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Function and Regulation of the Postsynaptic Exchange Factor, Kalirin-7 Megan B University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 4-2-2015 Function and Regulation of the Postsynaptic Exchange Factor, Kalirin-7 Megan B. Miller University of Connecticut, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Miller, Megan B., "Function and Regulation of the Postsynaptic Exchange Factor, Kalirin-7" (2015). Doctoral Dissertations. 698. https://opencommons.uconn.edu/dissertations/698 Function and Regulation of the Postsynaptic Exchange Factor, Kalirin-7 Megan B. Miller, Ph.D. University of Connecticut, 2015 Abstract Through studies of a single RhoGEF, Kalirin-7, the work presented herein provides insight into several of the key mechanisms underlying postsynaptic neurotransmission in the central nervous system. Much of this work has focused on the significance of non-enzymatic functions of Kal7, highlighting the importance of spatial and temporal control of multi-domain proteins and the postsynaptic protein/ lipid interactome. Our work on Kalrn promoter usage and phosphorylation (Chapters 5 & 6) expands upon our understanding of synaptic protein diversity and regulation. Additionally, Kalrn promoter switching has implications for the poorly-understood mechanisms by which cytoskeletal dynamics and membrane trafficking are coordinated at the postsynaptic density (PSD). Combined with our biochemical characterization of the Sec14 domain (Chapter 4), our exploration of Kalrn promoter usage also touches upon the importance of synaptic lipid metabolism. Thus, by enhancing our knowledge of the functions and regulatory mechanisms of Kal7, we highlight the extraordinary intricacies of postsynaptic signaling and the necessity for precise control of multiple cellular events. Furthermore, we provide a large body of evidence to support the notion that large, multi-domain proteins serve diverse roles at the synapse, which are crucial for normal brain function. i Function and Regulation of the Postsynaptic Exchange Factor, Kalirin-7 Megan Bridget Miller B.S., The Ohio State University, 2005 Ph.D., University of Connecticut, 2015 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2015 ii APPROVAL PAGE Doctor of Philosophy Dissertation Function and Regulation of the Postsynaptic Exchange Factor, Kalirin-7 Presented by: Megan B. Miller, B.S., Ph.D. Major Advisor _________________________________________________________________ Betty A. Eipper, Ph.D. Associate Advisor ______________________________________________________________ Kimberly L. Dodge-Kafka, Ph.D. Associate Advisor ______________________________________________________________ Eric S. Levine, Ph.D. Associate Advisor ______________________________________________________________ Richard E. Mains, Ph.D. University of Connecticut 2015 iii Acknowledgments First and foremost, I would like to thank my outstanding thesis advisors, Dr. Betty Eipper and Dr. Richard Mains. They have shown me unrivaled support and guidance every step of the way, while still allowing me to pursue my own intellectual curiosities. Looking back, I think one of the greatest lessons they have taught me is the value of an open-minded and enthusiastic approach to science. One would be hard-pressed to find two people who love what they do more than Dick and Betty. In addition to being excellent scientists, Dick and Betty are also model mentors. Their dedication to the well-being and career development of their trainees is apparent. Because of them, I feel confident in my ability to pursue interesting questions with the highest level of integrity, and for that I am beyond appreciative. I must also thank all of the other members of my lab for their support and comradery along the way. I feel extremely fortunate to have been a part of such a positive and enriching group. I would especially like to thank Yanping Wang and Darlene D’Amato for their incredible technical support and Dr. K.S. Vishwanatha who was integral to the biophysical studies presented in this dissertation. I would also like to thank my thesis committee members, Dr. Levine and Dr. Dodge-Kafka, for their honest feedback over the years. Thanks also to Dr. Yi Wu and the people at the KECK Neuroproteomics facility at Yale for their continued support and ongoing collaborations. Of course none of this would have been possible without the UConn Health Center Biomedical Science PhD program, the Graduate Programs Committee, the Department of Neuroscience and graduate program and all of the individuals who work so hard to assure an iv excellent educational experience for students. In particular, I would like to thank Dr. Barbara Kream, Stephanie Rauch, Swapna Das and Jody Gridley for their laudable dedication. Finally, I thank all of my friends and family for their immeasurable support and understanding over the years. To my parents, Mark and Lou Miller: I would have never had the courage to pursue this degree if it were not for the lifetime of unwavering support I have received from you. It was you who first instilled in me the value of hard work, and you have never discouraged me from chasing outlandish dreams. I owe this and all future success to you. To my sister (brother) Molly, thanks for reminding me to see the humor in life when I get too serious. You will never know how much our silly conversations or absurd brotherly memories have meant to my sanity. And lastly, to my extraordinary fiancé, Giulio Chiuini: I honestly could not have done this without you. From late-night study snacks and gentle reminders to eat vegetables to nearly constant words of encouragement and an all-too-frequent shoulder to cry on, you were ALWAYS there for me. The level support you consistently show me is beyond selfless, and I truly hope that I can somehow begin to return the favor. Words cannot begin to describe how eternally grateful I am to have you in my life. From the bottom of my heart: thank you and I love you. v Table of Contents Chapter 1: .................................................................................................................................................... 1 Introduction ................................................................................................................................................... 1 Spine dynamics are controlled by RhoGTPases and their regulators ....................................................... 3 The postsynaptic RhoGEF, Kalirin-7........................................................................................................ 4 Chapter 2 ..................................................................................................................................................... 7 Rho GEFs in synaptic physiology and behavior ........................................................................................... 7 Abstract .................................................................................................................................................... 7 Introduction ............................................................................................................................................. 7 Dual Rho GEFs (Kalirin and Trio) ....................................................................................................... 8 Discovery and expression ..................................................................................................................... 8 Protein structure and signaling .......................................................................................................... 10 Behavior .............................................................................................................................................. 11 Electrophysiology and synaptic function ............................................................................................ 14 Disease relevance ............................................................................................................................... 15 T-cell lymphoma invasion and metastasis family (Tiam1 and Tiam2) ............................................ 16 Discovery and expression ................................................................................................................... 16 Protein structure and function ............................................................................................................ 16 Behavior .............................................................................................................................................. 18 Electrophysiology and synaptic function ............................................................................................ 18 Disease relevance ................................................................................................................................ 21 Phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac Exchange Factor Family (P-Rex1 and P-Rex2) ................................................................................................................................................... 21 Discovery and expression ................................................................................................................... 21 Protein structure and function ............................................................................................................ 22 Behavior .............................................................................................................................................
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