Ror1是一個具有酪胺酸激酶蛋白區塊的膜接受器，在中樞神經系統的發育具有重要的功能。我利用生物資訊的工具預測出一些膜酪胺酸激酶包括Ror1有分佈在細胞核之傾向，除了正常的細胞膜分佈。在胃癌細胞裡表現cytoplasmic Ror1並使用螢光顯微鏡去觀察Ror1的分佈，結果觀察到Ror1主要分布在細胞核的現象。 進一步在細胞裡表現不同片段的Ror1，分析出Ror1 juxtamebrane domain，胺基酸序列471-513的區域會影響Ror1進細胞核的能力。在細胞裡表現RanGTP可以增加Ror1進入細胞核的比例，反之表現突變型RanQ69L則減少Ror1的細胞核佔有率。這指出RanGTP可能扮演輸送Ror1進入細胞核的角色。為了研究Ror1在細胞核的功能，我建構了兩種FKBP-fused Ror1表現質體。一種為細胞膜指向（M-Ror1），另一種為細胞核指向 (N-Ror1)。這兩種質體送到HeLa TF細胞裡穩定表現，並且可受到Doxycycline來控制Ror1的表現，也可加入AP20187來連結FKBP domain，藉此活化orphan receptor Ror1。抽取Ror1 活化前後的細胞RNA來作人類微陣列晶片的分析，分析結果顯示M-Ror1可活化Wnt signaling ，而N-Ror1可影響細胞骨架重新分佈。觀察N-Ror1對細胞行為的影響也驗證微陣列晶片的分析結果。N-Ror1可以改變細胞骨架的分佈也會增加細胞的移動能力。希冀本論文能對未來研究者研究膜接受器進入細胞核的生理現象與功能有所貢獻。

The goal of this thesis is to investigate the phenomena, the mechanism and the function of nuclear-localized membrane receptor, Ror1. Ror1 is a receptor protein tyrosine kinase that modulates neurite growth in the central nervous system development. Following transfection, the nuclear distribution patterns of transiently expressed cytoplasmic Ror1 were observed. Serial deletion constructs were then used to map the juxtamembrane domain of Ror1 (aa_471–513) for this nuclear translocation activity. Subsequent immunofluorescence analysis by cotransfection of Ran and Ror1 implied that the nuclear translocation event of Ror1 might be mediated through the Ran pathway. The two FKBP fusion Ror1 including membrane-localized (M-Ror1) and nuclear-localized (N-Ror1) Ror1 were stably expressed under doxycycline regulation in HeLa TF cells. The whole-genome microarray expression analysis of gene expression indicated activated M-Ror1 could affect genes involved in Wnt signaling pathway, and activated N-Ror1 could regulate actin cytoskeleton. The observation of phenotype also showed that activated N-Ror1 would result in the formation of F-actin stress fiber and the increased cell motility. This study might be beneficial in future research to understand the Ror1 biological signaling pathway.

1. Schlessinger J: Cell signaling by receptor tyrosine kinases. Cell 2000, 103(2):211-225.
2. Simon MA: Receptor tyrosine kinases: specific outcomes from general signals. Cell 2000, 103(1):13-15.
3. Carpenter G: Nuclear localization and possible functions of receptor tyrosine kinases. Curr Opin Cell Biol 2003, 15(2):143-148.
4. Mukherjee S, Tessema M, Wandinger-Ness A: Vesicular trafficking of tyrosine kinase receptors and associated proteins in the regulation of signaling and vascular function. Circ Res 2006, 98(6):743-756.
5. Arnoys EJ, Wang JL: Dual localization: proteins in extracellular and intracellular compartments. Acta Histochem 2007, 109(2):89-110.
6. Hsu SC, Hung MC: Characterization of a novel tripartite nuclear localization sequence in the EGFR family. J Biol Chem 2007, 282(14):10432-10440.
7. Hung LY, Tseng JT, Lee YC, Xia W, Wang YN, Wu ML, Chuang YH, Lai CH, Chang WC: Nuclear epidermal growth factor receptor (EGFR) interacts with signal transducer and activator of transcription 5 (STAT5) in activating Aurora-A gene expression. Nucleic Acids Res 2008, 36(13):4337-4351.
8. Xie Y, Hung MC: Nuclear localization of p185neu tyrosine kinase and its association with transcriptional transactivation. Biochem Biophys Res Commun 1994, 203(3):1589-1598.
9. Giri DK, Ali-Seyed M, Li LY, Lee DF, Ling P, Bartholomeusz G, Wang SC, Hung MC: Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor. Mol Cell Biol 2005, 25(24):11005-11018.
10. Cheng CJ, Ye XC, Vakar-Lopez F, Kim J, Tu SM, Chen DT, Navone NM, Yu-Lee LY, Lin SH, Hu MC: Bone microenvironment and androgen status modulate subcellular localization of ErbB3 in prostate cancer cells. Mol Cancer Res 2007, 5(7):675-684.
11. Ni CY, Yuan H, Carpenter G: Role of the ErbB-4 carboxyl terminus in gamma-secretase cleavage. J Biol Chem 2003, 278(7):4561-4565.
12. Williams CC, Allison JG, Vidal GA, Burow ME, Beckman BS, Marrero L, Jones FE: The ERBB4/HER4 receptor tyrosine kinase regulates gene expression by functioning as a STAT5A nuclear chaperone. J Cell Biol 2004, 167(3):469-478.
13. Feng SM, Muraoka-Cook RS, Hunter D, Sandahl MA, Caskey LS, Miyazawa K, Atfi A, Earp HS, 3rd: The E3 ubiquitin ligase WWP1 selectively targets HER4 and its proteolytically derived signaling isoforms for degradation. Mol Cell Biol 2009, 29(3):892-906.
14. Kilkenny DM, Hill DJ: Perinuclear localization of an intracellular binding protein related to the fibroblast growth factor (FGF) receptor 1 is temporally associated with the nuclear trafficking of FGF-2 in proliferating epiphyseal growth plate chondrocytes. Endocrinology 1996, 137(11):5078-5089.
15. Peng H, Myers J, Fang X, Stachowiak EK, Maher PA, Martins GG, Popescu G, Berezney R, Stachowiak MK: Integrative nuclear FGFR1 signaling (INFS) pathway mediates activation of the tyrosine hydroxylase gene by angiotensin II, depolarization and protein kinase C. J Neurochem 2002, 81(3):506-524.
16. Myers JM, Martins GG, Ostrowski J, Stachowiak MK: Nuclear trafficking of FGFR1: a role for the transmembrane domain. J Cell Biochem 2003, 88(6):1273-1291.
17. Johnston CL, Cox HC, Gomm JJ, Coombes RC: Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. A splice variant of FGFR-3 localizes to the nucleus. J Biol Chem 1995, 270(51):30643-30650.
18. Lee TH, Kato H, Pan LH, Ryu JH, Kogure K, Itoyama Y: Localization of nerve growth factor, trkA and P75 immunoreactivity in the hippocampal formation and basal forebrain of adult rats. Neuroscience 1998, 83(2):335-349.
19. Bonacchi A, Taddei ML, Petrai I, Efsen E, Defranco R, Nosi D, Torcia M, Rosini P, Formigli L, Rombouts K et al: Nuclear localization of TRK-A in liver cells. Histol Histopathol 2008, 23(3):327-340.
20. Pillai G, Cook N, Turley H, Leek RD, Blasquez C, Pezzella F, Harris AL, Gatter KC: The expression and cellular localization of phosphorylated VEGFR2 in lymphoma and non-neoplastic lymphadenopathy: an immunohistochemical study. Histopathology 2005, 46(2):209-216.
21. Zhang Y, Pillai G, Gatter K, Blazquez C, Turley H, Pezzella F, Watt SM: Expression and cellular localization of vascular endothelial growth factor A and its receptors in acute and chronic leukemias: an immunohistochemical study. Hum Pathol 2005, 36(7):797-805.
22. Lo HW, Hsu SC, Ali-Seyed M, Gunduz M, Xia W, Wei Y, Bartholomeusz G, Shih JY, Hung MC: Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway. Cancer Cell 2005, 7(6):575-589.
23. Wang SC, Hung MC: Nuclear translocation of the epidermal growth factor receptor family membrane tyrosine kinase receptors. Clin Cancer Res 2009, 15(21):6484-6489.
24. Wang SC, Lien HC, Xia W, Chen IF, Lo HW, Wang Z, Ali-Seyed M, Lee DF, Bartholomeusz G, Ou-Yang F et al: Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2. Cancer Cell 2004, 6(3):251-261.
25. Wang SC, Nakajima Y, Yu YL, Xia W, Chen CT, Yang CC, McIntush EW, Li LY, Hawke DH, Kobayashi R et al: Tyrosine phosphorylation controls PCNA function through protein stability. Nat Cell Biol 2006, 8(12):1359-1368.
26. Psyrri A, Yu Z, Weinberger PM, Sasaki C, Haffty B, Camp R, Rimm D, Burtness BA: Quantitative determination of nuclear and cytoplasmic epidermal growth factor receptor expression in oropharyngeal squamous cell cancer by using automated quantitative analysis. Clin Cancer Res 2005, 11(16):5856-5862.
27. Xia W, Wei Y, Du Y, Liu J, Chang B, Yu YL, Huo LF, Miller S, Hung MC: Nuclear expression of epidermal growth factor receptor is a novel prognostic value in patients with ovarian cancer. Mol Carcinog 2009, 48(7):610-617.
28. Hadzisejdic I, Mustac E, Jonjic N, Petkovic M, Grahovac B: Nuclear EGFR in ductal invasive breast cancer: correlation with cyclin-D1 and prognosis. Mod Pathol 2010, 23(3):392-403.
29. Lo HW, Xia W, Wei Y, Ali-Seyed M, Huang SF, Hung MC: Novel prognostic value of nuclear epidermal growth factor receptor in breast cancer. Cancer Res 2005, 65(1):338-348.
30. Psyrri A, Egleston B, Weinberger P, Yu Z, Kowalski D, Sasaki C, Haffty B, Rimm D, Burtness B: Correlates and determinants of nuclear epidermal growth factor receptor content in an oropharyngeal cancer tissue microarray. Cancer Epidemiol Biomarkers Prev 2008, 17(6):1486-1492.
31. Masiakowski P, Carroll RD: A novel family of cell surface receptors with tyrosine kinase-like domain. J Biol Chem 1992, 267(36):26181-26190.
32. Katoh M: Comparative genomics on ROR1 and ROR2 orthologs. Oncol Rep 2005, 14(5):1381-1384.
33. Katoh M: Identification and characterization of rat Ror1 and Ror2 genes in silico. Int J Mol Med 2005, 15(3):533-538.
34. Reddy UR, Phatak S, Pleasure D: Human neural tissues express a truncated Ror1 receptor tyrosine kinase, lacking both extracellular and transmembrane domains. Oncogene 1996, 13(7):1555-1559.
35. Oishi I, Takeuchi S, Hashimoto R, Nagabukuro A, Ueda T, Liu ZJ, Hatta T, Akira S, Matsuda Y, Yamamura H et al: Spatio-temporally regulated expression of receptor tyrosine kinases, mRor1, mRor2, during mouse development: implications in development and function of the nervous system. Genes Cells 1999, 4(1):41-56.
36. Al-Shawi R, Ashton SV, Underwood C, Simons JP: Expression of the Ror1 and Ror2 receptor tyrosine kinase genes during mouse development. Dev Genes Evol 2001, 211(4):161-171.
37. Matsuda T, Nomi M, Ikeya M, Kani S, Oishi I, Terashima T, Takada S, Minami Y: Expression of the receptor tyrosine kinase genes, Ror1 and Ror2, during mouse development. Mech Dev 2001, 105(1-2):153-156.
38. Paganoni S, Ferreira A: Expression and subcellular localization of Ror tyrosine kinase receptors are developmentally regulated in cultured hippocampal neurons. J Neurosci Res 2003, 73(4):429-440.
39. Paganoni S, Anderson KL, Ferreira A: Differential subcellular localization of Ror tyrosine kinase receptors in cultured astrocytes. Glia 2004, 46(4):456-466.
40. Shabani M, Asgarian-Omran H, Jeddi-Tehrani M, Vossough P, Faranoush M, Sharifian RA, Toughe GR, Kordmahin M, Khoshnoodi J, Roohi A et al: Overexpression of orphan receptor tyrosine kinase Ror1 as a putative tumor-associated antigen in Iranian patients with acute lymphoblastic leukemia. Tumour Biol 2007, 28(6):318-326.
41. Shabani M, Asgarian-Omran H, Vossough P, Sharifian RA, Faranoush M, Ghragozlou S, Khoshnoodi J, Roohi A, Jeddi-Tehrani M, Mellstedt H et al: Expression profile of orphan receptor tyrosine kinase (ROR1) and Wilms' tumor gene 1 (WT1) in different subsets of B-cell acute lymphoblastic leukemia. Leuk Lymphoma 2008, 49(7):1360-1367.
42. Robinson DR, Wu YM, Lin SF: The protein tyrosine kinase family of the human genome. Oncogene 2000, 19(49):5548-5557.
43. Nakai K, Horton P: PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 1999, 24(1):34-36.
44. Reinhardt A, Hubbard T: Using neural networks for prediction of the subcellular location of proteins. Nucleic Acids Res 1998, 26(9):2230-2236.
45. Lei Z, Dai Y: An SVM-based system for predicting protein subnuclear localizations. BMC Bioinformatics 2005, 6:291.
46. Bhasin M, Raghava GP: ESLpred: SVM-based method for subcellular localization of eukaryotic proteins using dipeptide composition and PSI-BLAST. Nucleic Acids Res 2004, 32(Web Server issue):W414-419.
47. Chen H, Huang N, Sun Z: SubLoc: a server/client suite for protein subcellular location based on SOAP. Bioinformatics 2006, 22(3):376-377.
48. Kumar M, Raghava GP: Prediction of nuclear proteins using SVM and HMM models. BMC Bioinformatics 2009, 10:22.
49. Rexach M, Blobel G: Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 1995, 83(5):683-692.
50. Ni CY, Murphy MP, Golde TE, Carpenter G: gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase. Science 2001, 294(5549):2179-2181.
51. Blaumueller CM, Qi H, Zagouras P, Artavanis-Tsakonas S: Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane. Cell 1997, 90(2):281-291.
52. Rosen MK, Standaert RF, Galat A, Nakatsuka M, Schreiber SL: Inhibition of FKBP rotamase activity by immunosuppressant FK506: twisted amide surrogate. Science 1990, 248(4957):863-866.
53. Allocca M, Di Vicino U, Petrillo M, Carlomagno F, Domenici L, Auricchio A: Constitutive and AP20187-induced Ret activation in photoreceptors does not protect from light-induced damage. Invest Ophthalmol Vis Sci 2007, 48(11):5199-5206.
54. Cotugno G, Formisano P, Giacco F, Colella P, Beguinot F, Auricchio A: AP20187-mediated activation of a chimeric insulin receptor results in insulin-like actions in skeletal muscle and liver of diabetic mice. Hum Gene Ther 2007, 18(2):106-117.
55. Welm BE, Freeman KW, Chen M, Contreras A, Spencer DM, Rosen JM: Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland. J Cell Biol 2002, 157(4):703-714.
56. Pownall ME, Welm BE, Freeman KW, Spencer DM, Rosen JM, Isaacs HV: An inducible system for the study of FGF signalling in early amphibian development. Dev Biol 2003, 256(1):89-99.
57. Alfa RW, Tuszynski MH, Blesch A: A novel inducible tyrosine kinase receptor to regulate signal transduction and neurite outgrowth. J Neurosci Res 2009, 87(12):2624-2631.
58. Cramer LP, Mitchison TJ, Theriot JA: Actin-dependent motile forces and cell motility. Curr Opin Cell Biol 1994, 6(1):82-86.
59. Pollard TD: Cellular motility powered by actin filament assembly and disassembly. Harvey Lect 2002, 98:1-17.
60. Paganoni S, Ferreira A: Neurite extension in central neurons: a novel role for the receptor tyrosine kinases Ror1 and Ror2. J Cell Sci 2005, 118(Pt 2):433-446.
61. Bretscher A: Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr Opin Cell Biol 1999, 11(1):109-116.
62. Oishi I, Sugiyama S, Liu ZJ, Yamamura H, Nishida Y, Minami Y: A novel Drosophila receptor tyrosine kinase expressed specifically in the nervous system. Unique structural features and implication in developmental signaling. J Biol Chem 1997, 272(18):11916-11923.
63. Forrester WC: The Ror receptor tyrosine kinase family. Cell Mol Life Sci 2002, 59(1):83-96.
64. Higuchi R, Krummel B, Saiki RK: A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 1988, 16(15):7351-7367.