Cloning of Receptor Tyrosine Kinases From ANvsia Jonathan Hislop Neurology and Neurosurgery, McGill University, Montreal. August, 1998 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Master of Science @Jonathan Hislop, 1998 National Library Biblioth$ue nationale du Cana a Acquisitions and Acquisitions et Bibbgraphic Semices m~icesbibliographiques 395 Wdlngton Street 395, rue Weilinglori OItawaON K1AW OnawaON K1AON4 CariPds Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence ailowing the exclusive permettant a la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in rnicrofonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substaatial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othenirise de celle-ci ne doivent être imprimes reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract I have cloned two putative receptor tyrosine kinases (RTKs) fiom Aplysia californica, designated Aror and Anif. Aror is homologous to RTKs fiom the Ror subfarnily. Furthemore, Aror contains an extracellular immunoglobulin-like domain, cysteine-rich region, and kringie domain. These domains are d characteristic of Ror recepton. Amf is homologous to members of the Ret and FGF receptor subfamilies of RTKs. However, Aruf does not have the extracellular domains typical of those receptors. Furthemore the degree of identity between Anif and either the Ret or FG F recepton is closer to that between members of different RTK subfamilies, han between membea of the sarne subfamily. Therefore, Amf encodes an RTK belonging to a novel subfamily related to the Ret/FGFR subfamily. J'ai cloné deux molécules qui sont de presum6s récepteurs tyrosine kinase (RTK) provenant de Aplysia cal@rrnica, et désignées Aror et Ad. Aror est homologue aw membres de la sous-famille Ror des RTK. De plus, Aror comprend un domaine extra- cellulaire apparenté aux immunoglobulines, une région riche en cystéine et un domaine kringle. Ces domaines sont tous caractéristiques des récepteurs Ror. Anif est homologue aux membres des sous-familles Ret et FGFR des RTK. Cependant, Anif n'a pas le domaine extra-cellulaire qui est typique de ces récepteurs. De plus, le degré d'identité entre Anif et l'un ou l'autre de Ret ou FGFR est plus prb du degré d'identité partagé entre les membres des diffdrentes sous-familles RTK que de celui partagé entre les membres d'une même sous-famille. Donc, Adencode un RTK appartenant à une nouvelle sous-famille reliée à celles de Ret et de FGFR, Acknowledgements 1 would like to thank my supervisor Wayne Sossin for his time and patience as a teacher. I'd also like to thank the other members of my advisory cornmittee, Phi1 Barker and David Kaplan, for their helpful comments and direction throughout this project. Thanks also go to David Ragsdale, and Peter MacPhearson for their encouragement and support. My biggest surprise about doing research has ken how much i've enjoyed workine- with everyone in our lab. John Dyer showed me how to perfom many of the procedures for the project, and was a great teacher. 1 enjoyed working with him tremendously, and listening to Monty Python with him. A number of other people €rom the lab also helped me learn various procedures, including Tony Pepio, Xiao-Tang Fan, and Stefanie Yanow. 1 had a great tirne working with al1 of them as well as others from the lab, such as Hedy Chung, Tina Vouloumanos, Darcy Scott, and Arash Nakhost. Poking fun at Arash was particularly rewarding. My family and fnends were also always supportive of me during the project. But my cat Checkers objected to this entire flair and wants lots of attention to make up for it. He also wants to try one of the Aplysia. III Statement of Originalitv The cloning of each of the receptoa discussed in this study (designated Aror, and Anif) constitute original scholmhip. Neither of these receptors have been previously cloned. 1 pexformed aii the procedures discussed in the study with the exception that most of the DNA sequencing was perfomed by our laboratory technician Xiao-Tang Fan, and by Bio S&T Inc., and The W.M. Keck Biotechnology Resource Laboratory. The rationale for the project was devised by Dr. Wayne Sossin. Table of Contents Abstract ................................................................................................................................. 1 Résumé ................................................................................................................................ II Acknow ledgements............................................................................................................. 111 Staternent of Originality ......................................................................................................iV Table of Contents ................ ... ............................................................................................. V List of Tables and Figures .......................................................................................... VI1 List of Abbreviations ......................................................................................................VI11 introduction .......................................................................................................................... 1 Receptor Tyrosine Kinases: Structure and Function ........................................................ 1 Oved Structure............................................................................................................ 1 The Extracellular Domain ..............................................................................................-3 The Kinase Domain ....................................................................................................... 3 RTK Activation and Signal Transduction .....................................................................4 RTKs Have Diverse Roles in Many Organisms ...........................................................7 RTKs in Invertebrates .................................................................................................1 O RTKs and Memory Formation in Aplysia californica .......................................... 1 O Serotonin Induces Translation and Intemediate Memory in Aplysia ........................ I 1 RTKs and Intermediate Memory in Aplysia .............................................................. 12 Rationale and Objectives ............................................................................................. 1 2 Cloning of RTKs fiom Aplysia ................................................................................. 1 3 Materials and Methods ..................................................................................................... 1 8 Construction of cDNA Library ...................................................................................... 18 PCR Based Screen and Design of Oligodegenerate bers............................................ 18 Screening for Full Length Clows ....................................................................................20 Cloning of Aplysia RTKs ................................................................................................25 Amplification of PCR Eragments ................................................................................. 25 Aror Hy bridization Screen .......................................................................................... 26 Aror encodes a putative RTK related to the Ror subfamily ....................................... 27 Anif Hybridization Screen ........................................ .....................,..........*..*,...*,.*.*....30 Anif encodes a putative RTK related to the RetIFGF receptor subfamilies ...............31 Discussion .......................................................................................................................... 45 Summary .........................................................................................................................45 Additional RTK Fragments Amplified ..........................................................................A5 Featwes of Aror ..............................................................................................................46 Extracellular Domain.................................................................................................... 46 Intracellular Domain ................................ .. ..............................................................47 Features of Amf ............................................................................................................. -49 Extraceilular Domain ....................................................................................................49 intracellular Domain ...................................................................................................-50 Future Directions ............................................................................................................51 References .........................................................................................................................