The Involvement of Rho GTPases in Plexin Mediated Signal Transduction Laura Turner A thesis submitted to the University of London for the degree of Doctor of Philosophy November 2003 MRC Laboratory for Molecular Cell Biology University of London Gower Street London WCIE 6BT ProQuest Number: U642461 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642461 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Plexins are a family of conserved transmembrane proteins. Together with neuropilins, they act as receptors for the semaphorin family of growth cone guidance molecules. Whilst it is clear that guidance involves cytoskeletal changes, little is known of the mechanisms via which plexins regulate growth cone morphology. Hints suggesting the involvement of Rho GTPases stem from observations of localised actin rearrangements elicited by plexins and semaphorins. This feeling is consolidated by the demonstration of the Rac dependent nature of plexin induced cellular responses. To investigate plexin mediated signal transduction, a heterologous assay for semaphorin induced collapse has been developed and characterised. Studies using jasplakinolide indicate that Sema3A-Fc induced collapse requires actin dissassembly. Interestingly, a similar collapsed phenotype is observed in cells treated with latrunculin A. It is possible that Sema3A-Fc and latrunculin A may utilise comparable intracellular machinery to achieve similar morphological outcomes. Experiments using GTPase mutants demonstrated that Sema3A-Fc induced collapse required Rac and Cdc42, but not Rho A. In addition, Sema3A-Fc stimulation led to Rac activation prior to morphological collapse. Interestingly, collapse induced by constitutively active Plexin-A1 did not require Rac. This suggests that Rac may act upstream of Plexin-Al, perhaps regulating the activity of Plexin-Al itself. Evidence that Plexin-Al interacts directly with Rac.GTP supports this theory. A putative inter- or intramolecular interaction was identified within the cytoplasmic tails of Plexin-Al and Plexin-B 1. As the critical residues required for these interactions are also essential for Rac binding, these interactions and the association between Rac and plexin may be mutually exclusive. Finally, the cytoplasmic tail of Plexin-Al was shown to dimerise, but antibody mediated clustering of Plexin-Al was not sufficient to induce morphological collapse. In the light of these results, Rac may regulate plexin activity by modulating either conformational changes or receptor aggregation states in response to semaphorin stimulation. CONTENTS ABSTRACT........................................................................................................................... 2 CONTENTS...........................................................................................................................3 LIST OF FIGURES.............................................................................................................10 LIST OF TABLES...............................................................................................................13 CHAPTER 1 - INTRODUCTION................................................................................... 14 1.1 INTRODUCTION..................................................................................................... 15 1.2 PLEXINS, SEMAPHORINS AND NEUROPILINS.............................................19 1.2.1 The discovery of semaphorins ....................................................... 19 1.2.2 The identification of plexins ...........................................................19 1.2.3 Semaphorins as ligands for plexin receptors ................................ 20 1.2.4 Neuropilin - a co-receptor for plexins ...........................................20 1.2.5 Molecular characteristics of the semaphorin superfamily ........... 22 1.3 EXPRESSION AND FUNCTION........................................................................... 26 1.3.1 The nervous system ........................................................................ 26 1.3.2 The immune system........................................................................ 29 1.3.3 The vascular system .......................................................................30 1.3.4 Developmental patterning..............................................................32 1.4 SENSING THE ENVIRONMENT.......................................................................... 32 1.5 THE ROLE OF THE CYTOSKELETON.............................................................. 33 1.5.1 The growth cone cytoskeleton ....................................................... 34 1.5.2 Actin binding proteins ....................................................................35 1.5.3 Changing direction ......................................................................... 36 1.6 SEMAPHORIN INDUCED SIGNAL TRANSDUCTION................................... 38 1.6.1 Rho GTPases...................................................................................38 1.6.2 Rho GTPases are essential for semaphorin signalling ................ 44 1.6.3 The semaphorin receptor eomplex ................................................ 52 1.6.4 Integrins ........................................................................................... 52 1.6.5 The immunoglobulin superfamily of cell adhesion molecules... .54 1.6.6 Tyrosine kinases ..............................................................................56 1.6.7 MICAL (molecule interacting with cas ligand) ............................ 59 1.6.8 LIM kinase ....................................................................................... 60 1.6.9 CRMPs (collapsin response mediator proteins) ........................... 61 1.6.10 GSR-3 (glycogen synthase kinase-3) ............................................62 1.6.11 Bi-directional signalling .................................................................63 1.7 THE REGULATION OF SEMAPHORIN ACTIVITY ..........................................64 1.7.1 Transcriptional regulation ..............................................................65 1.7.2 Protein translation and degradation ...............................................66 1.7.3 Endocytosis ...................................................................................... 67 1.7.4 Receptor autoinhibition ..................................................................69 1.7.5 Proteolysis .......................................................................................70 1.7.6 Receptor oligomerisation ................................................................72 1.7.7 Combinatorial complexes ...............................................................72 1.7.8 Cyclic nucleotides ...........................................................................73 1.8 SEMAPHORINS AND DISEASE ........................................................................... 74 1.8.1 Cancer ...............................................................................................75 1.8.2 Disorders of the nervous system .................................................... 76 1.8.3 Regeneration following neuronal injury ........................................76 1.9 CONCLUSION..........................................................................................................77 CHAPTER 2 - MATERIALS AND METHODS.......................................................... 80 2.1 MOLECULAR BIOLOGY......................................................................................81 2.1.1 Polymerase chain reaction ............................................................. 81 2.1.2 Proteinase K digestion of PGR products ...................................... 81 2.1.3 Restriction digests ..........................................................................81 2.1.4 Agarose gel electrophoresis ........................................................... 82 2.1.5 Extraction of DNA from agarose gels .......................................... 82 2.1.6 Ligations ..........................................................................................82 2.1.7 Preparation of CaCli competent Esherichia coli ...........................82 2.1.8 Transformation of E. coli by ‘heat shock’....................................83 2.1.9 Bacterial DNA mini preps............................................................. 83 2.1.10 Bacterial DNA maxi preps............................................................ 84 2.1.11 DNA quantitation ...........................................................................84 2.1.12 Site directed mutagenesis ...............................................................84 2.1.13 DNA constructs ..............................................................................84 2.1.14 DNA sequencing ............................................................................88 2.2 CELL BIOLOGY....................................................................................................