Expression and characterization of protocadherin LKC in mammalian cells

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1 Aus dem Institut für Physiologische Chemie der Tierärztlichen Hochschule Hannover Expression and characterization of protocadherin LKC in mammalian cells INAUGURAL-DISSERTATION zur Erlangung des Grades eines DOKTORS DER VETERINÄRMEDIZIN (Dr. med. vet.) durch die Tierärztliche Hochschule Hannover Vorgelegt von Michael Peter Rolf Krahn aus Everswinkel Hannover 2006

2 TABLE OF CONTENTS 1 INTRODUCTION 11 2 REVIEW OF THE LITERATURE THE EPITHELIA - GAIN AND MAINTENANCE OF POLARITY CELL JUNCTIONS CADHERINS IN CELL CONTACTS Diversity of the cadherin superfamily INTRACELLULAR SIGNALLING OF CADHERINS Classical cadherins Protocadherin signaling CADHERINS IN DISEASES Mutations in genes coding for cadherins Autoimmune diseases Implications of cadherins in carcinogenesis PROTOCADHERIN LKC 38 3 MATERIALS AND METHODS MATERIALS Chemical reagents Media and Solutions Antibodies Hostbacteria Plasmids Mammalian cell lines METHODS Semiquantitative PCR of mrna Isolation of mrna Reverse transcriptase PCR Competitive PCR Construction of cdna Clones AmplificationofcDNAfragment Subcloning of the PLKC-PCR-fragment in Topo TA2.1 vector Transformation of E.coli Isolation of plasmid DNA from E.coli Identification of positive clones byfragment restriction analysis Lange scale DNA preparation 47

3 Cloning of PLKC into the eukaryotic expression vector peyfp-n Cloning of PLKC into pcdna 3.1 myc-his vector Construction of domain deletion mutants Cellculture Media Cell culture procedures Transient transfection Establishment of stable cell lines Isolation and detection of proteins Immunoprecipitation Cell surface trypsin digestion Western blot Cell surface biotinylation Chemical cross linking of cell surface proteins Immunofluorescence Confocal laser microscopy Isolation of cytoskeleton-associated proteins and inhibition of actin polymerization Extraction of detergent resistant lipid microdomains Density gradient centrifugation Cell aggregation assay Two-dimensional polyacrylamide gel electrophoresis 67 RESULTS PLKC IS UPREGULATED DURING CELL DEVELOPMENT EXPRESSION AND SUBCELLULAR DISTRIBUTION IN MAMMALIAN CELLS Expression in different cell lines Redistribution of PLKC Compartment cell surface biotinylation PLKC is localized neither in adherens junctions nor in zonula occludens Association with the actin cytoskeleton Influence of calcium on subcellular distribution of PLKC Biochemical analysis of PLKC Biosynthesis and intracellular transport Effect of glycosylation on protein transport and cell surface expression Trypsin-sensitivity assay of cell surface proteins Expression of PLKC in glycosylation deficient CHO/Lec cells Biochemical analysis Analysis of subcellular distribution by confocal laser microscopy Dimerization of PLKC Seperationofcross-linkedPLKCmoleculesbySDS-PAGE 96

4 Identification of dimeric PLKC by sucrose density gradient centrifugation Determination of the structural type of PLKC-dimers Association with lipid microdomains FUNCTIONAL ASPECTS OF PLKC EXPRESSION Altered cell morphology of CHO cells Transepithelial resistance Cell proliferation assay Cell aggregation competency, Knockingdown PLKC by small interfering RNA INFLUENCE OF DIFFERENT DOMAINS ON TRANSPORT AND FUNCTION OF PLKC Deletion of extracellular cadherin repeats Roleof the PDZ-binding Site in transport and subcellular distribution Deletion of the cytoplasmic tail Deletion of the transmembrane domain D-PAGE ANALYSIS OF POTENTIAL INTRACELLULAR INTERACTION PARTNERS OF PLKC 128 DISCUSSION MARKERS OF POLARIZATION ARE UPREGULATED DURING CELL DIFFERENTIATION SUBCELLULAR DISTRIBUTION OF PLKC AND OTHER CADHERINS Role of PDZ binding sequences in protein transport Function of the cytoplasmic tail of transmembrane proteins The transmembrane domain Biosynthesis and transport kinetics The role of actin cytoskeleton in transport and localization of cadherins INFLUENCE OF GLYCOSYLATION ON TRANSPORT AND FUNCTION OLIGOMERIZATION OF MEMBERS OF THE CADHERIN FAMILY Expression and function of oligomeric protein complexes Oligomerization of cadherins mediates cell adhesion Calcium ions facilitate dimerization and function of cadherins DETERGENT RESISTANT MICRODOMAINS PLKC INVOLVED IN CELL-CELL CONTACTS Cell aggregation Alteration of cell morphology Influence of calcium on PLKC expression and adhesion activity Löss of function experiments IDENTIFICATION NEW POTENTIAL INTRACELLULAR INTERACTING PROTEINS 163 SUMMARY 167

5 7 ZUSAMMENFASSUNG REFERENCES APPENDIX INDEX OFFIGURES INDEX OFTABLES ACKNOWLEDGEMENTS 200

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