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I Post-Translational Modifications Of POST-TRANSLATIONAL MODIFICATIONS OF THROMBOXANE RECEPTOR G-PROTEIN ALPHA Q COMPLEX IN HYPOXIC PPHN By ANURAG SINGH SIKARWAR A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES OF THE UNIVERSITY OF MANITOBA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGY & PATHOPHYSIOLOGY UNIVERSITY OF MANITOBA WINNIPEG, MANITOBA CANADA COPYRIGHT © 2016 BY ANURAG SINGH SIKARWAR I DISCLAIMER This modified sandwich thesis comprises three multi-authored manuscripts, all of which contain a majority of original work contributed by the author of this thesis, but which also contain varying portions of work contributed by collaborating lab members. The Gαq palmitoylation and Thromboxane receptor (TP) phosphorylation studies in this thesis are published in American Journal of Respiratory Cell and Molecular Biology and British Journal of Pharmacology respectively; the adenylyl cyclase (AC) activity study is in currently preparation for publication. Individual author contributions are given below. 1. Palmitoylation of Gαq determines its association with the thromboxane receptor in hypoxic pulmonary hypertension. Am J Respir Cell Mol Biol. 2014 Jan;50(1):135-43. Anurag Singh Sikarwar is the first author of the Gαq palmitoylation study and was primarily responsible for study design, experimentation and data analysis. Within this manuscript, the ATP dose-response (in pulmonary artery smooth muscle cells) and U46619 dose-response in aortic myocytes was done with help from Martha Hinton. Santhosh Thomas performed the inositol 1,4,5 triphosphate assay. 14C-palmitate and 3H-leucine uptake was done by Anurag Singh Sikarwar with help from Santhosh Thomas. Force myography, Fura-2AM calcium imaging on pulmonary artery smooth muscle cells and all work on the HEK cell line was performed and analyzed by Anurag Singh Sikarwar. 2. Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324. Br J Pharmacol. 2014 Feb;171(3):676-87. Anurag Singh Sikarwar and Santhosh Thomas equally contributed as first authors of this study toward the study design, experimentation and data analysis. Anurag Singh Sikarwar and Santhosh Thomas designed thromboxane receptor α (TPα) serine mutants and performed the calcium assay experiments on stable and transiently transfected cells. Anurag Singh Sikarwar prepared the stable cell lines for human and porcine TPα overexpression, and carried out the force myography experiments. Santhosh Thomas performed radioligand binding studies, and protein kinase A and protein kinase C activity experiments. TP serine immunoprecipitation experiments were done by Martha Hinton. 3. Adenylyl cyclase activity is impaired by pulmonary arterial hypoxia (unpublished) Santhosh Thomas initially measured basal and stimulated AC activity, and completed the phosphodiesterase activity assays. Subsequent experimental design, data collection and data analysis were the responsibility of Anurag Singh Sikarwar. Anurag Singh Sikarwar did the force myography experiments, and performed AC activity assays following treatment with pertussis toxin and BAPTA. Anurag Singh Sikarwar and Martha Hinton jointly carried out the Western blot and immunoprecipitation experiments. Martha Hinton did the expression profile of AC enzyme isoforms. II Abstract Introduction: Persistent pulmonary hypertension of the newborn (PPHN) is associated with an elevated thromboxane to prostacyclin ratio, pulmonary artery (PA) hyperreactivity and hypersensitivity. Thromboxane receptor (TP), coupling with G-protein Gαq causes pulmonary vasoconstriction; whereas prostacyclin receptor (IP), coupling with Gαs, causes vasodilation and TP phosphorylation via adenylyl cyclase (AC)-cAMP-protein kinase A (PKA), desensitizes TP. Both TP phosphorylation and Gαq palmitoylation play major roles in regulation of signaling through the TP-Gαq complex. We hypothesized that increased Gαq palmitoylation and decreased AC activity could cause hypoxic TP hyperresponsiveness. We studied the impact of hypoxia on selected post-translational modifications of the receptor-G-protein complex, determining TP vasoconstriction: Gαq palmitoylation, TP phosphorylation and upstream AC activity. Methods: Force responses to thromboxane mimetic U46619, palmitoylation inhibition by 2- bromopalmitate (2-BP) and AC activation (forskolin) were studied by myography in hypoxic PPHN and control newborn swine pulmonary artery. Ca2+ mobilization was studied by fluorescent calcium indicators fura-2AM in pulmonary myocytes (PASMC), and fluo-4NW in HEK293 cells. Effects of hypoxia on Gαq palmitoylation were studied by metabolic labeling. Gαq cysteines and TP serines were mutated to determine sites of post-translational modifications. Protein expression and receptor-G-protein coupling were studied by Western blot and co-immunoprecipitation. PKA activity was assayed; and AC activity quantified. Results: Hypoxia increases Gαq palmitoylation, without increasing total palmitate uptake. Palmitoylation inhibition decreases U46619-stimulated force generation as well as Ca2+ III mobilization in PPHN PA rings and hypoxic PASMC. Mutation of palmitoylable cysteine and palmitoylation inhibition proportionately decrease U46619-mediated Ca2+ mobilization in HEK293 cells. TP serine phosphorylation is decreased by hypoxia due to decreased PKA activity; this causes TP hypersensitivity and hyper-reactivity. Serine 324 of TPα is the target of PKA-mediated desensitization. AC activator-induced relaxation is reduced in PPHN PA. Basal and receptor-stimulated AC activity are decreased in hypoxic PASMC. Decreased AC activity is not due to decreased AC expression, ATP availability nor increased Gαi activation. IP coupling with Gαs is increased in hypoxia. Conclusion: Increased Gαq palmitoylation plays a role in TPα hyper-responsiveness in hypoxic PPHN. Hypoxia also reduces responses to agents acting through AC, unleashing TP-mediated vasoconstriction. Reactivation of pulmonary AC might be useful therapeutically to promote vasodilation and TP desensitization. IV Acknowledgements First and foremost I would like to express my deepest gratitude to my supervisor Dr. Shyamala Dakshinamurti for accepting me her student and guiding my research all the way up to this stage. Dr. Dakshinamurti provided an excellent research and learning environment to me in her lab, and it is due to her excellent clinical, scientific and teaching skills, I learnt planning and successful execution of research experiments. Regular feedback, discussion and teaching in lab meetings by Dr. Dakshinamurti helped me a lot to understand, retain and apply scientific literature to solve research questions. I am very thankful to Dr. Dakshinamurti for her help and motivation in applying graduate studentships. Finally, I thank her for proofreading and revising the thesis chapters wherever required. I am very fortunate and thankful to have Dr. Andrew Halayko, Dr. Prashen Chelikani and Dr. Gary Shen as my advisory committee members. It is due to their constant motivation, critical feedback and discussions during committee meetings, I was able to complete my research projects successfully. I thank my committee members for providing me reference letters for studentships and awards. I owe my deepest gratitude to our lab managers Nora Nolette and Martha Hinton. From the very first day to the final year of my studies, your knowledge and expertise in using various laboratory apparatus were instrumental in successful accomplishment of experiments. I appreciate their friendly nature and patience while teaching me various laboratory techniques and helping me carry out experiments. I would like to thank my labmate and friend Jena fediuk for all the help, encouragement and company she gave me during her stay in the lab. I would like to acknowledge the company and support from Dr. Alex Gutsol (my friend and myograph teacher) and Dr. Santhosh Thomas Kallivallapil with whom I worked on many projects. I would like to thank Mr. Gerald Stelmack for teaching me calcium imaging and Richard Hemming for helping me with metabolic labeling experiments. I would like to extend my thanks to Dr. Halayko and his present and past lab members for the cheerful company and scientific discussions during various talks and everyday lab life. My sincere thanks to Dr. Chelikani and his lab members such as Raja Chakraborty, Sai Prasad Pydi, Thi lee, Nisha Singh, Jasbir Deol and Appalaraju Jaggupilli for helping me with HEK cell line work and using research facilities in the lab. Special thanks to my friends Raja Chakraborty and Sai Prasad Pydi for all the help and company I got during my stay at Dr. Chelikani’s lab. I am also very grateful to the Department of Physiology and Pathophysiology’s present and former head of the departments for all the support and encouragement and especially Gail McIndless and Judith Olfert, our graduate program advisors for helping with the administrative process and providing me various support letters that an international student needs time to time. V I gratefully acknowledge the funding sources Research Manitoba, and Children’s Hospital Research Institute of Manitoba for the graduate studentship, University of Manitoba, Faculty of Graduate Studies for international graduate student scholarship and travel awards that assisted my Ph.D. studies. I can’t put my thankfulness into words for my dear wife Nisha whose unconditional love, support and encouragement enabled me to complete my Ph.D. studies. I would like to thank my sweet daughter
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