CONTEXT SPECIFIC SIGNALING OF TGF-β RECEPTOR II DISSERTATION ZUR ERLANGUNG DES AKADEMISCHEN GRADES DES DOKTORS DER NATURWISSENSCHAFTEN (DR. RER. NAT.) EINGEREICHT IM FACHBEREICH BIOLOGIE,CHEMIE,PHARMAZIE DER FREIEN UNIVERSITÄT BERLIN vorgelegt von DANIEL WALTER HORBELT AUS ERLENBACH AM MAIN 2010 Diese Arbeit wurde von Oktober 2004 bis August 2010 in der Arbeitsgruppe von Prof. Petra Knaus am Institut für Chemie-Biochemie der Freien Universität Berlin angefertigt. 1. GUTACHTER:PROF.DR.PETRA KNAUS 2. GUTACHTER: PD DR.PETER N. ROBINSON Disputation am 12.11.2010 . Contents List of Figures V List of Tables VII Summary IX Zusammenfassung XI 1 Introduction 1 1.1 The TGF-β superfamily . .1 1.2 Mechanisms of TGF-β signaling . .3 1.2.1 Secretion and activation of TGF-β ...................3 1.2.2 Ligand binding and receptor activation of TGF-β receptors . .3 1.2.3 TGF-β isoform specificity . .6 1.2.4 The TGF-β receptor II isoform TβRII-B and aim of the project: "Antibodies against TβRII-B" . .8 1.2.5 Activation of Smad signaling by TGF-β receptors . .9 1.2.6 TGF-β signaling through Smads . 12 1.2.7 Non-Smad signaling by TGF-β ..................... 13 1.2.8 Endocytic regulation of TGF-β signaling . 15 1.3 Marfan Syndrome and MFS-related diseases . 17 1.3.1 Classical Marfan Syndrome . 17 1.3.2 Marfan syndrome type II, Loeys-Dietz syndrome and Thoracic Aortic Aneurysms and Dissections syndrome . 19 1.4 Pathological remodeling in the aortic media and the development of aortic aneurysms . 21 1.5 Role of TGF-β in vascular remodeling . 23 1.6 State of research on TGF-β type II receptor mutations and aim of the project: "TGFBR2 mutations in MFS related diseases" . 25 2 Materials and Methods 27 2.1 Molecular biology . 27 2.1.1 Cloning, mutagenesis, plasmid preparation . 27 I 2.1.2 RNA preparation, reverse transcription . 27 2.1.3 Quantitative RT-PCR . 28 2.2 Protein biochemistry . 29 2.2.1 SDS-PAGE and Western blotting . 29 2.2.2 Protein quantification using the BCA protein assay . 30 2.2.3 Immunoprecipitation . 31 2.2.4 ELISA . 31 2.2.5 In-vitro kinase assays . 32 2.2.6 Antibody Generation . 33 2.2.6.1 Affinity purification . 33 2.2.6.2 AlexaFluor labeling . 33 2.3 Cell biology and cell based assays . 34 2.3.1 Cell Culture . 34 2.3.1.1 Maintenance of cell lines . 34 2.3.1.2 Transfection of cell lines . 34 2.3.1.3 Hybridoma cell culture and subcloning . 35 2.3.2 Luciferase reporter gene assays . 35 2.3.3 Subcellular fractionation . 36 2.3.4 Immunocytochemistry . 36 2.3.4.1 Surface staining . 36 2.3.4.2 Smad nuclear translocation . 37 2.3.5 Phosphoprotein assays . 37 2.3.6 Flow cytometry . 38 2.3.6.1 Receptor expression and internalization . 38 2.3.6.2 Apoptosis and cell cycle flow cytometry . 38 3 Results: TGFBR2 mutations in MFS related diseases 41 3.1 Mutations in the TGF-β receptor II kinase domain . 41 3.2 Expression of mutant receptors . 41 3.3 Receptor internalization . 44 3.4 Autokinase activity of mutated receptors . 46 3.5 Reporter gene activation by TβRII mutants . 48 3.6 Activation of Smad- and non-Smad pathways . 56 3.7 TGF-β induced cell cycle arrest . 56 4 Discussion: TGFBR2 mutations in MFS related diseases 61 4.1 TGFBR mutations in MFS and related diseases . 61 4.2 Differential expression of mutant TGF-β receptors . 61 4.3 Internalization of mutant TGF-β receptors . 63 II 4.4 TGF-β pathway activity of TβRII mutants . 66 4.5 Dominant negative effect of mutant TβRII . 67 4.6 Smad and MAPK signaling of TβRII mutants . 68 4.7 TGF-β induced cell cycle arrest . 70 4.8 Residual signaling activity by R460C . 70 4.9 Correlation of signaling activities with disease phenotypes . 73 4.10 Paradoxal activation of TGF-β signaling . 74 4.11 Conclusions . 74 5 Results: Antibodies against TβRII-B 77 5.1 Generation and selection of hybridoma clones . 77 5.2 Purification and evaluation of monoclonal antibodies . 79 5.3 Anti-TβRII-B mAbs 4D9-s8 and 4B4 in immunological applications . 79 5.3.1 Anti-TβRII-B mAb in immunoprecipitation and Western blot . 79 5.3.2 Anti-TβRII-B in flow cytometry and immunocytochemistry . 83 5.4 Inhibitory potential of anti-TβRII-B antibodies . 83 5.4.1 Inhibition of TβRI:TβRII-B heterocomplex formation . 83 5.4.2 Inhibition of Smad nuclear translocation . 85 5.4.3 Repression of Smad transcriptional activity . 86 6 Discussion: Antibodies against TβRII-B 89 6.1 Antibodies against TβRII-B: previous approaches . 89 6.2 Purification and evaluation of TβRII-B mAbs . 89 6.3 Inhibitory potential of TβRII-B mAbs . 90 6.4 Implications for binding of TGF-β to TβRII-B . 91 7 Side Project: The TβRII interactome 95 Bibliography 101 Abbreviations 127 Appendix 130 A Nucleotide sequences and plasmid charts 131 A.1 Plasmid charts . 131 A.2 Mutagenesis primer sequences . 134 A.3 qPCR Primer sequences . 135 B In-silico analysis results for the TβRII-B insert 137 B.1 Results from PSIPRED . 137 III B.2 Results from PredictProtein . 139 Statement of authorship 143 Publications 145 Acknowledgements 149 IV List of Figures 1 Pathways in TGF-β signaling . .2 2 Structure of the TGF-β receptor signaling complex . .5 3 Binding of TGF-β1 to TβRII . .7 4 N-terminal peptide sequences of TβRII and TβRII-B . .8 5 Composition of the aortic wall . 22 6 TGF-β control of vascular remodeling . 24 7 Gating strategy for internalization measurements . 39 8 Mapping of mutations in the kinase domain . 42 9 Amino acid sequence alignment for human TGF-β superfamily type I and type II receptors . 43 10 Expression of TβRII mutants . 44 11 Surface expression of TβRII mutants . 45 12 Internalization of wildtype TβRII . 46 13 Endocytosis of wildtype and mutant TβRII . 47 14 Kinase activity of mutated TβRII. 48 15 Effect of dominant negative mutations of TβRII on reporter gene activity in HEK293T cells . 49 16 Smad2 phosphorylation and translocation induced by R528C . 49 17 Smad2 nuclear translocation induced by R528C . 50 18 Effect of dominant negative mutations of TβRII on reporter gene activity in DR26 cells . 51 19 Induction of 3TP-luc and (CAGA)12-luc reporters after titration of wildtype versus R537C or R537P plasmids . 52 20 Induction of 3TP-luc and (CAGA)12-luc reporters after titration of wildtype versus R528H or R528C plasmids . 53 21 Induction of 3TP-luc and (CAGA)12-luc reporters after titration of wildtype versus R460H or R460C plasmids . 54 22 Signaling activity of mutants L308P, Y336N, and S449F . 55 23 Activation of Smad and ERK pathways by TβRII mutations . 57 24 Activation of the p38-MAPK pathway by TβRII mutations . 58 25 Proliferation arrest induced by TβRII mutations . 59 V 26 Clinical manifestations of familial TAAD, MFS2 and LDS . 62 27 Structure of the catalytic subunit of PKA . 65 28 TβRII kinase structure with mutants of R460 . 71 29 Proposed model for the correlation of disease phenotypes with Smad and ERK signaling activities . 76 30 Binding specificity of supernatants from hybridoma clones and subclones 78 31 Affintiy purification of TβRII-B antibodies . 80 32 Binding of purified α-TβRII-B mAb . 81 33 Immunoprecipitation and Western blot with 4D9-s8 and 4B4 mAb . 82 34 Flow cytometry using the 4B4 mAb . 84 35 Immunocytochemistry using the 4B4 mAb . 85 36 Inhibition of TβRI:TβRII-B heterocomplex formation . 86 37 Inhibition of Smad nuclear translokation in L6 cells . 87 38 Inhibition of TGF-β induced reporter gene activity . 88 39 Spatial dimensions in the TGF-β receptor signaling complex . 93 40 FAF1 domain structure . 96 41 Overexpression of FAF1 represses TGF-β signaling . 96 42 Overexpression of FAF1 represses TβRII signaling . 97 43 Knockdown of FAF1 enhances TGF-β responsiveness . 98 44 TβRII surface levels are not affected by FAF1 overexpression . 99 VI List of Tables 1 Reported clinical phenotypes of patients with the TGFBR2 mutations stud- ied in this work . 21 2 Primary antibodies for Western blotting . 30 3 Quantitative differences of the nine TGFBR2 mutations studied in this work 75 4 Hybridoma clones screening results . 77 5 Antibody.