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Interaction of a G Protein-Coupled Receptor (Ste2p) of Saccharomyces Cerevisiae with Its Ligand and Its G-Protein Alpha Subunit

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Interaction of a G Protein-Coupled Receptor (Ste2p) of Saccharomyces Cerevisiae with Its Ligand and Its G-Protein Alpha Subunit University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2011 Interaction of a G protein-coupled receptor (Ste2p) of Saccharomyces cerevisiae with its ligand and its G-protein alpha subunit Li-Yin Huang [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Microbiology Commons Recommended Citation Huang, Li-Yin, "Interaction of a G protein-coupled receptor (Ste2p) of Saccharomyces cerevisiae with its ligand and its G-protein alpha subunit. " PhD diss., University of Tennessee, 2011. https://trace.tennessee.edu/utk_graddiss/1192 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 Li-Yin Huang entitled "Interaction of a G protein-coupled receptor (Ste2p) of Saccharomyces cerevisiae with its ligand and its G-protein alpha subunit." 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 equirr ements 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: Chunlei Su, Todd B. Reynolds, Elizabeth E. Howell, Timothy E. Sparer 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.) Interaction of a G protein-coupled receptor (Ste2p) of Saccharomyces cerevisiae with its ligand and its G-protein alpha subunit A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Li-Yin Huang December 2011 Copyright © 2011 by Li-Yin Huang All rights reserved. ii Dedication This dissertation is dedicated to my parents, Zen-Jun Huang and Zon-Hua Tsai, for their endless love and support. I would not be able to achieve any of this without the sacrifices they made in order for me to receive the best education possible. iii Acknowledgments I would like to thank many people who helped me throughout my studies. My first and deepest appreciation goes to my mentor Dr. Jeffrey M. Becker. He is such an enthusiastic scientist who always has great insights about research. 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 setbacks that I would not even try without him. It is a great pleasure and honor to work for him. I also would like to give my very appreciation to my committee members: Dr. Chunlei Su, Dr. Todd B. Reynolds, Dr. Elizabeth E. Howell, Dr. Timothy E. Sparer, and Dr. Ana Kitazono. Their extraordinary patience and encouragement motivated me. With their guidance and support, I finally learned how to become a good scientist. 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 on my study. I also want to thank Sarah Kauffman, Elena Ganusova, Steve Minkin, Heejung Kim, Giljun Park, Kyung Sik, Amanda Deyo, Madelyn Crawford, Jordan Kim, Brandon Sidney, and Seraj Uddin, who made Becker lab such an enjoyable and fun place to work at. I appreciate the help from Julie Maccarone in the biological experiments. My very special thanks belong to George Umanah for being my best friend and a great partner in the lab. Our reseach thrived on all the arguments and good discussions. iv I also want to thank my favorite teachers: Lisa Meidl and Julie Moore, who helped me a lot to improve my English and lead me to know God. Thanks to Meidl boys (Bill, Peter, Nate, and Timothy), Ron and Marti Holder, Life Focus group at Sunday school, and the choir in Cedar Springs Presbyterian Church. I really appreciate all the cares from the church family, who made it feel just like home. Last, but not the least, I would like to thank my very best friend, Jui-He Tsai (Ray). Since college to graduate school, we had been studying together for a long time. Ray is optimistic and has great personalities. It was very nice to always have him to share my struggles, laughters, tears, memories, and all the ups and downs in life. I really appreciate him. v Abstract The G protein-coupled receptor (GPCR) family is composed of hundreds of members and is expressed in eukaryotes. Each GPCR has seven transmembrane domains and is in charge of sensing changes from the environment, transducing signals, and activating a series of biological responses. The signal transduction pathway of the receptor starts from sensing outside signal and then activates G proteins. This signaling requires a tight control for activation without which impaired cellular function leads to pathology. We have used the pheromone alpha-factor receptor (Ste2p) of the yeast Saccharomyces cerevisiae as a model system to understand ligand binding, receptor activation, and G protein interaction. One method we have used to study ligand binding is to incorporate the photo-reactive crosslinker p-benzoyl-L-phenylalanine (Bpa) into Ste2p to capture alpha-factor. This powerful tool requires the incorporation of Bpa, an unnatural amino acid, into Ste2p by a special genetic manipulation designed in the lab of Peter Schulz (Scripps Institute) and adapted by our lab for Ste2p. Another method to study ligand binding that we have adapted for use in our system is to incorporate a chemical crosslinker [3,4-dihydroxylphenylacetyl (DHPA)] into alpha-factor for periodate- mediated crosslinking to Ste2p. The interacting domain between alpha-factor and transmembrane domain 2 to 3 of Ste2p was identified after DOPAC crosslinking, cyanogen bromide digestion and MALDI-TOF mass spectrometry. After ligand binding, signal transduction is mediated by the interaction of activated Ste2p with its G protein (Gpa1p). We studied this interaction by replacing natural residues in the intracellular loop 3 of Ste2p and C-terminal end of Gpa1p with cysteine and then determining disulfide crosslinking between Ste2p and Gpa1p. Some residues were found to be in close proximity and displayed different interacting patterns due to conformational changes of the receptor upon ligand binding. The information we gathered here allows us to understand more about the physical interactions of alpha-factor, Ste2p, and Gpa1p and provides us insights about the initiation and activation of the signal transduction pathway of a peptide ligand receptor. vi TABLE OF CONTENTS CHAPTER PAGE PART I: General Introduction 1 G Protein-Coupled Receptors: An Overview ............................................................ 2 2 Alpha-factor Pheromone and its G Protein-Coupled Receptor (Ste2p) of Saccharomyces cerevisiae ......................................................................................... 7 References for Part I ................................................................................................ 11 PART II: Unnatural Amino Acid Replacement in a Yeast G Protein-Coupled Receptor in its Native Environment (Published previously as: TYPE IN FULL REF) Abstract for Part II ................................................................................................... 22 1 Introduction .............................................................................................................. 23 2 Materials and Methods ............................................................................................. 26 3 Results ...................................................................................................................... 32 4 Discussion ................................................................................................................ 49 References for Part II ............................................................................................... 55 CHAPTER III: Changes in Conformation at the Cytoplasmic Ends of the Fifth and Sixth Transmembrane Helices of a Yeast G Protein-Coupled Receptor in Response to Ligand Binding (Published previously as: TYPE IN FULL REF) Abstract for Part III .................................................................................................. 66 1 Introduction .............................................................................................................. 68 2 Materials and Methods ............................................................................................. 71 3 Results ...................................................................................................................... 79 4 Discussion ................................................................................................................ 93 References for Part III ............................................................................................ 102 vii CHAPTER IV: Residue-to-Residue Interactions Between a G Protein-Coupled Receptor (GPCR)
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