Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2007 Targeted Disruption of Lynx2 Reveals Distinct Functions for Lynx Homologues in Learning and Behavior Ayse Begum Tekinay Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Tekinay, Ayse Begum, "Targeted Disruption of Lynx2 Reveals Distinct Functions for Lynx Homologues in Learning and Behavior" (2007). Student Theses and Dissertations. Paper 28. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. TARGETED DISRUPTION OF LYNX2 REVEALS DISTINCT FUNCTIONS FOR LYNX HOMOLOGUES IN LEARNING AND BEHAVIOR A Thesis Presented to the Faculty of The Rockefeller University In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Ayse Begum Tekinay June 2007 © Copyright by Ayse Begum Tekinay 2007 TARGETED DISRUPTION OF LYNX2 REVEALS DISTINCT FUNCTIONS FOR LYNX HOMOLOGUES IN LEARNING AND BEHAVIOR Ayse Begum Tekinay, Ph.D. The Rockefeller University 2007 Endogenous short peptide modulators of ion channels provide a new level of regulation of nervous function. Lynx1 was identified as an endogenous mammalian homologue of snake venom peptide neurotoxins capable of binding to and functionally modulating nicotinic acetylcholine receptors (nAChR). Lynx1 is a member of the Ly6- α−neurotoxin superfamily (Ly6SF) of genes. Through extensive database searches, I identified 85 members of this superfamily including previously unidentified vertebrate and invertebrate family members. I show that these proteins are very divergent in their sequences, and identify two conserved subfamilies, snake toxins and immune system expressed Ly6 genes through phylogenetic inference. I also discovered conserved sequences among Ly6SF proteins additional to the cysteines that characterize the three-dimensional topology of Ly6 domain. From these Ly6SF molecules, I characterize three lynx1 homologues: lynx2, lynx3 and Ly6H. These have different and specific expression patterns in the central and peripheral nervous systems. Importantly, like lynx1, lynx2 and lynx3 are able to bind specific nAChR combinations and modulate their desensitization properties, while Ly6H does not bind nor modulate the function of nAChRs. I have also analyzed the in vivo function of lynx2, through the targeted deletion of lynx2 in mice. Lynx2 null mutant (lynx2-/-) mice exhibited increased anxiety, better associative learning and better motor coordination and learning than wild type (WT) mice. Nicotine and nicotinic receptor antagonist, mecamylamine, show distinct effects on motor learning in lynx2-/- mice and WT mice. The properties of this new family of lynx1-like molecules, taken together with those of lynx1 and the secreted Ly6SF member SLURP1 (secreted Ly6/UPAR related protein), suggest a general role for Ly6SF proteins in the modulation of nAChRs and other receptor proteins. Turgay’a iii Acknowledgements I would like to thank my advisor, Nathaniel Heintz, for helping me whenever I had a tough time during my studies and giving me valuable advice whenever I needed it. I also would like to thank MaryBeth Hatten, David Gadsby and Joseph LeDoux for providing valuable scientific advice and introducing me to their lab members, who were very helpful. Joseph P. Doyle was extremely nice and patient with my never-ending questions in the first years of grad school and continued to be very supportive till the end. I’m grateful to Julie Miwa, Laura Kus, Tanya Stevens, Inez Ibanez-Tallon and Shiaoching Gong for their great scientific advice and helping me with my experiments. Laura Kus was incredibly helpful on anatomical annotations and characterizing expression pattern of lynx3. I’m grateful to Mayte Farinas, who has helped me with the statistical analysis of the behavioral data. I would like to thank Wendy Lee for spending a lot of time doing transfections for my co-immunoprecipitation studies and Jie Xing for making maxi-preps. I also would like to thank members of Heintz lab and GENSAT project, who made graduate school tolerable. Aylin Aslantaş was a great help with her summer work. Ivo Lieberam was a very helpful collaborator. I owe thanks to my college professors Mehmet Öztürk and Tayfun Özçelik for their advice and guidance, which they continued to provide long after college. I’m indebted to my parents, my sisters Aysun and Aylin, my nephew Batuhan and my family for raising me, supporting my education and taking care of my daughter for 19 months staying in a city, where they don’t even know the language. Minnettarım… iv My precious daughter, Sarah Hümeyra, has beared with her mommy working late for two years with her beautiful smile. Seni seviyorum kızım. I also would like to thank my husband, Turgay, from the bottom of my heart, for being my best friend, helping me when I felt down, supporting me and believing in me. I’ll always be grateful. v Table of Contents CHAPTER 1: .................................................................................................1 Introduction....................................................................................................1 Summary......................................................................................................................... 2 Ly6 Superfamily of genes .............................................................................................. 3 Structure, Evolution and Diversity ............................................................................. 3 Expression Patterns..................................................................................................... 8 Activity and Function ............................................................................................... 12 Nicotinic Acetylcholine Receptors............................................................................... 16 Diversity and Structure ............................................................................................. 16 Expression patterns ................................................................................................... 19 Function .................................................................................................................... 21 Lynx1 ............................................................................................................................ 28 Sequence and Structure............................................................................................. 28 Expression................................................................................................................. 28 Function .................................................................................................................... 29 Chapter 2:.....................................................................................................33 Phylogenetic Inference of Ly6 Superfamily of Proteins ..........................33 Introduction.................................................................................................................. 34 Identification of Ly6SF proteins and Alignment of Sequences ................................. 34 Phylogenetic Inference ................................................................................................ 42 Lynx Subfamily of Proteins...................................................................................... 43 vi Exon-Intron Structures of Ly6SF genes ................................................................... 46 CHAPTER 3: ...............................................................................................50 Expression and Functional Analysis of Lynx1 Homologues ...................50 Summary....................................................................................................................... 51 Expression Analysis of Lynx1 Homologues ............................................................... 52 Database analysis for expression of human and mouse Ly6 genes .......................... 52 RT-PCR Analysis of Ly6 Genes and Northern Blots ............................................... 56 BAC Transgenic Mice Show Expression Patterns of Lynx Homologues ................ 61 Western Blot Analysis .............................................................................................. 78 Lynx2 is a membrane protein ................................................................................... 82 Lynx2 is a membrane protein ................................................................................... 83 Immunohistochemistry ............................................................................................. 83 Functional Analysis of Lynx1 homologues ................................................................ 87 Co-immunoprecipitation analysis ............................................................................. 87 Electrophysiology ..................................................................................................... 92 CHAPTER 4: ...............................................................................................95 Gene-targeted Deletion Analysis for Lynx2 and Lynx3 ..........................95 Summary......................................................................................................................