Molecular dissection of the functional specificity of glycophosphatidylinositol anchors By Thomas B. Nicholson Department of Biochemistry McGill University, Montreal September 2007 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Thomas B. Nicholson 2007 Thesis Abstract Carcinoembryonic antigen (CEA) is a cell surface protein attached to the membrane by a glycophosphatidylinositol (GPI) anchor, a common modification of cell surface proteins. CEA is overexpressed in many human cancers, and plays a role in tumor progression through its ability to activate certain integrins, thereby blocking cellular differentiation, inhibiting anoikis, and disrupting normal tissue architecture. Previous work established that the CEA GPI anchor contains important and specific information directing these functions, which served as the basis for an investigation of the underlying mechanisms involved. The ability of the GPI anchor to determine protein function was examined using a chimera that consisted of the CEA GPI anchor attached to neural cell adhesion molecule (NCAM) self-adhesive external domains; this chimera, NCB, possessed CEA-like, rather than NCAM-like, functions. The CEA anchor targets the protein to specific domains on the cell surface, resulting in an association with specific signaling molecules. This targeting was employed to modify CEA function, as the presence of a protein with non-functional external domains but the same anchor led to a complete and specific loss of biological function of the CEA-like protein. GPI anchor addition is determined by a specific carboxy-terminal signal sequence, which we hypothesized contained information directing the addition of a particular GPI anchor with functional specificity. To identify this signal, chimeras were generated exchanging amino acids in this signal sequence between CEA and NCAM, a protein with different functional properties. A stretch of 6 amino acids within the signal sequence was found to be necessary and sufficient for the addition of the CEA-specific anchor. Since this region is well conserved, but not identical, in the CEA family members CC6 and CC7, we examined whether these proteins were also attached to the same GPI anchors. Surprisingly, while the anchors of these proteins are functionally equivalent to that of CEA, they are not completely identical. This work therefore explores the molecular basis for functional specificity of GPI anchors, demonstrating how specificity of GPI-anchored proteins is determined and the resulting functional consequences, while offering a novel method to inhibit the function of proteins with this type of anchorage. i Résumé de Thèse L’antigène carcinoembryonnaire humain (CEA) est un membre d’une famille de protéines de surface cellulaire fixées à la membrane par un ancrage glycophosphatidylinositol (GPI), une modification commune des protéines de la membrane plasmique. CEA est surexprimé dans plusieurs cancers humains et joue un rôle dans la progression tumorale par sa capacité à activer certaines intégrines, bloquant de ce fait la différentiation cellulaire et l’anoikis, et perturbant l’architecture tissulaire normale. Plusieurs recherches antérieures ont établi que l’ancre GPI de CEA contient de l’information importante et spécifique dirigeant ces fonctions. Ces travaux ont servi de base pour l’étude biologique et moléculaire des mécanismes impliqués. Afin de démontrer les fonctions biologiques de CEA, nous avons étudié les capacités de l’ancre GPI à modifier la fonction des protéines en utilisant une protéine hybride (NCB) composée de l’ancre GPI de CEA attachée aux domaines externes auto-adhésifs de la molécule d’adhésion cellulaire neuronale (NCAM). La protéine chimérique NCB possède des fonctions similaires à CEA. L’ancre de CEA cible la protéine à des domaines spécifiques de la surface cellulaire, ce qui mène à l’association de la protéine avec des éléments de signalisation spécifiques. Ce ciblage a été utilisé pour modifier la fonction de CEA, car la présence d’une protéine dont les domaines extracellulaires sont non fonctionnels, mais dont l’ancre est la même, a causé la perte complète et spécifique des fonctions biologiques de NCB. L’ajout d’ancres GPI est déterminé par une séquence spécifique située à l’extrémité carboxyle-terminale. Nous avons présumé que cette séquence contiendrait l’information nécessaire à l’addition sélective d’une ancre GPI de fonction spécifique. Afin d’identifier ce signal, des protéines hybrides ont été produites en échangeant des acides aminés entre CEA et NCAM, deux protéines de fonctions distinctes. La caractérisation de ces hybrides a démontré que l’ajout de l’ancre spécifique de CEA est déterminé par une séquence nécessaire et suffisante de 6 acides aminés. Puisque cette région est bien conservée, mais non identique, chez les membres CC6 et CC7 de la famille CEA, nous avons mené des recherches pour déterminer si ces protéines sont également fixées à la même ancre GPI. Quoique les ancres de ces protéines soient équivalentes à celle de CEA en termes de fonction, elles ne sont pas complètement identiques. Cette thèse présente de nouvelles informations sur la spécificité fonctionnelle des ancres GPI, en démontrant comment leur spécificité est déterminée. ii Ce travail discute les conséquences fonctionnelles des ancres GPI et présente une nouvelle méthode pour altérer la fonction de protéines associées à ce type d’ancrage. iii Table of Contents Abstract i Résumé de Thèse ii Table of contents iv List of figures vii List of Tables x Acknowledgements xi Abbreviations xiii Chapter 1: The specificity of the GPI anchor of CEA 1 1. General Introduction 2 2. The Carcinoembryonic antigen family 3 2.1 Expression of CEA family members 3 2.2 Structure of CEA family proteins 5 2.3 The CEA family in other organisms 7 3. In vitro functions of CEA family Members 8 3.1 Intercellular adhesion 8 3.2 Cellular differentiation 10 3.3 Apoptosis 11 3.4 Tissue architecture 12 3.5 Signaling 13 3.6 Importance of the membrane anchor of CEA family members 14 3.7 Other functions 15 4. The CEA family in cancer 17 4.1 Expression pattern in cancers 17 4.2 Clinical relevance of CEA 19 iv 4.3 CEA as a cancer target 20 5. CEA: in vivo studies 21 5.1 Tissue implantation studies 21 5.2 CEA transgenic mice 21 6. The plasma membrane 23 6.1 Plasma membrane composition 23 6.2 Membrane rafts 24 6.3 Raft heterogeneity 27 6.4 Functions of rafts 28 6.5 Other membrane domains 29 7. GPI-anchored proteins 32 7.1 GPI anchors 32 7.2 GPI anchor signal sequence 33 7.3 GPI anchor addition to preproteins 36 7.4 GPI anchor structure 37 7.5 Functional consequences of GPI anchorage 39 7.6 GPI anchor heterogeneity 40 7.7 GPI-anchored proteins and disease 41 8. Scope of the current work 42 Publications status of the research chapters presented in this thesis 44 Contribution of authors 44 Preface to Chapter 2 45 Chapter 2: Specific inhibition of GPI-anchored protein function by homing and self-association of specific GPI anchors 46 v Addendum to Chapter 2 79 Preface to Chapter 3 81 Chapter 3: Identification of a novel functional specificity signal within the GPI anchor signal sequence of carcinoembryonic antigen 82 Preface to Chapter 4 103 Chapter 4: Exploring the biological properties of the GPI anchors of CEACAM6 and CEACAM7 104 Chapter 5: General discussion 128 Preface to the Research Appendix: 147 Research Appendix: The GPI anchor of CEA mediates external membrane incorporation (“painting”) 148 Original contributions to knowledge 160 Bibliography 161 Appendices 196 vi List of Figures Chapter 1 Figure 1 Structure of the human CEA family 6 Figure 2 The composition of membrane rafts 25 Figure 3 Type of membrane domains formed in plasma membrane 30 Figure 4 The GPI anchor signal sequence 35 Figure 5 GPI anchor biosynthesis 37 Figure 6 The structure of the GPI anchor 38 Chapter 2 Figure 1 Surface expression of CEA and NCAM proteins on L6 myoblasts 56 Figure 2 ∆NCEA exists in close proximity to NCB but not to NCAM 57 Figure 3 ∆NCEA restores differentiation to NCB- expressing cells 61 Figure 4 Increased ECM binding by NCB transfectants is lost in the presence of ∆NCEA 63 Figure 5 NCB membrane raft association is unaltered in the presence of ∆NCEA 65 Figure 6 ∆NCEA interferes with NCB-mediated intercellular adhesion 69 Figure 7 ∆NCEA increases the size of NCB-containing rafts 71 Figure 8 Antibody crosslinking restores integrin activation 73 vii Addendum to Chapter 2 Figure 1 Effect of targeting the GPI anchor on anoikis resistance 80 Chapter 3 Figure 1 Reducing the CEA-derived sequence in NCB 91 Figure 2 Replacing 5 amino acid stretches in the GPI anchor signal sequence of CEA 94 Figure 3 Introducing shorter NCAM sequences into the CEA sequence of 1C 95 Figure 4 Inserting 5 amino acid sequences into NCAM is insufficient to create a protein with CEA-like properties 97 Figure 5 The upstream proline is required to give CEA-like properties 98 Figure 6 Scrambling the amino acid sequence in the identified key region 100 Chapter 4 Figure 1 Effect of ∆NCEA on the differentiation of L6 transfectants expressing CEA 113 Figure 2 Differentiation under co-culture conditions of the co-expressing transfectants 115 Figure 3 Characterization of the NCAM chimeras with the GPI anchors of CC6 and CC7 117 Figure 4 Integrin modulation by N6 and N7 chimeras 119 Figure 5 The effect of N6 and N7 on differentiation 121 viii General discussion Figure 1 Schematic of shank-defective and shank-less anchors 142 Figure