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Extracellular Regulation of Nitric Oxide Signaling Via Soluble Guanylate Cyclase

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Extracellular Regulation of Nitric Oxide Signaling Via Soluble Guanylate Cyclase Extracellular Regulation of Nitric Oxide Signaling via Soluble Guanylate Cyclase Item Type text; Electronic Dissertation Authors Ramanathan, Saumya Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 08/10/2021 13:19:27 Link to Item http://hdl.handle.net/10150/223312 EXTRACELLULAR REGULATION OF NITRIC OXIDE SIGNALING VIA SOLUBLE GUANYLATE CYCLASE by Saumya Ramanathan _____________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF MOLECULAR AND CELLULAR BIOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2012 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Saumya Ramanathan entitled EXTRACELLULAR REGULATION OF NITRIC OXIDE SIGNALING VIA SOLUBLE GUANYLATE CYCLASE and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of PHILOSOPHY _______________________________________________________________________ Date: April 11, 2012 Dr. William R. Montfort _______________________________________________________________________ Date: April 11, 2012 Dr. Todd Camenisch _______________________________________________________________________ Date: April 11, 2012 Dr. Roger Miesfeld _______________________________________________________________________ Date: April 11, 2012 Dr. Tsu-Shuen Tsao _______________________________________________________________________ Date: April 11, 2012 Dr. Ted Weinert Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: April 11, 2012 Dissertation Director: Dr. William R. Montfort 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or part maybe granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: SAUMYA RAMANATHAN 4 ACKNOWLEDGEMENTS Dr. Bill Montfort- without him I’d have no career. His enthusiasm for science inspires me. He welcomes people of different cultures and backgrounds into his lab, making it a truly enriching experience to work for him. For him, there is no good data or bad data, there is just interesting science. I loved working for you Bill!! J Committee members-Dr. Miesfeld, Dr. Weinert, Dr. Camenisch and Dr. Tsao- You all have been a part of my graduate career and I always felt that I could approach any of you without hesitation. Thank you for that and for timely advice on experiments, exams and life!! Dr. Scott Boitano-Thank you for all your help with the calcium experiments. I couldn’t have done any of this without your help. Montfort Lab-WOW!! You guys are the best. Jacquie-you keep the lab running like a machine, but more importantly, you care! In these five years, you have transitioned into someone so integral to my life that I do not know what I will do when I do not have your shoulder to lean on? Dr. Weichsel-Your wit and humor makes us all pale in comparison. Breezy Zegeer- for being the epitome of a powerful woman. Eman-you are the nicest addition to our lab. Thank you for being there for me during difficult times. The gang- Stacy, Jules, Bradley and Johnny- You are the best friends a girl can ask for. Good days, bad days, awful days and wonderful days-we’ve been through it all. I love you guys! MHW-Thank you!! You are a true cheerleader! Rahul- For all the apnapan in lab- Thank you! Aaron-You are one of the nicest kids around, please stay the same always (except stop making fun of me J). Marilyn Kramer-I wouldn’t be here in Tucson without your help. You helped me realize that I had options. Family-My parents-Amma and Appa-Thank you for making me the person I am, thank you for all the opportunities you have given me. Thank you for being the best parents in the world. My dear Annipanni-my blood brother-from that day in the hospital to now, its been 30 years and I can say for sure that you have never left my side and have been a part of every big decision I have made, good or bad. I wish all little girls had such elder brothers. My darling husband-Hemant-I love you. I am here today because of you. You have seen me at my best and my worst. You make me feel safe and inspire me to be a better human being-Thank you! My In-law’s-Amma and Appa Badgandi-Thank you for all your support in these last crucial months and for welcoming me into a your wonderful family!. Paro-my best friend, Thank you for welcoming me to Tucson with so much enthusiasm that I could not help but fall in love and thank you for being you. 5 DEDICATION To Amma-for teaching me that unconditional love exists To Appa-for teaching me the meaning of will-power by epitomizing it To Annipanni-for never allowing me to feel like I am alone To Hemant-Destiny brought me to you, love will keep me with you. “The greatest glory in living lies not in never falling, but in rising every time we fall.” ― Nelson Mandela 6 TABLE OF CONTENTS LIST OF TABLES……………………………………………………………………….11 LIST OF FIGURES……………………………………………………………………...12 ABSTRACT……………………………………………………………………………...15 CHAPTER 1: INTRODUCTION………………………………………………………..16 1.1. Nitric oxide biology and synthesis……………………………………………….16 1.2. Soluble guanylate cyclase: The nitric oxide receptor protein…………………….17 1.2.1. Regulation of soluble guanylate cyclase……………………………………..20 1.2.1.1. Isoforms and splice variants…………………………………………….20 1.2.1.2. Transcriptional and post-transcriptional regulation……………………..20 1.2.1.3. Subcellular localization………………………………………………….21 1.2.1.4. Hetero-dimerization vs. homo-dimerization…………………………….22 1.2.1.5. Protein-protein interaction………………………………………………22 1.2.1.6. Phosphorylation…………………………………………………………23 1.2.1.7. Calcium………………………………………………………………….23 1.2.1.8. Allosteric regulators of sGC…………………………………………….24 1.3. Thrombospondin-1: Regulator of nitric oxide signaling…………………………26 1.3.1. Discovery of thrombospondin-1 as a modulator of NO signaling…………..26 1.3.2. Thrombospondins-A family of extracellular matrix proteins……………….27 1.3.2.1. Domain organization of TSP-1………………………………………….27 1.3.3. TSP-1 inhibits NO signaling via CD47……………………………………...28 1.4. CD47 (Integrin Associated Protein)……………………………………………...30 1.4.1. Ligands of CD47…………………………………………………………….30 1.4.1.1. Signal inhibitory receptor protein α (SIRPα)…………………………..30 1.4.1.2. Thrombospondin-1……………………………………………………...32 7 TABLE OF CONTENTS-continued 1.4.2. CD47: a non-canonical GPCR………………………………………………32 1.5. Research topic and dissertation outline…………………………………………..33 CHAPTER 2: DEVELOPMENT OF A LIVE CELL SYSTEM FOR STUDYING TSP-1 SIGNALING…………………………………………………….36 2.1. Defining the model cell line……………………………………………………..36 2.2. Materials and Methods…………………………………………………………..37 2.2.1. Materials…………………………………………………………………….37 2.2.2. Cell culture………………………………………………………………….37 2.2.3. Expression and purification of E3CaG1…………………………………….38 2.2.4. Cloning and transient transfection of human sGC…………………………..39 2.2.5. sGC activity and cGMP accumulation in intact cells……………………….40 2.2.6. Flow cytometry………………………………………………………………41 2.2.7. Expression and purification of the G1 domain of human TSP-1……………42 2.3. Results……………………………………………………………………………42 2.3.1. Expression of sGC and measurement of NO-inducible sGC activity……….42 2.3.2. CD47 expression on cell surface……………………………………………43 2.3.4. E3CaG1……………………………………………………………………..43 2.3.5. CD47 is necessary but insufficient for E3CaG1 binding to Jurkat T cells and inhibition of sGC……………………………………………………….49 2.3.6. Role of integrin αv in E3CaG1 signaling via CD47………………………..50 2.3.7. G1 domain of TSP-1………………………………………………………..56 2.4. Conclusions and Discussion……………………………………………………..56 CHAPTER 3: E3CaG1 AND ANGIOTENSIN II INHIBIT sGC VIA INCREASE IN INTRACELLULAR CALCIUM……………………………………59 3.1. Hypothesis………………………………………………………………………59 8 TABLE OF CONTENTS-continued 3.2. Materials and methods………………………………………………………….60 3.2.1. Materials……………………………………………………………………60 3.2.2. Cell culture………………………………………………………………….60 3.2.3. Calcium imaging……………………………………………………………61 3.2.4. Flow cytometry……………………………………………………………..61 3.2.5. sGC activity and cGMP accumulation in intact cells………………………62 3.3. Results…………………………………………………………………………..64 2+ 3.3.1. E3CaG1 induces an increase in [Ca ]i…………………………………….64 2+ 3.3.2. E3CaG1-dependent increases in [Ca ]i requires CD47……………………64 2+ 3.3.3. E3CaG1 mediates increase in [Ca ]i and inhibits sGC in human microvascular
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