University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2013 The N-terminus of the Saccharomyces cerevisiae G protein- coupled receptor Ste2p: formation of dimer interfaces and negative regulation Mohammad Seraj Uddin [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Biochemistry Commons, Molecular Biology Commons, and the Structural Biology Commons Recommended Citation Uddin, Mohammad Seraj, "The N-terminus of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p: formation of dimer interfaces and negative regulation. " PhD diss., University of Tennessee, 2013. https://trace.tennessee.edu/utk_graddiss/2490 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Mohammad Seraj Uddin entitled "The N- terminus of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p: formation of dimer interfaces and negative regulation." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Microbiology. Jeffrey M Becker, Major Professor We have read this dissertation and recommend its acceptance: Tim E. Sparer, Todd B. Reynolds, Cynthia B. Peterson Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) The N-terminus of the Saccharomyces cerevisiae G protein-coupled receptor Ste2p: formation of dimer interfaces and negative regulation A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Mohammad Seraj Uddin August 2013 Copyright©2013 by Mohammad Seraj Uddin All rights reserved ii Dedication This dissertation is dedicated to my parents, Rashid Ahmed and Laila Begum, for their endless love and support. I would also dedicate this work to all family members, my wife Fahmida Chowdhury,my son Zarif and my parents-in-law for their encouragement and love. iii Acknowledgments I would like to thank many people who, without their help, this dissertation would not be possible. My first and deepest appreciation goes to my mentor Dr. Jeffrey M. Becker. It was really a great pleasure to work for such an enthusiastic scientist whose knowledge, guidance, support and directions will be with me for the rest of my career. I could not have done it without him. I am thankful that he was so patient and supportive during the time I was struggling with my projects. His critical suggestions and guidance helped me overcome lots of stumbling blocks that I would not even try without him. It is a great pleasure and honor to work for such a great scientist. I also would like to give my very appreciation to my committee members Dr. Timothy E. Sparer, Dr. Todd B. Reynolds, and Dr. Cynthia Peterson for their suggestions, critical reviews and guidance during my studies. Their extraordinary patience and encouragement motivated me. I would like to thank Dr. Fred Naider for his support and valuable ideas on critical decisions throughout these studies. I also want to thank all the Dr. Naider laboratory members for the peptide syntheses that made this study possible. I really appreciate all the lab members in Dr. Becker’s laboratory: Special thanks to Dr. Melinda Hauser, Dr. Byung-Kwon Lee, and Dr. Tom Masi for their guidance and help during my study. I also want to thank Sarah Kauffman, George Umanah, Liyin Huang, Elena Ganusova, Steve Minkin, Heejung Kim, Giljun Park, Kyung Sik, Amanda Deyo, Madelyn Crawford, Jordan Kim, Brandon Sidney, Julie Maccarone, John Lockart, who made Becker lab such an enjoyable and fun place to work at. iv Finally I am greatly indebted to my family in Bangladesh, Fahmida Chowdhury and my little son Zarif for all the love and support they gave me during all these years. And a big thank you to everyone who has been a friend and colleague throughout this process. I could not have it without you all. v Abstract G protein-coupled receptors (GPCRs), the largest family of membrane proteins on the cell surface, play essential roles in signal transduction in all eukaryotic organisms. These proteins are responsible for sensing and detecting a wide range of extracellular stimuli and translating them to intracellular responses. This signaling requires a tight control for receptor activation without which abnormal signal leads to diseases. In fact, malfunctions of these receptors are associated with numerous pathological conditions and currently an estimated 40-50% of therapeutic drugs are designed to target these receptors suggesting that further increases in understanding of GPCRs and the signaling pathways they initiate will lead to new and more specific drug targets. We have used Saccharomyces cerevisiae GPCR Ste2p as a model system to understand structure-function relationships of these receptors. In this study, the role of the extracellular N-terminus has been examined using various biophysical methods with the anticipation to uncover its role in receptor function. It was found that some residues in the extracellular N-terminus were not accessible to a sulfhydryl reagent and that the alternating pattern of accessibility is consistent with the structure of a beta strand. This beta strand was found to be involved in dimer formation. Moreover, a conserved tyrosine residue in the middle of the beta strand was found to interact with two residues in the extracellular loop 1. It was also found that the N-terminus is involved in negative regulation and important for cell surface expression. vi Table of Contents Chapter 1: General introduction ................................................................................................. 1 G protein-coupled receptors: an overview ..................................................................... 2 Classification of GPCRs ................................................................................................... 4 GPCR crystal structures .................................................................................................. 9 Fungal GPCRs ................................................................................................................. 12 S. cerevisiae GPCRs ........................................................................................................ 15 The use of Ste2p as a modeL GPCR ............................................................................. 20 References ........................................................................................................................ 27 Chapter 2 : Identification of residues involved in homodimer formation located within a β- strand region of the N-terminus of a yeast G protein-coupled receptor. ............................... 48 Abstract ............................................................................................................................ 50 Introduction ..................................................................................................................... 51 Methods ............................................................................................................................ 53 Results .............................................................................................................................. 58 Discussion ........................................................................................................................ 74 Conclusion ....................................................................................................................... 78 Acknowledgements ......................................................................................................... 79 References ........................................................................................................................ 80 vii Chapter 3: The N-terminus of Saccharomyces cerevisiae G protein-coupled receptor Ste2p is involved in negative regulation .............................................................................................. 89 Abstract ............................................................................................................................ 90 Introduction ..................................................................................................................... 91 Methods ............................................................................................................................ 94 Results ............................................................................................................................ 100 Discussion ...................................................................................................................... 124 Acknowledgments ......................................................................................................... 129 References ...................................................................................................................... 130 Chapter 4: Formation of ste2p dimers by a conserved tyrosine residue in the N-terminus that has