novel markers of coronary collateralization.
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Chittenden TW, Sherman JA, Xiong F, Hall AE, Lanahan AA, Taylor JM, Duan H, Pearlman JD, Moore JH, Schwartz SM, Simons M, Chittenden, Thomas W, Sherman, Jonathan A, Xiong, Fei, Hall, Amy E, Lanahan, Anthony A, Taylor, Jennifer M, Duan, Hangjun, Pearlman, Justin D, and Moore, Jason H
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- 2006
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3. Expression of NGF receptor in the developing and adult primate central nervous system
- Author
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Schatteman, GC, primary, Gibbs, L, additional, Lanahan, AA, additional, Claude, P, additional, and Bothwell, M, additional
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- 1988
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4. Ethanol tolerance in engineered strains of Clostridium thermocellum.
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Olson DG, Maloney MI, Lanahan AA, Cervenka ND, Xia Y, Pech-Canul A, Hon S, Tian L, Ziegler SJ, Bomble YJ, and Lynd LR
- Abstract
Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75-80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production., (© 2023. BioMed Central Ltd., part of Springer Nature.)
- Published
- 2023
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5. Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production.
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Holwerda EK, Olson DG, Ruppertsberger NM, Stevenson DM, Murphy SJL, Maloney MI, Lanahan AA, Amador-Noguez D, and Lynd LR
- Abstract
Background: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains., Results: Contrary to lactate dehydrogenase, deleting phosphotransacetylase ( pta , acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A , ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively., Conclusions: Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB . Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings., Competing Interests: Competing interestsLRL is a shareholder in a start-up company focusing on cellulosic biofuel production and conversion. There are no other competing interests., (© The Author(s) 2020.)
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- 2020
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6. Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum .
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Walker JE, Lanahan AA, Zheng T, Toruno C, Lynd LR, Cameron JC, Olson DG, and Eckert CA
- Abstract
The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum , we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus , is active in C. thermocellum . We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF . For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo / beta homologs, isolated from Acidithiobacillus caldus , are functional in C. thermocellum . For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lee Lynd is a co-founder of the Enchi Corporation, which has a financial interest in C. thermocellum. Work within was filed as a provisional patent, Provisional Patent Application No. 62/896,555 titled Novel Recombineering Machinery to Increase Homology Directed Genome Editing in Thermophilic Microbes.
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- 2019
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7. The redox-sensing protein Rex modulates ethanol production in Thermoanaerobacterium saccharolyticum.
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Zheng T, Lanahan AA, Lynd LR, and Olson DG
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- Adaptation, Biological, Alcohol Dehydrogenase metabolism, Fermentation, Gene Deletion, Gene Expression Regulation, Bacterial, Genetic Complementation Test, Mutation, Oxidation-Reduction, Whole Genome Sequencing, Ethanol metabolism, Gene Products, rex genetics, Gene Products, rex metabolism, Thermoanaerobacterium genetics, Thermoanaerobacterium metabolism
- Abstract
Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.
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- 2018
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8. Metabolome analysis reveals a role for glyceraldehyde 3-phosphate dehydrogenase in the inhibition of C. thermocellum by ethanol.
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Tian L, Perot SJ, Stevenson D, Jacobson T, Lanahan AA, Amador-Noguez D, Olson DG, and Lynd LR
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Background: Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application., Results: Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum , we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum . We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum ., Conclusions: We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum . This enzyme is a promising target for future metabolic engineering work.
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- 2017
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9. Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases ( adhE s).
- Author
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Hon S, Lanahan AA, Tian L, Giannone RJ, Hettich RL, Olson DG, and Lynd LR
- Abstract
Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE . To explore the effects of overexpressing wild-type, mutant, and exogenous adhE s, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum . As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.
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- 2016
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10. Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485.
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Zhou J, Olson DG, Lanahan AA, Tian L, Murphy SJ, Lo J, and Lynd LR
- Abstract
Background: Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30-70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities., Results: It was found that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield., Conclusion: PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.
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- 2015
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11. Identifying promoters for gene expression in Clostridium thermocellum .
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Olson DG, Maloney M, Lanahan AA, Hon S, Hauser LJ, and Lynd LR
- Abstract
A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes β-galactosidase ( lacZ ) and NADPH-alcohol dehydrogenase ( adhB ) in Clostridium thermocellum , an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum , gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ ), and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB )., (© 2015 The Authors.)
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- 2015
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12. PTP1b is a physiologic regulator of vascular endothelial growth factor signaling in endothelial cells.
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Lanahan AA, Lech D, Dubrac A, Zhang J, Zhuang ZW, Eichmann A, and Simons M
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- Animals, Aorta cytology, Cell Movement physiology, Cell Proliferation, Disease Models, Animal, Endothelial Cells cytology, Female, Hindlimb blood supply, Human Umbilical Vein Endothelial Cells, Ischemia metabolism, Ischemia physiopathology, Male, Mice, Mice, Mutant Strains, Neovascularization, Physiologic physiology, Primary Cell Culture, Protein Tyrosine Phosphatase, Non-Receptor Type 1 genetics, RNA, Small Interfering genetics, Vascular Endothelial Growth Factor A metabolism, Endothelial Cells physiology, Protein Tyrosine Phosphatase, Non-Receptor Type 1 metabolism, Signal Transduction physiology, Vascular Endothelial Growth Factor Receptor-2 metabolism
- Abstract
Background: Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis., Methods and Results: We generated mice carrying an endothelial-specific deletion of PTP1b and examined the effect of this knockout on VEGF signaling, angiogenesis, and arteriogenesis in vitro and in vivo. PTP1b knockout endothelial cells had increased VEGF-dependent activation of extracellular signal-regulated kinase signaling, sprouting, migration, and proliferation compared with controls. Endothelial PTP1b null mice had increased retinal and Matrigel implant angiogenesis and accelerated wound healing, pointing to enhanced angiogenesis. Increased arteriogenesis was demonstrated by observations of faster recovery of arterial blood flow and large numbers of newly formed arterioles in the hindlimb ischemia mouse model. PTP1b endothelial knockout also rescued impaired blood flow recovery after common femoral artery ligation in synectin null mice., Conclusions: PTP1b is a key regulator of endothelial VEGFR2 signaling and plays an important role in regulation of the extent of vascular tree formation., (© 2014 American Heart Association, Inc.)
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- 2014
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13. Angiopoietin-2 secretion by endothelial cell exosomes: regulation by the phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) and syndecan-4/syntenin pathways.
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Ju R, Zhuang ZW, Zhang J, Lanahan AA, Kyriakides T, Sessa WC, and Simons M
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- Angiopoietin-2 genetics, Animals, Cells, Cultured, Endothelial Cells cytology, Exosomes genetics, Mice, Mice, Knockout, Neovascularization, Physiologic physiology, Nitric Oxide Synthase Type III genetics, Phosphatidylinositol 3-Kinases genetics, Proto-Oncogene Proteins c-akt genetics, Syndecan-4 genetics, Syntenins genetics, Angiopoietin-2 metabolism, Endothelial Cells metabolism, Exosomes metabolism, Nitric Oxide Synthase Type III metabolism, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-akt metabolism, Signal Transduction physiology, Syndecan-4 metabolism, Syntenins metabolism
- Abstract
Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.
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- 2014
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14. Endothelial cell-dependent regulation of arteriogenesis.
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Moraes F, Paye J, Mac Gabhann F, Zhuang ZW, Zhang J, Lanahan AA, and Simons M
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- Adaptor Proteins, Signal Transducing deficiency, Adaptor Proteins, Signal Transducing genetics, Animals, Cells, Cultured, Genotype, Mice, Mice, 129 Strain, Mice, Inbred C57BL, Mice, Knockout, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle metabolism, Phenotype, Time Factors, Adaptor Proteins, Signal Transducing metabolism, Arteries metabolism, Endothelial Cells metabolism, Neovascularization, Physiologic
- Abstract
Rationale: Arteriogenesis is the process of formation of arterial conduits. Its promotion is an attractive therapeutic strategy in occlusive atherosclerotic diseases. Despite the functional and clinical importance of arteriogenesis, the biology of the process is poorly understood. Synectin, a gene previously implicated in the regulation of vascular endothelial cell growth factor signaling, offers a unique opportunity to determine relative contributions of various cell types to arteriogenesis., Objective: We investigated the cell-autonomous effects of a synectin knockout in arterial morphogenesis., Methods and Results: A floxed synectin knockin mouse line was crossbred with endothelial-specific (Tie2, Cdh5, Pdgfb) and smooth muscle myosin heavy chain-specific Cre driver mouse lines to produce cell type-specific deletions. Ablation of synectin expression in endothelial, but not smooth muscle cells resulted in the presence of developmental arterial morphogenetic defects (smaller size of the arterial tree, reduced number of arterial branches and collaterals) and impaired arteriogenesis in adult mice., Conclusions: Synectin modulates developmental and adult arteriogenesis in an endothelial cell-autonomous fashion. These findings show for the first time that endothelial cells are central to both developmental and adult arteriogenesis and provide a model for future studies of factors involved in this process.
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- 2013
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15. VEGFR2 trafficking: speed doesn't kill.
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Zhang X, Lanahan AA, and Simons M
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- Animals, Arteries cytology, Arteries growth & development, Endosomes metabolism, Endothelial Cells cytology, Endothelium, Vascular cytology, Endothelium, Vascular growth & development, Extracellular Signal-Regulated MAP Kinases genetics, Extracellular Signal-Regulated MAP Kinases metabolism, Humans, Mice, Mice, Transgenic, Morphogenesis genetics, Neuropilin-1 metabolism, Protein Isoforms genetics, Protein Isoforms metabolism, Protein Transport, Signal Transduction, Vascular Endothelial Growth Factor A metabolism, Vascular Endothelial Growth Factor Receptor-2 metabolism, Arteries metabolism, Endothelial Cells metabolism, Endothelium, Vascular metabolism, Gene Expression Regulation, Developmental, Neuropilin-1 genetics, Vascular Endothelial Growth Factor A genetics, Vascular Endothelial Growth Factor Receptor-2 genetics
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- 2013
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16. Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.
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Bono F, De Smet F, Herbert C, De Bock K, Georgiadou M, Fons P, Tjwa M, Alcouffe C, Ny A, Bianciotto M, Jonckx B, Murakami M, Lanahan AA, Michielsen C, Sibrac D, Dol-Gleizes F, Mazzone M, Zacchigna S, Herault JP, Fischer C, Rigon P, Ruiz de Almodovar C, Claes F, Blanc I, Poesen K, Zhang J, Segura I, Gueguen G, Bordes MF, Lambrechts D, Broussy R, van de Wouwer M, Michaux C, Shimada T, Jean I, Blacher S, Noel A, Motte P, Rom E, Rakic JM, Katsuma S, Schaeffer P, Yayon A, Van Schepdael A, Schwalbe H, Gervasio FL, Carmeliet G, Rozensky J, Dewerchin M, Simons M, Christopoulos A, Herbert JM, and Carmeliet P
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- Allosteric Regulation, Animals, Antibodies, Monoclonal pharmacology, Arthritis, Experimental drug therapy, Bone Resorption drug therapy, Carcinoma, Lewis Lung drug therapy, Carcinoma, Lewis Lung metabolism, Carcinoma, Lewis Lung pathology, Fibroblast Growth Factors antagonists & inhibitors, Fibroblast Growth Factors metabolism, HEK293 Cells, Human Umbilical Vein Endothelial Cells cytology, Human Umbilical Vein Endothelial Cells drug effects, Human Umbilical Vein Endothelial Cells metabolism, Humans, Mice, Neovascularization, Pathologic drug therapy, Pancreatic Neoplasms drug therapy, Pancreatic Neoplasms metabolism, Pancreatic Neoplasms pathology, Phosphorylation drug effects, Protein Kinase Inhibitors metabolism, Receptor Protein-Tyrosine Kinases antagonists & inhibitors, Signal Transduction, Xenograft Model Antitumor Assays, Protein Kinase Inhibitors pharmacology, Receptors, Fibroblast Growth Factor antagonists & inhibitors, Receptors, Fibroblast Growth Factor metabolism, Small Molecule Libraries pharmacology
- Abstract
Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment., (Copyright © 2013 Elsevier Inc. All rights reserved.)
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- 2013
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17. Fibroblast growth factor signaling potentiates VE-cadherin stability at adherens junctions by regulating SHP2.
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Hatanaka K, Lanahan AA, Murakami M, and Simons M
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- Animals, Capillary Permeability, Cattle, Cells, Cultured, Endothelial Cells cytology, Endothelium, Vascular cytology, Endothelium, Vascular metabolism, Human Umbilical Vein Endothelial Cells, Humans, Phosphorylation, Adherens Junctions metabolism, Antigens, CD metabolism, Cadherins metabolism, Endothelial Cells metabolism, Fibroblast Growth Factors metabolism, Protein Tyrosine Phosphatase, Non-Receptor Type 11 metabolism, Signal Transduction physiology
- Abstract
Background: The fibroblast growth factor (FGF) system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization., Methods and Findings: In vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs) known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity., Conclusions: These results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability.
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- 2012
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18. Therapeutic implications of GIPC1 silencing in cancer.
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Chittenden TW, Pak J, Rubio R, Cheng H, Holton K, Prendergast N, Glinskii V, Cai Y, Culhane A, Bentink S, Schwede M, Mar JC, Howe EA, Aryee M, Sultana R, Lanahan AA, Taylor JM, Holmes C, Hahn WC, Zhao JJ, Iglehart JD, and Quackenbush J
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- Antineoplastic Agents pharmacology, Breast Neoplasms metabolism, Cell Transformation, Neoplastic, Colorectal Neoplasms metabolism, Disease Progression, Epithelial Cells cytology, Female, Humans, Oligonucleotide Array Sequence Analysis, Polymerase Chain Reaction methods, RNA Interference, Adaptor Proteins, Signal Transducing genetics, Apoptosis, Breast Neoplasms genetics, Colorectal Neoplasms genetics, Gene Expression Regulation, Neoplastic, Gene Silencing
- Abstract
GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers.
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- 2010
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19. VEGF receptor 2 endocytic trafficking regulates arterial morphogenesis.
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Lanahan AA, Hermans K, Claes F, Kerley-Hamilton JS, Zhuang ZW, Giordano FJ, Carmeliet P, and Simons M
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- Adaptor Proteins, Signal Transducing, Animals, Arteries growth & development, Carrier Proteins genetics, Cell Membrane physiology, Endocytosis, Endothelium, Vascular embryology, Gene Silencing, Mice, Mice, Knockout, Myosin Heavy Chains deficiency, Myosin Heavy Chains genetics, Myosin Heavy Chains physiology, Neuropeptides genetics, Phosphorylation, Vascular Endothelial Growth Factor A physiology, Zebrafish, Arteries embryology, Endothelium, Vascular physiology, Morphogenesis physiology, Neuropeptides deficiency, Vascular Endothelial Growth Factor Receptor-2 physiology
- Abstract
VEGF is the key growth factor regulating arterial morphogenesis. However, molecular events involved in this process have not been elucidated. Synectin null mice demonstrate impaired VEGF signaling and a marked reduction in arterial morphogenesis. Here, we show that this occurs due to delayed trafficking of VEGFR2-containing endosomes that exposes internalized VEGFR2 to selective dephosphorylation by PTP1b on Y(1175) site. Synectin involvement in VEGFR2 intracellular trafficking requires myosin-VI, and myosin-VI knockout in mice or knockdown in zebrafish phenocopy the synectin null phenotype. Silencing of PTP1b restores VEGFR2 activation and significantly recovers arterial morphogenesis in myosin-VI(-/-) knockdown zebrafish and synectin(-/-) mice. We conclude that activation of the VEGF-mediated arterial morphogenesis cascade requires phosphorylation of the VEGFR2 Y(1175) site that is dependent on trafficking of internalized VEGFR2 away from the plasma membrane via a synectin-myosin-VI complex. This key event in VEGF signaling occurs at an intracellular site and is regulated by a novel endosomal trafficking-dependent process., (Copyright 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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20. ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish.
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Ren B, Deng Y, Mukhopadhyay A, Lanahan AA, Zhuang ZW, Moodie KL, Mulligan-Kehoe MJ, Byzova TV, Peterson RT, and Simons M
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- Adaptor Proteins, Signal Transducing, Animals, Carrier Proteins physiology, Male, Mice, Neovascularization, Physiologic, Neuropeptides physiology, Phosphatidylinositol 3-Kinases physiology, Signal Transduction physiology, Vascular Endothelial Growth Factor A pharmacology, Vascular Endothelial Growth Factor Receptor-2 physiology, Zebrafish, Zebrafish Proteins, Arteries growth & development, Mitogen-Activated Protein Kinase 1 physiology, Mitogen-Activated Protein Kinase 3 physiology, Morphogenesis, Proto-Oncogene Proteins c-akt physiology
- Abstract
Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis.
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- 2010
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21. Heterozygous deficiency of PHD2 restores tumor oxygenation and inhibits metastasis via endothelial normalization.
- Author
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Mazzone M, Dettori D, de Oliveira RL, Loges S, Schmidt T, Jonckx B, Tian YM, Lanahan AA, Pollard P, de Almodovar CR, De Smet F, Vinckier S, Aragonés J, Debackere K, Luttun A, Wyns S, Jordan B, Pisacane A, Gallez B, Lampugnani MG, Dejana E, Simons M, Ratcliffe P, Maxwell P, and Carmeliet P
- Subjects
- Animals, Blood Vessels embryology, Blood Vessels metabolism, Cell Shape, DNA-Binding Proteins genetics, Endothelial Cells cytology, Glycolysis, Heterozygote, Hypoxia metabolism, Hypoxia-Inducible Factor-Proline Dioxygenases, Immediate-Early Proteins genetics, Mice, Neoplasms pathology, Procollagen-Proline Dioxygenase, Blood Vessels cytology, DNA-Binding Proteins metabolism, Endothelial Cells metabolism, Immediate-Early Proteins metabolism, Neoplasm Metastasis, Neoplasms blood supply, Oxygen metabolism
- Abstract
A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.
- Published
- 2009
- Full Text
- View/download PDF
22. Vascular endothelial growth factor and semaphorin induce neuropilin-1 endocytosis via separate pathways.
- Author
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Salikhova A, Wang L, Lanahan AA, Liu M, Simons M, Leenders WP, Mukhopadhyay D, and Horowitz A
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Carrier Proteins genetics, Cells, Cultured, Chickens, Clathrin physiology, Humans, Ligands, Membrane Microdomains physiology, Mice, Mice, Knockout, Neuropeptides deficiency, Neuropeptides genetics, Protein Transport genetics, Protein Transport physiology, Signal Transduction genetics, Vascular Endothelial Growth Factor A metabolism, Endocytosis physiology, Neuropilin-1 metabolism, Semaphorins physiology, Signal Transduction physiology, Vascular Endothelial Growth Factor A physiology
- Abstract
The neuropilin (Nrp)1 receptor is essential for both nervous and vascular system development. Nrp1 is unusually versatile, because it transmits both chemoattractive and repulsive signals in response to vascular endothelial growth factor (VEGF)-A and class 3 semaphorins, respectively. Both Nrp1 and VEGF receptor 2 undergo ligand-dependent endocytosis. We sought to establish the endocytic pathway of Nrp1 and to determine whether uptake is required for its signaling. Whereas Nrp1 underwent clathrin-dependent endocytosis in response to VEGFA(165) treatment, semaphorin 3C (sema3C) induced lipid raft-dependent endocytosis. The myosin VI PDZ (postsynaptic density 95, Disk large, Zona occludens-1) adaptor protein synectin was essential for Nrp1 trafficking. Sema3C failed to inhibit migration of synectin(-/-) endothelial cells, mirroring the lower migratory response of these cells to VEGFA(165). These results show that the endocytic pathway of Nrp1 is determined by its ligand and that the trafficking of Nrp1 is essential for its signaling.
- Published
- 2008
- Full Text
- View/download PDF
23. Neuropilin-1-VEGFR-2 complexing requires the PDZ-binding domain of neuropilin-1.
- Author
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Prahst C, Héroult M, Lanahan AA, Uziel N, Kessler O, Shraga-Heled N, Simons M, Neufeld G, and Augustin HG
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Animals, Carrier Proteins chemistry, Cytoplasm metabolism, Humans, Mice, Models, Biological, Neuropeptides chemistry, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Signal Transduction, Swine, Vascular Endothelial Growth Factor A metabolism, Neuropilin-1 metabolism, Vascular Endothelial Growth Factor Receptor-2 metabolism
- Abstract
Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF(165) binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF(165) binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1.
- Published
- 2008
- Full Text
- View/download PDF
24. Synectin-dependent gene expression in endothelial cells.
- Author
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Lanahan AA, Chittenden TW, Mulvihill E, Smith K, Schwartz S, and Simons M
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Cell Adhesion, Cell Separation, Endothelial Cells cytology, Heart, Lung cytology, Mice, Mice, Knockout, Nucleic Acid Hybridization methods, Oligonucleotide Array Sequence Analysis, Receptor, Fibroblast Growth Factor, Type 2 pharmacology, Carrier Proteins genetics, Carrier Proteins physiology, Endothelial Cells physiology, Gene Expression Regulation drug effects, Neuropeptides genetics, Neuropeptides physiology
- Abstract
Synectin (GIPC1), a receptor scaffold protein, has been isolated by our laboratory as a syndecan-4 cytoplasmic domain binding partner that regulates important aspects of cell motility (Gao Y, Li M, Chen W, Simons M. J Cell Physiol 184: 373-379, 2000; Tkachenko E, Elfenbein A, Tirziu D, Simons M. Circ Res 98: 1398-1404, 2006). Moreover, synectin plays a major role in arterial morphogenesis and in growth factor signaling in arterial endothelial cells by regulating Rac1 activity (Chittenden TW, Claes F, Lanahan AA, Autiero M, Palac RT, Tkachenko EV, Elfenbein A, Ruiz de Almodovar C, Dedkov E, Tomanek R, Li W, Westmore M, Singh J, Horowitz A, Mulligan-Kehoe MJ, Moodie KL, Zhuang ZW, Carmeliet P, Simons M. Dev Cell 10: 783-795, 2006). The present study was carried out to characterize changes in synectin-dependent gene expression induced by homozygous disruption of the gene in endothelial cells. Using a combination of suppression subtraction hybridization and high throughput microarray technology, we have identified aberrant biological processes of transcriptional regulation in synectin(-/-) primary endothelial cells including abnormal basal regulation of genes associated with development, cell organization and biogenesis, intracellular tracking, and cell adhesion. Analysis of gene expression following FGF2 treatment demonstrated significant abnormalities in transcription, cytoskeletal organization and biogenesis, and protein modification and transport in synectin(-/-) compared with synectin(+/+) endothelial cells. These results confirm synectin involvement in FGF2-dependent signal transduction and provide insights into synectin-dependent gene expression in the endothelium.
- Published
- 2006
- Full Text
- View/download PDF
25. Selective regulation of arterial branching morphogenesis by synectin.
- Author
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Chittenden TW, Claes F, Lanahan AA, Autiero M, Palac RT, Tkachenko EV, Elfenbein A, Ruiz de Almodovar C, Dedkov E, Tomanek R, Li W, Westmore M, Singh JP, Horowitz A, Mulligan-Kehoe MJ, Moodie KL, Zhuang ZW, Carmeliet P, and Simons M
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Arteries abnormalities, Arteries cytology, Carrier Proteins chemistry, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Movement, Cell Proliferation, Cells, Cultured, Embryo, Nonmammalian, Endothelial Cells cytology, Endothelial Cells physiology, Endothelium, Vascular cytology, Female, Femoral Artery cytology, Gene Expression Regulation, Gene Expression Regulation, Developmental, Mice, Mice, Knockout, Myocardium cytology, Neuropeptides genetics, Pregnancy, Venae Cavae cytology, Zebrafish Proteins genetics, Arteries embryology, Arteries growth & development, Morphogenesis, Neuropeptides deficiency, Zebrafish embryology, Zebrafish Proteins metabolism
- Abstract
Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.
- Published
- 2006
- Full Text
- View/download PDF
26. Verge: a novel vascular early response gene.
- Author
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Regard JB, Scheek S, Borbiev T, Lanahan AA, Schneider A, Demetriades AM, Hiemisch H, Barnes CA, Verin AD, and Worley PF
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Brain Ischemia metabolism, Cell Hypoxia, Cell Membrane Permeability physiology, Cells, Cultured, Disease Models, Animal, Endothelium, Vascular cytology, Endothelium, Vascular drug effects, Enzyme Activators pharmacology, Gene Expression Regulation, Developmental physiology, Growth Substances pharmacology, Humans, Mice, Molecular Sequence Data, Myocardium metabolism, Neovascularization, Physiologic genetics, Organ Specificity, Protein Kinase C metabolism, RNA, Messenger biosynthesis, Rats, Seizures chemically induced, Seizures metabolism, Sequence Homology, Amino Acid, Transfection, Endothelium, Vascular metabolism, Genes, Immediate-Early genetics, Immediate-Early Proteins biosynthesis, Immediate-Early Proteins genetics
- Abstract
Vascular endothelium forms a continuous, semipermeable barrier that regulates the transvascular movement of hormones, macromolecules, and other solutes. Here, we describe a novel immediate early gene that is expressed selectively in vascular endothelial cells, verge (vascular early response gene). Verge protein includes an N-terminal region of approximately 70 amino acids with modest homology (approximately 30% identity) to Apolipoprotein L but is otherwise unique. Verge mRNA and protein are induced selectively in the endothelium of adult vasculature by electrical or chemical seizures. Verge expression appears to be responsive to local tissue conditions, because it is induced in the hemisphere ipsilateral to transient focal cerebral ischemia. In contrast to the transient expression in adult, Verge mRNA and protein are constitutively expressed at high levels in the endothelium of developing tissues (particularly heart) in association with angiogenesis. Verge mRNA is induced in cultured endothelial cells by defined growth factors and hypoxia. Verge protein is dramatically increased by cysteine proteinase inhibitors, suggesting rapid turnover, and is localized to focal regions near the periphery of the cells. Endothelial cell lines that stably express Verge form monolayers that show enhanced permeability in response to activation of protein kinase C by phorbol esters. This response is accompanied by reorganization of the actin cytoskeleton and the formation of paracellular gaps. These studies suggest that Verge functions as a dynamic regulator of endothelial cell signaling and vascular function.
- Published
- 2004
- Full Text
- View/download PDF
27. Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins.
- Author
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Tu JC, Xiao B, Naisbitt S, Yuan JP, Petralia RS, Brakeman P, Doan A, Aakalu VK, Lanahan AA, Sheng M, and Worley PF
- Subjects
- Animals, Binding Sites physiology, COS Cells, Calcium metabolism, Calcium Channels metabolism, Carrier Proteins chemistry, Carrier Proteins genetics, Disks Large Homolog 4 Protein, Homer Scaffolding Proteins, Humans, Inositol 1,4,5-Trisphosphate Receptors, Intracellular Signaling Peptides and Proteins, Kidney cytology, Membrane Proteins, Microscopy, Immunoelectron, Mutagenesis, Site-Directed physiology, Neurons metabolism, Neuropeptides chemistry, Proline metabolism, Protein Structure, Tertiary, Rabbits, Rats, Receptors, Cytoplasmic and Nuclear metabolism, Receptors, N-Methyl-D-Aspartate metabolism, SAP90-PSD95 Associated Proteins, Synapses chemistry, Synapses metabolism, Synapses ultrastructure, Transfection, Adaptor Proteins, Signal Transducing, Carrier Proteins metabolism, Nerve Tissue Proteins metabolism, Neurons chemistry, Neuropeptides metabolism, Receptors, Metabotropic Glutamate metabolism
- Abstract
Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.
- Published
- 1999
- Full Text
- View/download PDF
28. Neuronal activity induction of the stathmin-like gene RB3 in the rat hippocampus: possible role in neuronal plasticity.
- Author
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Beilharz EJ, Zhukovsky E, Lanahan AA, Worley PF, Nikolich K, and Goodman LJ
- Subjects
- Alternative Splicing physiology, Amino Acid Sequence, Animals, Base Sequence, COS Cells, DNA, Complementary, Exons genetics, Gene Expression Regulation drug effects, Gene Expression Regulation physiology, Introns genetics, Long-Term Potentiation physiology, Memory physiology, Molecular Sequence Data, Nerve Growth Factors pharmacology, PC12 Cells, RNA, Messenger analysis, Rats, Stathmin, Dentate Gyrus cytology, Microtubule Proteins, Neuronal Plasticity physiology, Neurons chemistry, Neurons physiology, Phosphoproteins genetics
- Abstract
Synaptic activity induces a rapid transcriptional response that is essential for the establishment of long-term neuronal plasticity. Using a differential cloning technique, we have identified a gene induced by seizure activity in the brain as RB3. RB3 is a recently cloned gene belonging to the stathmin family of phosphoproteins. Like SCG10, RB3 is brain-specific, although in situ hybridization results show that the expression of RB3 is more ubiquitous than is that of SCG10. Using genomic DNA sequencing, we show that the 27 amino acid sequence unique to the RB3" transcript is encoded by an alternatively spliced exon, exon 2'. Using a peptide antibody raised against exon 2' to detect RB3" and an anti-Flag antibody to detect an epitope-tagged version of RB3, we show that both proteins are localized to the Golgi apparatus of transfected COS7 cells. Of particular interest, RB3 mRNA, but not SCG10 mRNA, is rapidly induced in the dentate gyrus granule layer of the hippocampus after electrically induced seizure activity as well as stimuli leading to long-term potentiation (LTP). In addition, RB3 mRNA is induced in pheochromocytoma (PC12) cells treated with 250 ng/ml NGF. These results suggest that RB3 may play a role in activity-induced neuronal plasticity and neuronal differentiation.
- Published
- 1998
29. Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins.
- Author
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Xiao B, Tu JC, Petralia RS, Yuan JP, Doan A, Breder CD, Ruggiero A, Lanahan AA, Wenthold RJ, and Worley PF
- Subjects
- Amino Acid Sequence, Animals, Brain metabolism, Carrier Proteins antagonists & inhibitors, Carrier Proteins chemistry, Carrier Proteins genetics, Homer Scaffolding Proteins, Mice, Molecular Sequence Data, Multigene Family genetics, Neuropeptides antagonists & inhibitors, Neuropeptides chemistry, Neuropeptides genetics, Rats, Tissue Distribution, Carrier Proteins physiology, Nerve Tissue Proteins metabolism, Neuropeptides physiology, Receptors, Metabotropic Glutamate metabolism, Synapses metabolism
- Abstract
Homer is a neuronal immediate early gene (IEG) that is enriched at excitatory synapses and binds group 1 metabotropic glutamate receptors (mGluRs). Here, we characterize a family of Homer-related proteins derived from three distinct genes. Like Homer IEG (now termed Homer 1a), all new members bind group 1 mGluRs. In contrast to Homer 1a, new members are constitutively expressed and encode a C-terminal coiled-coil (CC) domain that mediates self-multimerization. CC-Homers form natural complexes that cross-link mGluRs and are enriched at the postsynaptic density. Homer 1a does not multimerize and blocks the association of mGluRs with CC-Homer complexes. These observations support a model in which the dynamic expression of Homer 1a competes with constitutively expressed CC-Homers to modify synaptic mGluR properties.
- Published
- 1998
- Full Text
- View/download PDF
30. Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors.
- Author
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Tu JC, Xiao B, Yuan JP, Lanahan AA, Leoffert K, Li M, Linden DJ, and Worley PF
- Subjects
- Amino Acid Sequence, Animals, Calcium metabolism, Calcium Channels genetics, Carrier Proteins genetics, Carrier Proteins physiology, Dynamins, GTP Phosphohydrolases genetics, Gene Expression, Genes, Immediate-Early, Glutamic Acid pharmacology, Homer Scaffolding Proteins, Inositol 1,4,5-Trisphosphate Receptors, Intracellular Membranes metabolism, Ligands, Molecular Sequence Data, Neuropeptides genetics, Neuropeptides physiology, Rats, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Metabotropic Glutamate antagonists & inhibitors, Time Factors, Calcium Channels metabolism, Carrier Proteins metabolism, Neuropeptides metabolism, Proline genetics, Receptors, Cytoplasmic and Nuclear metabolism, Receptors, Metabotropic Glutamate metabolism
- Abstract
Group I metabotropic glutamate receptors (mGluRs) activate PI turnover and thereby trigger intracellular calcium release. Previously, we demonstrated that mGluRs form natural complexes with members of a family of Homer-related synaptic proteins. Here, we present evidence that Homer proteins form a physical tether linking mGluRs with the inositol trisphosphate receptors (IP3R). A novel proline-rich "Homer ligand" (PPXXFr) is identified in group 1 mGluRs and IP3R, and these receptors coimmunoprecipitate as a complex with Homer from brain. Expression of the IEG form of Homer, which lacks the ability to cross-link, modulates mGluR-induced intracellular calcium release. These studies identify a novel mechanism in calcium signaling and provide evidence that an IEG, whose expression is driven by synaptic activity, can directly modify a specific synaptic function.
- Published
- 1998
- Full Text
- View/download PDF
31. Dynamic regulation of RGS2 suggests a novel mechanism in G-protein signaling and neuronal plasticity.
- Author
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Ingi T, Krumins AM, Chidiac P, Brothers GM, Chung S, Snow BE, Barnes CA, Lanahan AA, Siderovski DP, Ross EM, Gilman AG, and Worley PF
- Subjects
- Animals, COS Cells chemistry, COS Cells enzymology, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Cerebral Cortex chemistry, Cerebral Cortex cytology, Cerebral Cortex enzymology, Cocaine pharmacology, Dopamine Antagonists pharmacology, Dopamine Uptake Inhibitors pharmacology, Female, GTP Phosphohydrolases metabolism, Gene Expression drug effects, Gene Expression physiology, Genes, Immediate-Early physiology, Haloperidol pharmacology, Hippocampus chemistry, Hippocampus cytology, Hippocampus enzymology, Hydrolysis, Lipid Metabolism, Male, Neurons chemistry, Neurons drug effects, RNA, Messenger metabolism, Rats, Rats, Inbred F344, Rats, Sprague-Dawley, Receptors, Muscarinic physiology, Signal Transduction drug effects, Synaptic Transmission drug effects, Synaptic Transmission physiology, GTP-Binding Proteins physiology, Neuronal Plasticity physiology, Neurons enzymology, Signal Transduction physiology
- Abstract
Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein, and recent studies provide insight into the molecules involved in this response. Here, we demonstrate that mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction.
- Published
- 1998
32. GRIP: a synaptic PDZ domain-containing protein that interacts with AMPA receptors.
- Author
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Dong H, O'Brien RJ, Fung ET, Lanahan AA, Worley PF, and Huganir RL
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Brain metabolism, Carrier Proteins chemistry, Carrier Proteins genetics, Cell Line, Cloning, Molecular, Intracellular Signaling Peptides and Proteins, Molecular Sequence Data, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins genetics, Neurons metabolism, Rats, Receptor Aggregation, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae, Tissue Distribution, Transfection, Carrier Proteins metabolism, Nerve Tissue Proteins metabolism, Receptors, AMPA metabolism, Synapses metabolism
- Abstract
AMPA glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system and play a role in the synaptic plasticity underlying learning and memory. AMPA receptors are heteromeric complexes of four homologous subunits (GluR1-4) that differentially combine to form a variety of AMPA receptor subtypes. These subunits are thought to have a large extracellular amino-terminal domain, three transmembrane domains and an intracellular carboxy-terminal domain. AMPA receptors are localized at excitatory synapses and are not found on adjacent inhibitory synapses enriched in GABA(A) receptors. The targeting of neurotransmitter receptors, such as AMPA receptors, and ion channels to synapses is essential for efficient transmission. A protein motif called a PDZ domain is important in the targeting of a variety of membrane proteins to cell-cell junctions including synapses. Here we identify a synaptic PDZ domain-containing protein GRIP (glutamate receptor interacting protein) that specifically interacts with the C termini of AMPA receptors. GRIP is a new member of the PDZ domain-containing protein family which has seven PDZ domains and no catalytic domain. GRIP appears to serve as an adapter protein that links AMPA receptors to other proteins and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain.
- Published
- 1997
- Full Text
- View/download PDF
33. Homer: a protein that selectively binds metabotropic glutamate receptors.
- Author
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Brakeman PR, Lanahan AA, O'Brien R, Roche K, Barnes CA, Huganir RL, and Worley PF
- Subjects
- Animals, Binding Sites, Brain metabolism, Carrier Proteins chemistry, Carrier Proteins genetics, Cell Line, Cloning, Molecular, Gene Expression Regulation, Hippocampus metabolism, Homer Scaffolding Proteins, Humans, Immunoenzyme Techniques, Mice, Molecular Sequence Data, Nerve Tissue Proteins chemistry, Nerve Tissue Proteins genetics, Neurons metabolism, Neuropeptides chemistry, Neuropeptides genetics, RNA, Messenger metabolism, Rats, Sequence Deletion, Sequence Homology, Amino Acid, Signal Transduction, Carrier Proteins metabolism, Nerve Tissue Proteins metabolism, Neuropeptides metabolism, Receptors, Metabotropic Glutamate metabolism, Synapses metabolism
- Abstract
Spatial localization and clustering of membrane proteins is critical to neuronal development and synaptic plasticity. Recent studies have identified a family of proteins, the PDZ proteins, that contain modular PDZ domains and interact with synaptic ionotropic glutamate receptors and ion channels. PDZ proteins are thought to have a role in defining the cellular distribution of the proteins that interact with them. Here we report a novel dendritic protein, Homer, that contains a single, PDZ-like domain and binds specifically to the carboxy terminus of phosphoinositide-linked metabotropic glutamate receptors. Homer is highly divergent from known PDZ proteins and seems to represent a novel family. The Homer gene is also distinct from members of the PDZ family in that its expression is regulated as an immediate early gene and is dynamically responsive to physiological synaptic activity, particularly during cortical development. This dynamic transcriptional control suggests that Homer mediates a novel cellular mechanism that regulates metabotropic glutamate signalling.
- Published
- 1997
- Full Text
- View/download PDF
34. Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites.
- Author
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Lyford GL, Yamagata K, Kaufmann WE, Barnes CA, Sanders LK, Copeland NG, Gilbert DJ, Jenkins NA, Lanahan AA, and Worley PF
- Subjects
- Actins isolation & purification, Actins metabolism, Amino Acid Sequence, Animals, Base Sequence, Brain physiology, Chickens, DNA, Complementary, Gene Expression Regulation, Gene Library, Hippocampus metabolism, In Situ Hybridization, Male, Molecular Sequence Data, Neuronal Plasticity, Protein Biosynthesis, Rats, Rats, Inbred F344, Rats, Sprague-Dawley, Sequence Homology, Amino Acid, Spectrin genetics, Transcription, Genetic, Brain metabolism, Cytoskeletal Proteins biosynthesis, Cytoskeletal Proteins genetics, Cytoskeleton metabolism, Dendrites metabolism, Genes, Immediate-Early, Nerve Tissue Proteins biosynthesis, Nerve Tissue Proteins genetics, Neurons metabolism
- Abstract
Neuronal activity is an essential stimulus for induction of plasticity and normal development of the CNS. We have used differential cloning techniques to identify a novel immediate-early gene (IEG) cDNA that is rapidly induced in neurons by activity in models of adult and developmental plasticity. Both the mRNA and the encoded protein are enriched in neuronal dendrites. Analysis of the deduced amino acid sequence indicates a region of homology with alpha-spectrin, and the full-length protein, prepared by in vitro transcription/translation, coprecipitates with F-actin. Confocal microscopy of the native protein in hippocampal neurons demonstrates that the IEG-encoded protein is enriched in the subplasmalemmal cortex of the cell body and dendrites and thus colocalizes with the actin cytoskeletal matrix. Accordingly, we have termed the gene and encoded protein Arc (activity-regulated cytoskeleton-associated protein). Our observations suggest that Arc may play a role in activity-dependent plasticity of dendrites.
- Published
- 1995
- Full Text
- View/download PDF
35. Plaque formation and purification of BK virus in cultured human urinary cells.
- Author
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Sack GH, Felix JS, and Lanahan AA
- Subjects
- BK Virus analysis, BK Virus isolation & purification, DNA, Viral analysis, Humans, Urine microbiology, Viral Plaque Assay, Virus Cultivation, BK Virus growth & development, Polyomavirus growth & development, Urine cytology
- Abstract
Primary human urinary cells support growth and plaque formation by papovavirus BK, permitting plaque purification of the virus. After plaquing, the virus forms uniform plaques and its DNA has a homogeneous restriction enzyme fragment profile. Support of BK replication as well as morphological and cultural characteristics suggest an epithelial origin for the cells.
- Published
- 1980
- Full Text
- View/download PDF
36. Gene transfer and molecular cloning of the human NGF receptor.
- Author
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Chao MV, Bothwell MA, Ross AH, Koprowski H, Lanahan AA, Buck CR, and Sehgal A
- Subjects
- Animals, Cell Line, Cell Transformation, Neoplastic drug effects, DNA, Recombinant, Genes, Humans, Melanoma metabolism, Mice, Oncogenes, Rats, Receptors, Nerve Growth Factor, Repetitive Sequences, Nucleic Acid, Tunicamycin pharmacology, Cloning, Molecular, Receptors, Cell Surface genetics
- Abstract
Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.
- Published
- 1986
- Full Text
- View/download PDF
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