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Role of Patched-1 Intracellular Domains in Canonical and Non-Canonical Hedgehog Signalling Events

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Role of Patched-1 Intracellular Domains in Canonical and Non-Canonical Hedgehog Signalling Events Role of Patched-1 Intracellular Domains in Canonical and Non-Canonical Hedgehog Signalling Events by Malcolm Harvey A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Laboratory Medicine & Pathobiology University of Toronto © Copyright by Malcolm Harvey 2013 Role of Patched-1 Intracellular Domains in Canonical and Non- Canonical Hedgehog Signalling Events Malcolm Harvey Master of Science Department of Laboratory Medicine & Pathobiology University of Toronto 2013 Abstract Patched-1 (Ptch1) is the primary receptor for Hedgehog (Hh) ligands and mediates both canonical and non-canonical Hh signalling. Previously, our lab identified that mice possessing a Ptch1 C-terminal truncation display blocked mammary gland development at puberty that is overcome by overexpression of activated c-src. Testing the hypothesis that this involves a direct interaction between Ptch1 and c-src, we identified through co-immunoprecipitation that Ptch1 and c-src associate in an Hh-dependent manner, and that the Ptch1 C-terminus regulates activation of c-src in response to Hh ligand. Since the effects of Ptch1 intracellular domain deletions on canonical Hh signalling are ill-defined, we assayed this through luciferase reporter assays and qRT-PCR. Transient assays revealed that the Ptch1 middle intracellular loop is required for response to ligand, while qRT-PCR from primary cells showed that C-terminal truncation impairs canonical Ptch1 function. Together, this indicates that the intracellular domains of Ptch1 mediate distinct canonical and non-canonical functions. ii Acknowledgments First, I would like to thank my supervisor Dr. Paul A. Hamel for offering me the opportunity to pursue graduate studies in his lab and for his guidance, advice, support, and patience along the way. I also give my sincere thanks to Drs. Stephane Angers and Reinhart Reithmeier for being a part of my advisory committee and contributing their valuable insight, suggestions, and time. Additionally, I would like to thank Drs. Jorge Filmus and Herman Yeger for agreeing to participate in my thesis defense. I would also like to thank present and former Hamel lab members Dr. Laurent Balenci, Dr. Hong Chang, Andrew Fleet, Melissa Hicuburundi, Jennifer Lee, Nadia Okolowsky, and Aaliya Tamachi for their assistance, friendship, and kindness. I am especially grateful to Aaliya for assisting with cloning, Nadia for assisting with primary cell isolation, and both of them for always being there through the many ups and downs. And most importantly, I would like to thank my family for their endless love and support. Assuming this thesis is defended successfully, I may at last have an answer to the question of "When are you coming home!?" iii Table of Contents ABSTRACT .................................................................................................................................. II ACKNOWLEDGEMENTS ....................................................................................................... III TABLE OF CONTENTS ........................................................................................................... IV LIST OF FIGURES .................................................................................................................... VI ABBREVIATION KEY ............................................................................................................ VII INTRODUCTION ......................................................................................................................... 1 1 THE CANONICAL HEDGEHOG SIGNALLING PATHWAY .......................................... 1 1.1 LIGAND SYNTHESIS AND SECRETION ..................................................................................... 2 1.2 LIGAND RECEPTION ..................................................................................................................... 4 1.3 SIGNAL TRANSDUCION THROUGH SMOOTHENED .............................................................. 7 1.4 REGULATION OF GLI-MEDIATED TRANSCRIPTION ........................................................... 10 1.5 TRANSCRIPTION OF HEDGEHOG TARGET GENES .............................................................. 14 2 PTCH1 STRUCTURE AND FUNCTION ............................................................................. 14 2.1 MOLECULAR CHARACTERIZATION ....................................................................................... 14 2.1.1 The sterol sensing domain ...................................................................................................... 15 2.1.2 Ptch1 splice variants .............................................................................................................. 16 2.1.3 Function of the Ptch1 C-terminus .......................................................................................... 17 2.2 MECHANISM OF SMOOTHENED INHIBITION ....................................................................... 18 2.3 EFFECT OF PTCH1 MUTATION IN NEOPLASIA AND DEVELOPMENTAL DISORDER .. 20 2.3.1 Nevoid basal cell carcinoma syndrome .................................................................................. 20 2.3.2 Basal cell carcinoma .............................................................................................................. 22 2.3.3 Medulloblastoma .................................................................................................................... 24 2.3.4 Holoprosencephaly ................................................................................................................. 25 2.4 RELATION TO PTCH2 .................................................................................................................. 26 3 NON-CANONICAL HEDGEHOG SIGNALLING ............................................................. 27 iv 3.1 NON-CANONICAL HEDGEHOG SIGNALLING EVENTS ACTING THROUGH PTCH1 ..... 28 3.2 IDENTIFICATION OF A NOVEL PTCH1-MEDIATED SIGNALLING CASCADE INVOLVING C-SRC ...................................................................................................................... 30 RATIONALE .............................................................................................................................. 33 HYPOTHESIS & OBJECTIVES .............................................................................................. 33 MATERIALS & METHODS ..................................................................................................... 34 CELL CULTURE ........................................................................................................................................ 34 PRIMARY CELL CULTURE .................................................................................................................... 34 CLONING & EXPRESSION CONSTRUCTS ........................................................................................... 35 WESTERN BLOTTING AND IMMUNOPRECIPITATION .................................................................... 35 GLYCOSIDASE TREATMENT ................................................................................................................ 36 PREPARATION OF SHH-CONDITIONED MEDIA ................................................................................ 36 LUCIFERASE ASSAYS............................................................................................................................. 37 RT-PCR AND qRT-PCR ............................................................................................................................ 38 IMMUNOFLUORESCENCE AND IMAGING ......................................................................................... 39 IN VITRO BINDING ASSAYS ................................................................................................................... 39 RESULTS .................................................................................................................................... 41 DELETION MUTANT ANALYSIS OF THE PTCH1-C-SRC INTERACTION ...................................... 41 EFFECT OF HEDGEHOG LIGAND STIMULATION ON THE PTCH1-C-SRC INTERACTION ........ 45 DIFFERENTIAL EFFECTS OF PTCH1 AND THE MES MUTANT ON SIGNAL TRANSDUCTION CASCADES ................................................................................................................................................ 47 CANONICAL HEDGEHOG SIGNALLING CONSEQUENCES OF PTCH1 INTRACELLULAR DOMAIN DELETIONS .............................................................................................................................. 48 DISCUSSION .............................................................................................................................. 55 EXOGENOUS PTCH1 AND C-SRC INTERACT IN VITRO ................................................................... 55 MODULATION OF THE PTCH1-C-SRC ASSOCIATION IN RESPONSE TO SHH LIGAND ............ 58 THE MES ALLELE IS HYPOMORPHIC IN MAMMARY FIBROBLASTS .......................................... 59 LOSS OF THE PTCH1 MIDDLE INTRACELLULAR LOOP RESULTS IN AN INABILITY TO RESPOND TO SHH STIMULATION ....................................................................................................... 62 CONCLUSIONS ......................................................................................................................................... 64 v REFERENCES ...........................................................................................................................
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