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187 results on '"Koch, W. J."'

8. Connective tissue growth factor inhibition enhances cardiac repair and limits fibrosis after myocardial infarction

Author

Vainio, L. E. (Laura E.), Szabó, Z. (Zoltán), Lin, R. (Ruizhu), Ulvila, J. (Johanna), Yrjölä, R. (Raisa), Alakoski, T. (Tarja), Piuhola, J. (Jarkko), Koch, W. J. (Walter J.), Ruskoaho, H. (Heikki), Fouse, S. D. (Shaun D.), Seeley, T. W. (Todd W.), Gao, E. (Erhe), Signore, P. (Pierre), Lipson, K. E. (Kenneth E.), Magga, J. (Johanna), Kerkelä, R. (Risto), Vainio, L. E. (Laura E.), Szabó, Z. (Zoltán), Lin, R. (Ruizhu), Ulvila, J. (Johanna), Yrjölä, R. (Raisa), Alakoski, T. (Tarja), Piuhola, J. (Jarkko), Koch, W. J. (Walter J.), Ruskoaho, H. (Heikki), Fouse, S. D. (Shaun D.), Seeley, T. W. (Todd W.), Gao, E. (Erhe), Signore, P. (Pierre), Lipson, K. E. (Kenneth E.), Magga, J. (Johanna), and Kerkelä, R. (Risto)

Abstract

Summary Myocardial infarction (MI)−induced cardiac fibrosis attenuates cardiac contractile function, and predisposes to arrhythmias and sudden cardiac death. Expression of connective tissue growth factor (CTGF) is elevated in affected organs in virtually every fibrotic disorder and in the diseased human myocardium. Mice were subjected to treatment with a CTGF monoclonal antibody (mAb) during infarct repair, post-MI left ventricular (LV) remodeling, or acute ischemia−reperfusion injury. CTGF mAb therapy during infarct repair improved survival and reduced LV dysfunction, and reduced post-MI LV hypertrophy and fibrosis. Mechanistically, CTGF mAb therapy induced expression of cardiac developmental and/or repair genes and attenuated expression of inflammatory and/or fibrotic genes.

Published
2019

9. beta-2 adrenergic receptors overexpression promotes angiogenesis in the falling myocardium through activation of vascular endothelial growth factor/Akt transduction pathway

Author

C. Zincarelli, Marchese M., Fomminella D., Avallone E., Koch W. J., RENGO, GIUSEPPE, GOLINO, LUCA, ALTOBELLI, GIOVANNA GIUSEPPINA, CIMINI, VINCENZO, FORTUNATO, FRANCESCA, RENGO, ALESSANDRO, RENGO, FRANCO, LEOSCO, DARIO, C., Zincarelli, Rengo, Giuseppe, Golino, Luca, Altobelli, GIOVANNA GIUSEPPINA, Cimini, Vincenzo, Fortunato, Francesca, Marchese, M., Fomminella, D., Avallone, E., Rengo, Alessandro, Rengo, Franco, Koch, W. J., and Leosco, Dario

Subjects
Akt, angiogenesi, heart failure
Abstract

Possible role of VEGF/Akt transduction pathway after beta-2 adrenergic receptors overexpression-promoted angiogenesis.

Published
2008

10. Impaired neoangiogenesis in β₂-adrenoceptor gene-deficient mice: restoration by intravascular human β₂-adrenoceptor gene transfer and role of NFκB and CREB transcription factors

Author

Ciccarelli, Michele, Sorriento, D., Cipolletta, E., Santulli, G., Fusco, A., Zhou, R., Eckhart, A. D., Peppel, K., Koch, W. J., Trimarco, B., and Iaccarino, Guido

Subjects
Male, Vascular Endothelial Growth Factor A, Cells, Knockout, Genetic Vectors, Neovascularization, Physiologic, beta-2, Transfection, Mice, Radioligand Assay, Ischemia, Receptors, deficiency/genetics/metabolism, Animals, Humans, Physiologic, Luciferases, Neovascularization, Cells, Cultured, Mice, Knockout, Cultured, Animal, NF-kappa B, Endothelial Cells, Gene Therapy, Genetic Therapy, CREB-Binding Protein, Research Papers, Disease Models, Animal, Adrenergic, Animals, Blood Flow Velocity, CREB-Binding Protein, metabolism, Cells, Cultured, Disease Models, Animal, Endothelial Cells, metabolism, Gene Therapy, Genetic Vectors, Humans, Ischemia, physiopathology/therapy, Luciferases, metabolism, Male, Mice, Mice, Knockout, NF-kappa B, metabolism, Neovascularization, Physiologic, Radioligand Assay, Receptors, deficiency/genetics/metabolism, Signal Transduction, Transfection, Vascular Endothelial Growth Factor A, metabolism, Disease Models, physiopathology/therapy, Receptors, Adrenergic, beta-2, Blood Flow Velocity, Signal Transduction
Abstract

There is much evidence supporting the role of β₂-adrenoceptors (β₂AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of β₂AR knock-out mice (β₂AR-/-) in vivo and explored possible molecular mechanisms in vitro.Femoral artery resection (FAR) was performed in wild-type and β₂AR-/- mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis. Intrafemoral delivery of an adenovirus encoding the human β₂AR (ADβ₂AR) was used to reinstate β₂ARs in β₂AR-/- mice. Molecular mechanisms were investigated in mouse-derived aortic endothelial cells (EC) in vitro, focusing on NFκB activation and transcriptional activity.Angiogenesis was severely impaired in β₂AR-/- mice subjected to FAR, but was restored by gene therapy with ADβ₂AR. The proangiogenic responses to a variety of stimuli were impaired in β₂AR-/- EC in vitro. Moreover, removal of β₂ARs impaired the activation of NFκB, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates βARs) nor TNFα induced NFκB activation in β₂AR(-/-) EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NFκB, was constitutively increased in β₂AR(-/-) ECs. ADβ₂AR administration restored β₂AR membrane density, reduced CREB activity and reinstated the NFκB response to isoprenaline and TNFα.Our results suggest that β₂ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.

Published
2010

12. Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model

Author

Weber, C, primary, Neacsu, I, additional, Krautz, B, additional, Schlegel, P, additional, Sauer, S, additional, Raake, P, additional, Ritterhoff, J, additional, Jungmann, A, additional, Remppis, A B, additional, Stangassinger, M, additional, Koch, W J, additional, Katus, H A, additional, Müller, O J, additional, Most, P, additional, and Pleger, S T, additional

Published
2013
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14. Poster session 3

Author

Nanka, O., primary, Krejci, E., additional, Pesevski, Z., additional, Sedmera, D., additional, Smart, N., additional, Rossdeutsch, A., additional, Dube, K. N., additional, Riegler, J., additional, Price, A. N., additional, Taylor, A., additional, Muthurangu, V., additional, Turner, M., additional, Lythgoe, M. F., additional, Riley, P. R., additional, Kryvorot, S., additional, Vladimirskaya, T., additional, Shved, I., additional, Schwarzl, M., additional, Seiler, S., additional, Huber, S., additional, Steendijk, P., additional, Maechler, H., additional, Truschnig-Wilders, M., additional, Pieske, B., additional, Post, H., additional, Caprio, C., additional, Baldini, A., additional, Chiavacci, E., additional, Dolfi, L., additional, Verduci, L., additional, Meghini, F., additional, Cremisi, F., additional, Pitto, L., additional, Kuan, T.-C., additional, Chen, M.-C., additional, Yang, T.-H., additional, Wu, W.-T., additional, Lin, C. S., additional, Rai, H., additional, Kumar, S., additional, Sharma, A. K., additional, Mastana, S., additional, Kapoor, A., additional, Pandey, C. M., additional, Agrawal, S., additional, Sinha, N., additional, Orlowska-Baranowska, E. H., additional, Placha, G., additional, Gora, J., additional, Baranowski, R., additional, Abramczuk, E., additional, Hryniewiecki, T., additional, Gaciong, Z., additional, Verschuren, J. J. W., additional, Wessels, J. A. M., additional, Trompet, S., additional, Stott, D. J., additional, Sattar, N., additional, Buckley, B., additional, Guchelaar, H. J., additional, Jukema, J. W., additional, Gharanei, M., additional, Hussain, A., additional, Mee, C. J., additional, Maddock, H. L., additional, Wijnen, W. J., additional, Van Den Oever, S., additional, Van Der Made, I., additional, Hiller, M., additional, Tijsen, A. J., additional, Pinto, Y. M., additional, Creemers, E. E., additional, Nikulina, S. U. Y., additional, Chernova, A., additional, Petry, A., additional, Rzymski, T., additional, Kracun, D., additional, Riess, F., additional, Pike, L., additional, Harris, A. L., additional, Gorlach, A., additional, Katare, R., additional, Oikawa, A., additional, Riu, F., additional, Beltrami, A. P., additional, Cesseli, D., additional, Emanueli, C., additional, Madeddu, P., additional, Zaglia, T., additional, Milan, G., additional, Franzoso, M., additional, Pesce, P., additional, Sarais, C., additional, Sandri, M., additional, Mongillo, M., additional, Butler, T. J., additional, Seymour, A.-M. L., additional, Ashford, D., additional, Jaffre, F., additional, Bussen, M., additional, Flohrschutz, I., additional, Martin, G. R., additional, Engelhardt, S., additional, Kararigas, G., additional, Nguyen, B. T., additional, Jarry, H., additional, Regitz-Zagrosek, V., additional, Van Bilsen, M., additional, Daniels, A., additional, Munts, C., additional, Janssen, B. J. A., additional, Van Der Vusse, G. J., additional, Van Nieuwenhoven, F. A., additional, Montalvo, C., additional, Villar, A. V., additional, Merino, D., additional, Garcia, R., additional, Llano, M., additional, Ares, M., additional, Hurle, M. A., additional, Nistal, J. F., additional, Dembinska-Kiec, A., additional, Beata Kiec-Wilk, B. K. W., additional, Anna Polus, A. P., additional, Urszula Czech, U. C., additional, Tatiana Konovaleva, T. K., additional, Gerd Schmitz, G. S., additional, Bertrand, L., additional, Balteau, M., additional, Timmermans, A., additional, Viollet, B., additional, Sakamoto, K., additional, Feron, O., additional, Horman, S., additional, Vanoverschelde, J. L., additional, Beauloye, C., additional, De Meester, C., additional, Martinez, E., additional, Martin, R., additional, Miana, M., additional, Jurado, R., additional, Gomez-Hurtado, N., additional, Bartolome, M. V., additional, San Roman, J. A., additional, Lahera, V., additional, Nieto, M. L., additional, Cachofeiro, V., additional, Rochais, F., additional, Sturny, R., additional, Mesbah, K., additional, Miquerol, L., additional, Kelly, R. G., additional, Messaoudi, S., additional, Gravez, B., additional, Tarjus, A., additional, Pelloux, V., additional, Samuel, J. L., additional, Delcayre, C., additional, Launay, J. M., additional, Clement, K., additional, Farman, N., additional, Jaisser, F., additional, Hadyanto, L., additional, Castellani, C., additional, Vescovo, G., additional, Ravara, B., additional, Tavano, R., additional, Pozzobon, M., additional, De Coppi, P., additional, Papini, E., additional, Vettor, R., additional, Thiene, G., additional, Angelini, A., additional, Meloni, M., additional, Caporali, A., additional, Cesselli, D., additional, Fortunato, O., additional, Avolio, E., additional, Schindler, R., additional, Simrick, S., additional, Brand, T., additional, Smart, N. S., additional, Herman, A., additional, Roura Ferrer, S., additional, Rodriguez Bago, J., additional, Soler-Botija, C., additional, Pujal, J. M., additional, Galvez-Monton, C., additional, Prat-Vidal, C., additional, Llucia-Valldeperas, A., additional, Blanco, J., additional, Bayes-Genis, A., additional, Foldes, G., additional, Maxime, M., additional, Ali, N. N., additional, Schneider, M. D., additional, Harding, S. E., additional, Reni, C., additional, Mangialardi, G., additional, De Pauw, A., additional, Sekkali, B., additional, Friart, A., additional, Ding, H., additional, Graffeuil, A., additional, Catalucci, D., additional, Balligand, J. L., additional, Azibani, F., additional, Tournoux, F., additional, Schlossarek, S., additional, Polidano, E., additional, Fazal, L., additional, Merval, R., additional, Carrier, L., additional, Chatziantoniou, C., additional, Buyandelger, B., additional, Linke, W., additional, Zou, P., additional, Kostin, S., additional, Ku, C., additional, Felkin, L., additional, Birks, E., additional, Barton, P., additional, Sattler, M., additional, Knoell, R., additional, Schroder, K., additional, Benkhoff, S., additional, Shimokawa, H., additional, Grisk, O., additional, Brandes, R. P., additional, Parepa, I. R., additional, Mazilu, L., additional, Suceveanu, A. I., additional, Suceveanu, A., additional, Rusali, L., additional, Cojocaru, L., additional, Matei, L., additional, Toringhibel, M., additional, Craiu, E., additional, Pires, A. L., additional, Pinho, M., additional, Pinho, S., additional, Sena, C., additional, Seica, R., additional, Leite-Moreira, A., additional, Dabroi, F., additional, Schiaffino, S., additional, Kiseleva, E., additional, Krukov, N., additional, Nikitin, O., additional, Ardatova, L., additional, Mourouzis, I., additional, Pantos, C., additional, Kokkinos, A. D., additional, Cokkinos, D. V., additional, Scoditti, E., additional, Massaro, M., additional, Carluccio, M. A., additional, Pellegrino, M., additional, Calabriso, N., additional, Gastaldelli, A., additional, Storelli, C., additional, De Caterina, R., additional, Lindner, D., additional, Zietsch, C., additional, Schultheiss, H.-P., additional, Tschope, C., additional, Westermann, D., additional, Everaert, B. R., additional, Nijenhuis, V. J., additional, Reith, F. C. M., additional, Hoymans, V. Y., additional, Timmermans, J. P., additional, Vrints, C. J., additional, Simova, I., additional, Mateev, H., additional, Katova, T., additional, Haralanov, L., additional, Dimitrov, N., additional, Mironov, N., additional, Golitsyn, S. P., additional, Sokolov, S. F., additional, Yuricheva, Y. U. A., additional, Maikov, E. B., additional, Shlevkov, N. B., additional, Rosenstraukh, L. V., additional, Chazov, E. I., additional, Radosinska, J., additional, Knezl, V., additional, Benova, T., additional, Slezak, J., additional, Urban, L., additional, Tribulova, N., additional, Virag, L., additional, Kristof, A., additional, Kohajda, Z. S., additional, Szel, T., additional, Husti, Z., additional, Baczko, I., additional, Jost, N., additional, Varro, A., additional, Sarusi, A., additional, Farkas, A. S., additional, Orosz, S. Z., additional, Forster, T., additional, Farkas, A., additional, Zakhrabova-Zwiauer, O. M., additional, Hardziyenka, M., additional, Nieuwland, R., additional, Tan, H. L., additional, Raaijmakers, A. J. A., additional, Bourgonje, V. J. A., additional, Kok, G. J. M., additional, Van Veen, A. A. B., additional, Anderson, M. E., additional, Vos, M. A., additional, Bierhuizen, M. F. A., additional, Benes, J., additional, Sebestova, B., additional, Ghouri, I. A., additional, Kemi, O. J., additional, Kelly, A., additional, Burton, F. L., additional, Smith, G. L., additional, Ozdemir, S., additional, Acsai, K., additional, Doisne, N., additional, Van Der Nagel, R., additional, Beekman, H. D. M., additional, Van Veen, T. A. B., additional, Sipido, K. R., additional, Antoons, G., additional, Harmer, S. C., additional, Mohal, J. S., additional, Kemp, D., additional, Tinker, A., additional, Beech, D., additional, Burley, D. S., additional, Cox, C. D., additional, Wann, K. T., additional, Baxter, G. F., additional, Wilders, R., additional, Verkerk, A., additional, Fragkiadaki, P., additional, Germanakis, G., additional, Tsarouchas, K., additional, Tsitsimpikou, C., additional, Tsardi, M., additional, George, D., additional, Tsatsakis, A., additional, Rodrigues, P., additional, Barros, C., additional, Najmi, A. K., additional, Khan, V., additional, Akhtar, M., additional, Pillai, K. K., additional, Mujeeb, M., additional, Aqil, M., additional, Bayliss, C. R., additional, Messer, A. E., additional, Leung, M.-C., additional, Ward, D., additional, Van Der Velden, J., additional, Poggesi, C., additional, Redwood, C. S., additional, Marston, S., additional, Vite, A., additional, Gandjbakhch, E., additional, Gary, F., additional, Fressart, V., additional, Leprince, P., additional, Fontaine, G., additional, Komajda, M., additional, Charron, P., additional, Villard, E., additional, Falcao-Pires, I., additional, Gavina, C., additional, Hamdani, N., additional, Stienen, G. J. M., additional, Niessens, H. W. M., additional, Leite-Moreira, A. F., additional, Paulus, W. J., additional, Memo, M., additional, Marston, S. B., additional, Vafiadaki, E., additional, Qian, J., additional, Arvanitis, D. A., additional, Sanoudou, D., additional, Kranias, E. G., additional, Elmstedt, N., additional, Lind, B., additional, Ferm-Widlund, K., additional, Westgren, M., additional, Brodin, L.-A., additional, Mansfield, C., additional, West, T., additional, Ferenczi, M., additional, Wijnker, P. J. M., additional, Foster, D. B., additional, Coulter, A., additional, Frazier, A., additional, Murphy, A. M., additional, Shah, M., additional, Sikkel, M. B., additional, Desplantez, T., additional, Collins, T. P., additional, O' Gara, P., additional, Lyon, A. R., additional, Macleod, K. T., additional, Ottesen, A. H., additional, Louch, W. E., additional, Carlson, C., additional, Landsverk, O. J. B., additional, Stridsberg, M., additional, Sjaastad, I., additional, Oie, E., additional, Omland, T., additional, Christensen, G., additional, Rosjo, H., additional, Cartledge, J., additional, Clark, L. A., additional, Ibrahim, M., additional, Siedlecka, U., additional, Navaratnarajah, M., additional, Yacoub, M. H., additional, Camelliti, P., additional, Terracciano, C. M., additional, Chester, A., additional, Gonzalez-Tendero, A., additional, Torre, I., additional, Garcia-Garcia, F., additional, Dopazo, J., additional, Gratacos, E., additional, Taylor, D., additional, Bhandari, S., additional, Seymour, A.-M., additional, Fliegner, D., additional, Jost, J., additional, Bugger, H., additional, Ventura-Clapier, R., additional, Carpi, A., additional, Campesan, M., additional, Canton, M., additional, Menabo, R., additional, Pelicci, P. G., additional, Giorgio, M., additional, Di Lisa, F., additional, Hancock, M., additional, Venturini, A., additional, Al-Shanti, N., additional, Stewart, C., additional, Ascione, R., additional, Angelini, G., additional, Suleiman, M.-S., additional, Kravchuk, E., additional, Grineva, E., additional, Galagudza, M., additional, Kostareva, A., additional, Bairamov, A., additional, Krychtiuk, K. A., additional, Watzke, L., additional, Kaun, C., additional, Demyanets, S., additional, Pisoni, J., additional, Kastl, S. P., additional, Huber, K., additional, Maurer, G., additional, Wojta, J., additional, Speidl, W. S., additional, Varga, Z. V., additional, Farago, N., additional, Zvara, A., additional, Kocsis, G. F., additional, Pipicz, M., additional, Csonka, C., additional, Csont, T., additional, Puskas, G. L., additional, Ferdinandy, P., additional, Klevstigova, M., additional, Silhavy, J., additional, Manakov, D., additional, Papousek, F., additional, Novotny, J., additional, Pravenec, M., additional, Kolar, F., additional, Novakova, O., additional, Novak, F., additional, Neckar, J., additional, Barallobre-Barreiro, J., additional, Didangelos, A., additional, Yin, X., additional, Fernandez-Caggiano, M., additional, Drozdov, I., additional, Willeit, P., additional, Domenech, N., additional, Mayr, M., additional, Lemoine, S., additional, Allouche, S., additional, Coulbault, L., additional, Galera, P., additional, Gerard, J. L., additional, Hanouz, J. L., additional, Suveren, E., additional, Whiteman, M., additional, Studneva, I. M., additional, Pisarenko, O., additional, Shulzhenko, V., additional, Serebryakova, L., additional, Tskitishvili, O., additional, Timoshin, A., additional, Fauconnier, J., additional, Meli, A. C., additional, Thireau, J., additional, Roberge, S., additional, Lompre, A. M., additional, Jacotot, E., additional, Marks, A. M., additional, Lacampagne, A., additional, Dietel, B., additional, Altendorf, R., additional, Daniel, W. G., additional, Kollmar, R., additional, Garlichs, C. D., additional, Parente, V., additional, Balasso, S., additional, Pompilio, G., additional, Colombo, G., additional, Milano, G., additional, Squadroni, L., additional, Cotelli, F., additional, Pozzoli, O., additional, Capogrossi, M. C., additional, Ajiro, Y., additional, Saegusa, N., additional, Iwade, K., additional, Giles, W. R., additional, Stafforini, D. M., additional, Spitzer, K. W., additional, Sirohi, R., additional, Candilio, L., additional, Babu, G., additional, Roberts, N., additional, Lawrence, D., additional, Sheikh, A., additional, Kolvekar, S., additional, Yap, J., additional, Hausenloy, D. J., additional, Yellon, D. M., additional, Aslam, M., additional, Rohrbach, S., additional, Schlueter, K.-D., additional, Piper, H. M., additional, Noll, T., additional, Guenduez, D., additional, Malinova, L., additional, Ryabukho, V. P., additional, Lyakin, D. V., additional, Denisova, T. P., additional, Montoro-Garcia, S., additional, Shantsila, E., additional, Lip, G. Y. H., additional, Kalaska, B., additional, Sokolowska, E., additional, Kaminski, K., additional, Szczubialka, K., additional, Kramkowski, K., additional, Mogielnicki, A., additional, Nowakowska, M., additional, Buczko, W., additional, Stancheva, N., additional, Mekenyan, E., additional, Gospodinov, K., additional, Tisheva, S., additional, Darago, A., additional, Rutkai, I., additional, Kalasz, J., additional, Czikora, A., additional, Orosz, P., additional, Bjornson, H. D., additional, Edes, I., additional, Papp, Z., additional, Toth, A., additional, Riches, K., additional, Warburton, P., additional, O'regan, D. J., additional, Ball, S. G., additional, Turner, N. A., additional, Wood, I. C., additional, Porter, K. E., additional, Kogaki, S., additional, Ishida, H., additional, Nawa, N., additional, Takahashi, K., additional, Baden, H., additional, Ichimori, H., additional, Uchikawa, T., additional, Mihara, S., additional, Miura, K., additional, Ozono, K., additional, Lugano, R., additional, Padro, T., additional, Garcia-Arguinzonis, M., additional, Badimon, L., additional, Ferraro, F., additional, Viner, R., additional, Ho, J., additional, Cutler, D., additional, Matchkov, V., additional, Aalkjaer, C., additional, Krijnen, P. A. J., additional, Hahn, N. E., additional, Kholova, I., additional, Sipkens, J. A., additional, Van Alphen, F. P., additional, Simsek, S., additional, Schalkwijk, C. G., additional, Van Buul, J. D., additional, Van Hinsbergh, V. W. M., additional, Niessen, H. W. M., additional, Caro, C. G., additional, Seneviratne, A., additional, Monaco, C., additional, Hou, D., additional, Singh, J., additional, Gilson, P., additional, Burke, M. G., additional, Heraty, K. B., additional, Krams, R., additional, Coppola, G., additional, Albrecht, K., additional, Schgoer, W., additional, Wiedemann, D., additional, Bonaros, N., additional, Steger, C., additional, Theurl, M., additional, Stanzl, U., additional, Kirchmair, R., additional, Amadesi, S., additional, Spinetti, G., additional, Cangiano, E., additional, Valgimigli, M., additional, Miller, A. M., additional, Cardinali, A., additional, Vierlinger, K., additional, Pagano, G., additional, Liccardo, D., additional, Zincarelli, C., additional, Femminella, G. D., additional, Lymperopoulos, A., additional, De Lucia, C., additional, Koch, W. J., additional, Leosco, D., additional, Rengo, G., additional, Hinkel, R., additional, Husada, W., additional, Trenkwalder, T., additional, Di, Q., additional, Lee, S., additional, Petersen, B., additional, Bock-Marquette, I., additional, Niemann, H., additional, Di Maio, M., additional, Kupatt, C., additional, Nourian, M., additional, Yassin, Z., additional, Kelishadi, R., additional, Memarian, S. H., additional, Heidari, A., additional, Leuner, A., additional, Poitz, D. M., additional, Brunssen, C., additional, Ravens, U., additional, Strasser, R. H., additional, Morawietz, H., additional, Vogt, F., additional, Grahl, A., additional, Flege, C., additional, Marx, N., additional, Borinski, M., additional, De Geest, B., additional, Jacobs, F., additional, Muthuramu, I., additional, Gordts, S. C., additional, Van Craeyveld, E., additional, Herijgers, P., additional, Weinert, S., additional, Medunjanin, S., additional, Herold, J., additional, Schmeisser, A., additional, Braun-Dullaeus, R. C., additional, Wagner, A. H., additional, Moeller, K., additional, Adolph, O., additional, Schwarz, M., additional, Schwale, C., additional, Bruehl, C., additional, Nobiling, R., additional, Wieland, T., additional, Schneider, S. W., additional, Hecker, M., additional, Cross, A., additional, Strom, A., additional, Cole, J., additional, Goddard, M., additional, Hultgardh-Nilsson, A., additional, Nilsson, J., additional, Mauri, C., additional, Mitkovskaya, N. P., additional, Kurak, T. A., additional, Oganova, E. G., additional, Shkrebneva, E. I., additional, Kot, Z. H. N., additional, Statkevich, T. V., additional, Molica, F., additional, Burger, F., additional, Matter, C. M., additional, Thomas, A., additional, Staub, C., additional, Zimmer, A., additional, Cravatt, B., additional, Pacher, P., additional, Steffens, S., additional, Blanco, R., additional, Sarmiento, R., additional, Parisi, C., additional, Fandino, S., additional, Blanco, F., additional, Gigena, G., additional, Szarfer, J., additional, Rodriguez, A., additional, Garcia Escudero, A., additional, Riccitelli, M. A., additional, Wantha, S., additional, Simsekyilmaz, S., additional, Megens, R. T., additional, Van Zandvoort, M. A., additional, Liehn, E., additional, Zernecke, A., additional, Klee, D., additional, Weber, C., additional, Soehnlein, O., additional, Lima, L. M., additional, Carvalho, M. G., additional, Gomes, K. B., additional, Santos, I. R., additional, Sousa, M. O., additional, Morais, C. A. S., additional, Oliveira, S. H. V., additional, Gomes, I. F., additional, Brandao, F. C., additional, Lamego, M. R. A., additional, Fornai, L., additional, Kiss, A., additional, Giskes, F., additional, Eijkel, G., additional, Fedrigo, M., additional, Valente, M. L., additional, Heeren, R. M. A., additional, Grdinic, A., additional, Vojvodic, D., additional, Djukanovic, N., additional, Grdinic, A. G., additional, Obradovic, S., additional, Majstorovic, I., additional, Rusovic, S., additional, Vucinic, Z., additional, Tavciovski, D., additional, Ostojic, M., additional, Lai, S.-C., additional, Chen, M.-Y., additional, Wu, H.-T., additional, Gouweleeuw, L., additional, Oberdorf-Maass, S. U., additional, De Boer, R. A., additional, Van Gilst, W. H., additional, Maass, A. H., additional, Van Gelder, I. C., additional, Benard, L., additional, Li, C., additional, Warren, D., additional, Shanahan, C. M., additional, Zhang, Q. P., additional, Bye, A., additional, Vettukattil, R., additional, Aspenes, S. T., additional, Giskeodegaard, G., additional, Gribbestad, I. S., additional, Wisloff, U., additional, Bathen, T. F., additional, Cubedo, J., additional, Alonso, R., additional, Mata, P., additional, Ivic, I., additional, Vamos, Z., additional, Cseplo, P., additional, Kosa, D., additional, Torok, O., additional, Hamar, J., additional, Koller, A., additional, Norita, K., additional, De Noronha, S. V., additional, Sheppard, M. N., additional, Amat-Roldan, I., additional, Iruretagoiena, I., additional, Psilodimitrakopoulos, S., additional, Crispi, F., additional, Artigas, D., additional, Loza-Alvarez, P., additional, Harrison, J. C., additional, Smart, S. D., additional, Besely, E. H., additional, Kelly, J. R., additional, Yao, Y., additional, Sammut, I. A., additional, Hoepfner, M., additional, Kuzyniak, W., additional, Sekhosana, E., additional, Hoffmann, B., additional, Litwinski, C., additional, Pries, A., additional, Ermilov, E., additional, Fontoura, D., additional, Lourenco, A. P., additional, Vasques-Novoa, F., additional, Pinto, J. P., additional, Roncon-Albuquerque, R., additional, Oyeyipo, I. P., additional, Olatunji, L. A., additional, Usman, T. O., additional, Olatunji, V. A., additional, Bacova, B., additional, Viczenczova, C., additional, Dosenko, V., additional, Goncalvesova, E., additional, Vanrooyen, J., additional, Maulik, S. K., additional, Seth, S., additional, Dinda, A. K., additional, Jaiswal, A., additional, Mearini, G., additional, Khajetoorians, D., additional, Kraemer, E., additional, Gedicke-Hornung, C., additional, Precigout, G., additional, Eschenhagen, T., additional, Voit, T., additional, Garcia, L., additional, Lorain, S., additional, Mendes-Ferreira, P., additional, Maia-Rocha, C., additional, Adao, R., additional, Cerqueira, R. J., additional, Mendes, M. J., additional, Castro-Chaves, P., additional, De Keulenaer, G. W., additional, Bras-Silva, C., additional, Ruiter, G., additional, Wong, Y. 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J., additional, Llacer, A., additional, Galasso, G., additional, Ferrara, N., additional, Akhmedov, A., additional, Klingenberg, R., additional, Brokopp, C., additional, Hof, D., additional, Zoller, S., additional, Corti, R., additional, Gay, S., additional, Von Eckardstein, A., additional, Hoerstrup, S. P., additional, Luescher, T. F., additional, Heijman, J., additional, Zaza, A., additional, Johnson, D. M., additional, Rudy, Y., additional, Peeters, R. L. M., additional, Volders, P. G. A., additional, Westra, R. L., additional, Fujita, S., additional, Okamoto, R., additional, Taniguchi, M., additional, Konishi, K., additional, Goto, I., additional, Sugimoto, K., additional, Nakamura, M., additional, Shiraki, K., additional, Buechler, C., additional, and Ito, M., additional

Published
2012
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15. AAV6. ARKct cardiac gene therapy ameliorates cardiac function and normalizes the catecholaminergic axis in a clinically relevant large animal heart failure model

Author

Raake, P. W. J., primary, Schlegel, P., additional, Ksienzyk, J., additional, Reinkober, J., additional, Barthelmes, J., additional, Schinkel, S., additional, Pleger, S., additional, Mier, W., additional, Haberkorn, U., additional, Koch, W. J., additional, Katus, H. A., additional, Most, P., additional, and Muller, O. J., additional

Published
2012
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29. Adenovirus-mediated gene transfer of the β2-adrenergic receptor to donor hearts enhances cardiac function.

Author

Kypson, A P, Hendrickson, S C, Akhter, S A, Wilson, K, McDonald, P H, Lilly, R E, Dolber, P C, Glower, D D, Lefkowitz, R J, and Koch, W J

Subjects
BETA adrenoceptors, HEART transplantation, GENETIC transformation, ADENOVIRUS diseases, THERAPEUTICS
Abstract

Gene transfer to modify donor heart function during transplantation has significant therapeutic implications. Recent studies by our laboratory in transgenic mice have shown that overexpression of β2-adrenergic receptors (β2-ARs) leads to significantly enhanced cardiac function. Thus, we investigated the functional consequences of adenovirus-mediated gene transfer of the human β2-AR in a rat heterotopic heart transplant model. Donor hearts received 1 ml of solution containing 1 × 1010 p.f.u. of adenovirus encoding the β2-AR or an empty adenovirus as a control. Five days after transplantation, basal left ventricular (LV) pressure was measured using an isolated, isovolumic heart perfusion apparatus. A subset of hearts was stimulated with the β2-AR agonist, zinterol. Treatment with the β2-AR virus resulted in global myocardial gene transfer with a six-fold increase in mean β-AR density which corresponded to a significant increase in basal contractility (LV + dP/dtmax, control: 3152.1 ± 286 versus β2-AR, 6250.6* ± 432.5 mmHg/s; n = 10, *P < 0.02). β2-AR overexpressing hearts also had higher contractility after zinterol administration compared with control hearts. Our results indicate that myocardial function of the transplanted heart can be enhanced by the adenovirus-mediated delivery of β2-ARs. Thus, genetic manipulation may offer a novel therapeutic strategy to improve donor heart function in the post- operative setting. [ABSTRACT FROM AUTHOR]

Published
1999
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35. Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy.

Author

Naga Prasad, S V, Esposito, G, Mao, L, Koch, W J, and Rockman, H A

Abstract

Activation of phosphoinositide 3-kinases is coupled to both phosphotyrosine/growth factor and G protein-coupled receptors. We explored the role of phosphoinositide 3-kinase activation in myocardium during in vivo pressure overload hypertrophy in mice. Cytosolic extracts from wild type hypertrophied hearts showed a selective increase in the phosphoinositide 3-kinase gamma isoform. To address the role of G protein-coupled receptor-mediated activation of phosphoinositide 3-kinase, we used transgenic mice with cardiac-specific overexpression of a Gbetagamma sequestering peptide. Extracts from hypertrophied transgenic hearts showed complete loss of phosphoinositide 3-kinase activation, indicating a Gbetagamma-dependent process. To determine the class of G proteins that contribute Gbetagamma dimers for in vivo phosphoinositide 3-kinase activation, two strategies were used: 1) transgenic mice with cardiac-specific overexpression of a G(q) inhibitor peptide and 2) pertussis toxin treatment prior to pressure overload in wild type mice. Pressure overloaded G(q) inhibitor transgenic mice showed a complete absence of phosphoinositide 3-kinase activation, whereas pretreatment with pertussis toxin showed robust phosphoinositide 3-kinase activation. Taken together, these data demonstrate that activation of the phosphoinositide 3-kinase during in vivo pressure overload hypertrophy is Gbetagamma-dependent and the Gbetagamma dimers arise from stimulation of G(q)-coupled receptors.

Published
2000

37. Defective beta-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice with calsequestrin overexpression.

Author

Cho, M C, Rapacciuolo, A, Koch, W J, Kobayashi, Y, Jones, L R, and Rockman, H A

Abstract

Calsequestrin is a high capacity Ca(2+)-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca(2+)-induced Ca(2+) release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998) J. Clin. Invest. 101, 1385-1393). To investigate whether alterations in intracellular Ca(2+) trigger changes in the beta-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired beta-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total beta-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased beta-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in beta-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.

Published
1999

38. Control of myocardial contractile function by the level of beta-adrenergic receptor kinase 1 in gene-targeted mice.

Author

Rockman, H A, Choi, D J, Akhter, S A, Jaber, M, Giros, B, Lefkowitz, R J, Caron, M G, and Koch, W J

Abstract

We studied the effect of alterations in the level of myocardial beta-adrenergic receptor kinase betaARK1) in two types of genetically altered mice. The first group is heterozygous for betaARK1 gene ablation, betaARK1(+/-), and the second is not only heterozygous for betaARK1 gene ablation but is also transgenic for cardiac-specific overexpression of a betaARK1 COOH-terminal inhibitor peptide, betaARK1(+/-)betaARKct. In contrast to the embryonic lethal phenotype of the homozygous betaARK1 knockout (Jaber, M., Koch, W. J., Rockman, H. A., Smith, B., Bond, R. A., Sulik, K., Ross, J., Jr., Lefkowitz, R. J., Caron, M. G., and Giros, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12974-12979), betaARK1(+/-) mice develop normally. Cardiac catheterization was performed in mice and showed a stepwise increase in contractile function in the betaARK1(+/-) and betaARK1(+/-)betaARKct mice with the greatest level observed in the betaARK1(+/-)betaARKct animals. Contractile parameters were measured in adult myocytes isolated from both groups of gene-targeted animals. A significantly greater increase in percent cell shortening and rate of cell shortening following isoproterenol stimulation was observed in the betaARK1(+/-) and betaARK1(+/-)betaARKct myocytes compared with wild-type cells, indicating a progressive increase in intrinsic contractility. These data demonstrate that contractile function can be modulated by the level of betaARK1 activity. This has important implications in disease states such as heart failure (in which betaARK1 activity is increased) and suggests that betaARK1 should be considered as a therapeutic target in this situation. Even partial inhibition of betaARK1 activity enhances beta-adrenergic receptor signaling leading to improved functional catecholamine responsiveness.

Published
1998

39. Mechanism of beta-adrenergic receptor desensitization in cardiac hypertrophy is increased beta-adrenergic receptor kinase.

Author

Choi, D J, Koch, W J, Hunter, J J, and Rockman, H A

Abstract

Pressure overload cardiac hypertrophy in the mouse was achieved following 7 days of transverse aortic constriction. This was associated with marked beta-adrenergic receptor (beta-AR) desensitization in vivo, as determined by a blunted inotropic response to dobutamine. Extracts from hypertrophied hearts had approximately 3-fold increase in cytosolic and membrane G protein-coupled receptor kinase (GRK) activity. Incubation with specific monoclonal antibodies to inhibit different GRK subtypes showed that the increase in activity could be attributed predominately to the beta-adrenergic receptor kinase (betaARK). Although overexpression of a betaARK inhibitor in hearts of transgenic mice did not alter the development of cardiac hypertrophy, the beta-AR desensitization associated with pressure overload hypertrophy was prevented. To determine whether the induction of betaARK occurred because of a generalized response to cellular hypertrophy, betaARK activity was measured in transgenic mice homozygous for oncogenic ras overexpression in the heart. Despite marked cardiac hypertrophy, no difference in betaARK activity was found in these mice overexpressing oncogenic ras compared with controls. Taken together, these data suggest that betaARK is a central molecule involved in alterations of beta-AR signaling in pressure overload hypertrophy. The mechanism for the increase in betaARK activity appears not to be related to the induction of cellular hypertrophy but to possibly be related to neurohumoral activation.

Published
1997

40. Transgenic mice with cardiac overexpression of alpha1B-adrenergic receptors. In vivo alpha1-adrenergic receptor-mediated regulation of beta-adrenergic signaling.

Author

Akhter, S A, Milano, C A, Shotwell, K F, Cho, M C, Rockman, H A, Lefkowitz, R J, and Koch, W J

Abstract

Transgenic mice were generated with cardiac-specific overexpression of the wild-type (WT) alpha1B-adrenergic receptor (AR) using the murine alpha-myosin heavy chain gene promoter. Previously, we described transgenic mice with alpha-myosin heavy chain-directed expression of a constitutively active mutant alpha1B-AR that had a phenotype of myocardial hypertrophy (Milano, C. A., Dolber, P. C., Rockman, H. A., Bond, R. A., Venable M. E., Allen, L. F., and Lefkowitz, R. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10109-10113). In animals with >40-fold WT alpha1-AR overexpression, basal myocardial diacylglycerol content was significantly increased, indicating enhanced alpha1-adrenergic signaling and phospholipase C activity. In contrast to the mice overexpressing constitutively active mutant alpha1B-ARs, the hearts of these mice did not develop cardiac hypertrophy despite an 8-fold increase in ventricular mRNA for atrial natriuretic factor. In vivo physiology was studied in anesthetized intact animals and showed left ventricular contractility in response to the beta-agonist isoproterenol to be significantly depressed in animals overexpressing WT alpha1B-ARs. Membranes purified from the hearts of WT alpha1BAR-overexpressing mice demonstrated significantly attenuated adenylyl cyclase activity basally and after stimulation with isoproterenol, norepinephrine, or phenylephrine. Interestingly, these in vitro changes in signaling were reversed after treating the mice with pertussis toxin, suggesting that the extraordinarily high levels of WT alpha1B-ARs can lead to coupling to pertussis toxin-sensitive G proteins. Another potential contributor to the observed decreased myocardial signaling and function could be enhanced beta-AR desensitization as beta-adrenergic receptor kinase (betaARK1) activity was found to be significantly elevated (>3-fold) in myocardial extracts isolated from WT alpha1B-AR-overexpressing mice. This type of altered signal transduction may become critical in disease conditions such as heart failure where betaARK1 levels are elevated and beta-ARs are down-regulated, leading to a higher percentage of cardiac alpha1-ARs. Thus, these mice serve as a unique experimental model to study the in vivo interactions between alpha- and beta-ARs in the heart.

Published
1997

41. G(o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism.

Author

van Biesen, T, Hawes, B E, Raymond, J R, Luttrell, L M, Koch, W J, and Lefkowitz, R J

Abstract

Mitogen-activated protein kinase (MAPK) is activated in response to both receptor tyrosine kinases and G-protein-coupled receptors. Recently, Gi-coupled receptors, such as the alpha 2A adrenergic receptor, were shown to mediate Ras-dependent MAPK activation via a pathway requiring G-protein beta gamma subunits (G beta gamma) and many of the same intermediates involved in receptor tyrosine kinase signaling. In contrast, Gq-coupled receptors, such as the M1 muscarinic acetylcholine receptor (M1AChR), activate MAPK via a pathway that is Ras-independent but requires the activity of protein kinase C (PKC). Here we show that, in Chinese hamster ovary cells, the M1AChR and platelet-activating factor receptor (PAFR) mediate MAPK activation via the alpha-subunit of the G(o) protein. G(o)-mediated MAPK activation was sensitive to treatment with pertussis toxin but insensitive to inhibition by a G beta gamma-sequestering peptide (beta ARK1ct). M1AChR and PAFR catalyzed G(o) alpha-subunit GTP exchange, and MAPK activation could be partially rescued by a pertussis toxin-insensitive mutant of G(o) alpha but not by similar mutants of Gi. G(o)-mediated MAPK activation was insensitive to inhibition by a dominant negative mutant of Ras (N17Ras) but was completely blocked by cellular depletion of PKC. Thus, M1AChR and PAFR, which have previously been shown to couple to Gq, are also coupled to G(o) to activate a novel PKC-dependent mitogenic signaling pathway.

Published
1996

42. Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.

Author

Ferguson, S S, Ménard, L, Barak, L S, Koch, W J, Colapietro, A M, and Caron, M G

Abstract

The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta 2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration.

Published
1995

43. Phosphatidylinositol 4,5-bisphosphate (PIP2)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP2 binding site distinguishes the GRK subfamilies.

Author

Pitcher, J A, Fredericks, Z L, Stone, W C, Premont, R T, Stoffel, R H, Koch, W J, and Lefkowitz, R J

Abstract

The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. In this study we demonstrate that phosphatidylinositol 4, 5-bisphosphate (PIP2) enhances GRK5-mediated beta-adrenergic receptor (betaAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. We have shown previously that the membrane association of betaARK (beta-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the betagamma subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme (Pitcher, J. A., Touhara, K., Payne, E. S., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 11707-11710). Thus, five members of the GRK family bind PIP2, betaARK (GRK2), betaARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the betaARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies.

Published
1996

44. Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation.

Author

Hawes, B E, van Biesen, T, Koch, W J, Luttrell, L M, and Lefkowitz, R J

Abstract

Receptors that couple to the heterotrimeric G proteins, Gi or Gq, can stimulate phosphoinositide (PI) hydrolysis and mitogen-activated protein kinase (MAPK) activation. PI hydrolysis produces inositol 1,4,5-trisphosphate and diacylglycerol, leading to activation of protein kinase C (PKC), which can stimulate increased MAPK activity. However, the relationship between PI hydrolysis and MAPK activation in Gi and Gq signaling has not been clearly defined and is the subject of this study. The effects of several signaling inhibitors are assessed including expression of a peptide derived from the carboxyl terminus of the beta adrenergic receptor kinase 1 (beta ARKct), which specifically blocks signaling mediated by the beta gamma subunits of G proteins (G beta gamma), expression of dominant negative mutants of p21ras (RasN17) and p74raf-1 (N delta Raf), protein-tyrosine kinase (PTK) inhibitors and cellular depletion of PKC. The Gi-coupled alpha 2A adrenergic receptor (AR) stimulates MAPK activation which is blocked by expression of beta ARKct, RasN17, or N delta Raf, or by PTK inhibitors, but unaffected by cellular depletion of PKC. In contrast, MAPK activation stimulated by the Gq-coupled alpha 1B AR or M1 muscarinic cholinergic receptor is unaffected by expression of beta ARKct or RasN17 expression or by PTK inhibitors, but is blocked by expression of N delta Raf or by PKC depletion. These data demonstrate that Gi- and Gq-coupled receptors stimulate MAPK activation via distinct signaling pathways. G beta gamma is responsible for mediating Gi-coupled receptor-stimulated MAPK activation through a mechanism utilizing p21ras and p74raf independent of PKC. In contrast, G alpha mediates Gq-coupled receptor-stimulated MAPK activation using a p21ras-independent mechanism employing PKC and p74raf. To define the role of G beta gamma in Gi-coupled receptor-mediated PI hydrolysis and MAPK activation, direct stimulation with G beta gamma was used. Expression of G beta gamma resulted in MAPK activation that was sensitive to inhibition by expression of beta ARKct, RasN17, or N delta Raf or by PTK inhibitors, but insensitive to PKC depletion. By comparison, G beta gamma-mediated PI hydrolysis was not affected by beta ARKct, RasN17, or N delta Raf expression or by PTK inhibitors. Together, these results demonstrate that G beta gamma mediates MAPK activation and PI hydrolysis via independent signaling pathways.

Published
1995

45. Mutational analysis of the pleckstrin homology domain of the beta-adrenergic receptor kinase. Differential effects on G beta gamma and phosphatidylinositol 4,5-bisphosphate binding.

Author

Touhara, K, Koch, W J, Hawes, B E, and Lefkowitz, R J

Abstract

The beta gamma subunits of heterotrimeric G proteins (G beta gamma) play a variety of roles in cellular signaling, one of which is membrane targeting of the beta-adrenergic receptor kinase (beta ARK). This is accomplished via a physical interaction of G beta gamma and a domain within the carboxyl terminus of beta ARK which overlaps with a pleckstrin homology (PH) domain. The PH domain of beta ARK not only binds G beta gamma but also interacts with phosphatidylinositol 4,5-bisphosphate (PIP2). Based on previous mapping of the G beta gamma binding region of beta ARK, and conserved residues within the PH domain, we have constructed a series of mutants in the carboxyl terminus of beta ARK in order to determine important residues involved in G beta gamma and PIP2 binding. To examine the effects of mutations on G beta gamma binding, we employed three different methodologies: direct G beta gamma binding to GST fusion proteins; the ability of GST fusion proteins to inhibit G beta gamma-mediated beta ARK translocation to rhodopsin-enriched rod outer segments; and the ability of mutant peptides expressed in cells to inhibit G beta gamma-mediated inositol phosphate accumulation. Direct PIP2 binding was also assessed on mutant GST fusion proteins. Ala residue insertion following Trp643 completely abolished the ability of beta ARK to bind G beta gamma, suggesting that a proper alpha-helical conformation is necessary for the G beta gamma.beta ARK interaction. In contrast, this insertional mutation had no effect on PIP2 binding. Both G beta gamma binding and PIP2 binding were abolished following Ala replacement of Trp643, suggesting that this conserved residue within the last subdomain of the PH domain is crucial for both interactions. Other mutations also produced differential effects on the physical interactions of the beta ARK carboxyl terminus with G beta gamma and PIP2. These results suggest that the last PH subdomain and its neighboring sequences within the carboxyl terminus of beta ARK, including Trp643, Leu647, and residues Lys663-Arg669, are critical for G beta gamma binding while Trp643 and residues Asp635-Glu639 are important for the PH domain to form the correct structure for binding to PIP2.

Published
1995

46. G beta gamma subunits mediate mitogen-activated protein kinase activation by the tyrosine kinase insulin-like growth factor 1 receptor.

Author

Luttrell, L M, van Biesen, T, Hawes, B E, Koch, W J, Touhara, K, and Lefkowitz, R J

Abstract

The receptors for insulin-like growth factor 1 (IGF1) and insulin are related heterotetrameric proteins which, like the epidermal growth factor (EGF) receptor, possess intrinsic ligand-stimulated tyrosine protein kinase activity. In Rat 1 fibroblasts, stimulation of mitogen-activated protein (MAP) kinase via the IGF1 receptor and the Gi-coupled receptor for lysophosphatidic acid (LPA), but not via the EGF receptor, is sensitive both to pertussis toxin treatment and to cellular expression of a specific G beta gamma subunit-binding peptide. The IGF1, LPA, and EGF receptor-mediated signals are all sensitive to inhibitors of tyrosine protein kinases, require p21ras activation, and are independent of protein kinase C. These data suggest that some tyrosine kinase growth factor receptors (e.g. IGF1 receptor) and classical G protein-coupled receptors (e.g. LPA receptor) employ a similar mechanism for mitogenic signaling that involves both tyrosine phosphorylation and G beta gamma subunits derived from pertussis toxin-sensitive G proteins.

Published
1995

47. Effect of cellular expression of pleckstrin homology domains on Gi-coupled receptor signaling.

Author

Luttrell, L M, Hawes, B E, Touhara, K, van Biesen, T, Koch, W J, and Lefkowitz, R J

Abstract

Pleckstrin homology (PH) domains are 90-110 amino acid regions of protein sequence homology that are found in a variety of proteins involved in signal transduction and growth control. We have previously reported that the PH domains of several proteins, including beta ARK1, PLC gamma, IRS-1, Ras-GRF, and Ras-GAP, expressed as glutathione S-transferase fusion proteins, can reversibly bind purified bovine brain G beta gamma subunits in vitro with varying affinity. To determine whether PH domain peptides would behave as antagonists of G beta gamma subunit-mediated signal transduction in intact cells, plasmid minigene constructs encoding these PH domains were prepared, which permit transient cellular expression of the peptides. Pertussis toxin-sensitive, G beta gamma subunit-mediated inositol phosphate (IP) production was significantly inhibited in COS-7 cells transiently coexpressing the alpha 2-C10 adrenergic receptor (AR) and each of the PH domain peptides. Pertussis toxin-insensitive, Gq alpha subunit-mediated IP production via coexpressed M1 muscarinic acetylcholine receptor (M1 AChR) was attenuated only by the PLC gamma PH domain peptide, suggesting that the inhibitory effect of most of the PH domain peptides was G beta gamma subunit-specific. Stimulation of the mitogen-activated protein (MAP) kinase pathway by Gi-coupled receptors in COS-7 cells has been reported to require activation of p21ras and to be independent of protein kinase C. Since several proteins involved in activation contain PH domains, the effect of PH domain peptide expression on alpha 2-C10 AR-mediated p21ras-GTP exchange and MAP kinase activation as well as direct G beta gamma subunit-mediated activation of MAP kinase was determined. In each assay, coexpression of the PH domain peptides resulted in significant inhibition. Increasing G beta gamma subunit expression surmounted PH domain peptide-mediated inhibition of MAP kinase activation. These data suggest that the PH domain peptides behave as specific antagonists of G beta gamma-mediated signaling in intact cells and that interactions between PH domains and G beta gamma subunits or structurally related proteins may play a role in the activation of mitogenic signaling pathways by G protein-coupled receptors.

Published
1995

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