趨化素接受器為G蛋白耦合性受體(G protein-coupled receptor)並具有七個穿膜區域。至今，CXCR4在多種魚類中已被發現，但點帶石斑魚CXCR4 (grouper CXCR4；或簡稱gCXCR4)其特性尚未被探討，至今也仍未有相關研究。在本研究中我們選殖出gCXCR4之基因序列並發現gCXCR4具有367個胺基酸，其分子量大小推測為40.37 kDa。gCXCR4包含七個穿膜區域、四個膜外區域及四個膜內區域。在三級蛋白預測結構中，gCXCR4第三及第四膜外區域與人類CXCR4差異極大。以即時定量聚合酶鏈鎖反應進行分析，得知gCXCR4會在眼睛、鰓及頭腎組織大量表現，而由免疫組織染色也進一步證實gCXCR4的確會在鰓及頭腎中大量表現。我們也發現gCXCR4在點帶石斑魚生長發育中可能扮演重要調控角色。而當gCXCR4大量表現時， GF-1細胞株之生長會顯著受到影響。將gCXCR4轉染至GF-1細胞後，可發現gCXCR4主要會表現在細胞膜上，且可發現細胞型態會有所改變。在初代點帶石斑魚腦細胞中，內生性gCXCR4主要也表現在細胞膜上。從我們的研究中可知gCXCR4和其他物種之CXCR4具有同源性，我們也提供了gCXCR4基本的特性分析，在未來也可更進一步進行gCXCR4之相關研究。

Chemokine receptors are a group of G protein-coupled receptors with seven transmembrane domains. To date, CXCR4 has been identified in several fish species but no functional characterization of CXCR4 in grouper has been reported. In this study, we identification and characterization of the CXCR4 in Epinephelus coioides (gCXCR4). The open reading frame of gCXCR4 contained 367 amino acid residues with an estimated molecule mass of 40.37kDa. The putative gCXCR4 protein consisted of seven transmembrane regions, four extracellular regions and four cytoplasmic regions. Nonetheless, the predicted tertiary structure of the extracellular domains III and IV of gCXCR4 appeared different from that of human CXCR4. The gCXCR4 mRNA presented most abundantly in eye, gill and head kidney as revealed by RT-qPCR analysis. Analysis of the expression level of gCXCR4 indicated a regulatory role of the gene in the growth and development of grouper. Overexpression of gCXCR4 in GF-1 cells was shown to affect the proliferation of the cells. After transfection, the subcellular localization of gCXCR4 was found mainly at the cell membrane. Additionally, morphological changes of the transfected cells were observed soon after transfection. In primary grouper brain cells, endogenous gCXCR4 was also expressed mainly on cells membrane. From our studies, we can know gCXCR4 is homologous with CXCR4s from other species and provide basic information for further functional research of gCXCR4 in the future.

Aiuti, A., Turchetto, L., Cota, M., Cipponi, A., Brambilla, A., Arcelloni, C., Paroni, R., Vicenzi, E., Bordignon, C. and Poli, G. (1999) Human CD34+ cells express CXCR4 and its ligand stromal cell-derived factor-1. Implications for infection by T-cell tropic human immunodeficiency virus. Blood 94:62-73.

Alabyev, B. Y., Najakshin, A. M., Mechetina, L. V. and Taranin, A. V. (2000) Cloning of a CXCR4 homolog in chondrostean fish and characterization of the CXCR4-specific structural features. Dev Comp Immunol 24:765-70.

Alkhatib, G. (2009) The biology of CCR5 and CXCR4. Curr Opin HIV AIDS 4:96-103.

Balestrieri, M. L., Balestrieri, A., Mancini, F. P. and Napoli, C. (2008) Understanding the immunoangiostatic CXC chemokine network. Cardiovasc Res 78:250-6.

Balkwill, F. (2004) Cancer and the chemokine network. Nat Rev Cancer 4:540-50.

Belperio, J. A., Keane, M. P., Arenberg, D. A., Addison, C. L., Ehlert, J. E., Burdick, M. D. and Strieter, R. M. (2000) CXC chemokines in angiogenesis. J Leukoc Biol 68:1-8.

Berson, J. F., Long, D., Doranz, B. J., Rucker, J., Jirik, F. R. and Doms, R. W. (1996) A seven-transmembrane domain receptor involved in fusion and entry of T- cell-tropic human immunodeficiency virus type 1 strains. J Virol 70:6288-95.

Bhakta, S., Hong, P. and Koc, O. (2006) The surface adhesion molecule CXCR4 stimulates mesenchymal stem cell migration to stromal cell-derived factor-1 in vitro but does not decrease apoptosis under serum deprivation. Cardiovasc Revasc Med 7:19-24.

Bungo, F., Ahmed, M., Mathias, U. and Johng, S. R. (2010) CXCR4 and cancer. Pathol Int 60:497-505.

Busillo, J. M. and Benovic, J. L. (2007) Regulation of CXCR4 signaling. Biochim Biophys Acta (BBA) - Biomembranes 1768:952-63.

Cheng, M., Zhou, J., Zhu, W., Tanaka, T., Zeng, Q., Kishore, R., Losordo, D. and Qin, G. (2008) Abstract 1962: CXCR4-mediated bone marrow stem cell homing and niche maintenance is modulated by the functional status of c-kit. Circulation 118:S_412-d-3.
Chi, S. C., Lo, C. F., Kou, G. H., Chang, P. S., Peng, S. E. and Chen, S. N. (1997) Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel) . J Fish Dis 20:185-93.

Cho, H. H., Kyoung, K. M., Seo, M. J., Kim, Y. J., Bae, Y. C. and Jung, J. S. (2007) Overexpression of CXCR4 increases migration and proliferation of human adipose tissue stromal cells. Stem Cells Dev 15:853-64.

Chong, S. W., Emelyanov, A., Gong, Z. and Korzh, V. (2001) Expression pattern of two zebrafish genes, cxcr4a and cxcr4b. Mech Dev 109:347-54.

Daniels, G. D., Zou, J., Charlemagne, J., Partula, S., Cunningham, C. and Secombes, C. J. (1999) Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbow trout Oncorhynchus mykiss. J Leukoc Biol 65:684-90.

Do, C. A., Patricio, I., Cruz, M. T., Carvalheiro, H., Oliveira, C. R. and Lopes, M. C. (2010) CXCL12/CXCR4 promotes motility and proliferation of glioma cells. Cancer Biol Ther 9:56 - 65.

Egawa, T., Kawabata, K., Kawamoto, H., Amada, K., Okamoto, R., Fujii, N., Kishimoto, T., Katsura, Y. and Nagasawa, T. (2001) The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor. Immunity 15:323-34.

Farzan, M., Babcock, G. J., Vasilieva, N., Wright, P. L., Kiprilov, E., Mirzabekov, T. and Choe, H. (2002) The role of post-translational modifications of the CXCR4 amino terminus in stromal-derived factor 1 alpha association and HIV-1 entry. J Biol Chem 277:29484-9.

Federsppiel, B., Melhado, I. G., Duncan, A. M. V., Delaney, A., Schappert, K., Clark-Lewis, I. and Jirik, F. R. (1993) Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen. Genomics 16:707-12.

Feng, Y., Broder, C. C., Kennedy, P. E. and Berger, E. A. (1996) HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-7.

Gibson-Kueh, S., Netto, P., Ngoh-Lim, G. H., Chang, S. F., Ho, L. L., Qin, Q. W., Chua, F. H. C., Ng, M. L. and Ferguson, H. W. (2003) The pathology of systemic Iridoviral disease in Fish. J Comp Pathol 129:111-9.
Heesen, M., Berman, M. A., Benson, J. D., Gerard, C. and Dorf, M. E. (1996) Cloning of the mouse fusin gene, homologue to a human HIV-1 co-factor. J Immunol 157:5455-60.

Hernández-López, C., Valencia, J., Hidalgo, L., Martínez, V. G., Zapata, A. G., Sacedon, R., Varas, A. and Vicente, A. (2008) CXCL12/CXCR4 signaling promotes human thymic dendritic cell survival regulating the Bcl-2/Bax ratio. Immunol Lett 120:72-8.

Hirose, J., Kawashima, H., Yoshie, O., Tashiro, K. and Miyasaka, M. (2001) Versican interacts with chemokines and modulates cellular responses. J Biol Chem 276:5228-34.

Horuk, R. (2009) Chemokine receptor antagonists: overcoming developmental hurdles. Nat Rev Drug Discov 8:23-33.

Inoue, Y., Endo, M., Haruta, C., Taniuchi, T., Moritomo, T. and Nakanishi, T. (2003a) Molecular cloning and sequencing of the silver chimaera (Chimaera phantasma) interleukin-8 cDNA. Fish Shellfish Immunol 15:269-74.

Inoue, Y., Haruta, C., Usui, K., Moritomo, T. and Nakanishi, T. (2003b) Molecular cloning and sequencing of the banded dogfish (Triakis scyllia) interleukin-8 cDNA. Fish Shellfish Immunol 14:275-81.

Jia, A. and Zhang, X. H. (2009) Molecular cloning, characterization, and expression analysis of the CXCR4 gene from Turbot: Scophthalmus maximus. J Biomed Biotechnol doi:10.1155/2009/767893.

Kabashima, K., Shiraishi, N., Sugita, K., Mori, T., Onoue, A., Kobayashi, M., Sakabe, J.i., Yoshiki, R., Tamamura, H., Fujii, N., Inaba, K. and Tokura, Y. (2007) CXCL12-CXCR4 engagement is required for migration of cutaneous dendritic cells. Am J Pathol 171:1249-57.

Kahn, J., Byk, T., Jansson-Sjostrand, L., Petit, I., Shivtiel, S., Nagler, A., Hardan, I., Deutsch, V., Gazit, Z., Gazit, D., Karlsson, S. and Lapidot, T. (2004) Overexpression of CXCR4 on human CD34+ progenitors increases their proliferation, migration, and NOD/SCID repopulation. Blood 103:2942-9.

Kakinuma, T. and Hwang, S. T. (2006) Chemokines, chemokine receptors, and cancer metastasis. J Leukoc Biol 79:639-51.

Kenji, S., Masaki, M., Katsuhiro, S., Hiroshi, I., Prasenjit, M. and Graham, F. B. (2002) Regional expression of CXCL12/CXCR4 in liver and hepatocellular carcinoma and cell-cycle variation during in vitro differentiation. Cancer Sci 93:789-97.

Khan, M. Z., Brandimarti, R., Musser, B. J., Resue, D. M., Fatatis, A. and Meucci, O. (2003) The chemokine receptor CXCR4 regulates cell-cycle proteins in neurons. J Neurovirol 9:300-14.

Kim, C. H. and Broxmeyer, H. E. (1999) Chemokines: signal lamps for trafficking of T and B cells for development and effector function. J Leukoc Biol 65:6-15.

Kochetkova, M., Kumar, S. and McColl, S. R. (2009) Chemokine receptors CXCR4 and CCR7 promote metastasis by preventing anoikis in cancer cells. Cell Death Differ 16:664-73.

Kuroda, N., Uinukool, T. S., Sato, A., Samonte, I. E., Figueroa, F., Mayer, W. E. and Klein, J. (2003) Identification of chemokines and a chemokine receptor in cichlid fish, shark, and lamprey. Immunogenetics 54:884-95.

Kyte, J. and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105-32.

Laing, K. J., Zou, J. J., Wang, T., Bols, N., Hirono, I., Aoki, T. and Secombes, C. J. (2002) Identification and analysis of an interleukin 8-like molecule in rainbow trout Oncorhynchus mykiss. Dev Comp Immunol 26:433-44.

Lee, E. Y., Park, H. H., Kim, Y. T., Chung, J. K. and Choi, T. J. (2001) Cloning and sequence analysis of the interleukin-8 gene from flounder (Paralichthys olivaceous) . Gene 274:237-43.

Lee, H. J., Kim, S. W., Kim, H. Y., Li, S., Yun, H. J., Song, K. S., Kim, S. and Jo, D. Y. (2009) Chemokine receptor CXCR4 expression, function, and clinical implications in gastric cancer. Int J Oncol 34:473-80.

Ma, Q., Jones, D. and Springer, T. A. (1999) The chemokine receptor CXCR4 is required for the retention of B lineage and granulocytic precursors within the bone marrow microenvironment. Immunity 10:463-71.

Moepps, B., Braun, M., Knöpfle, K., Dillinger, K., Knöchel, W. and Gierschik, P. (2000) Characterization of a Xenopus laevis CXC chemokine receptor 4: implications for hemato-poietic cell development in the vertebrate embryo. Eur J Immunol 30:2924-34.
Müller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M. E., McClanahan, T., Murphy, E., Yuan, W., Wagner, S. N., Barrera, J. L., Mohar, A., Verastegui, E. and Zlotnik, A. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410:50-6.

Munday, B. L. and Nakai, T. (1997) Nodaviruses as pathogens in larval and juvenile marine finfish. World J Microbiol Biotechnol 13:375-81.

Murdoch, C., Monk, P. N. and Finn, A. (1999) Functional expression of chemokine receptor CXCR4 on human epithelial cells. Immunology 98:36-41.

Murphy, P. M., Baggiolini, M., Charo, I. F., Hebert, C. A., Horuk, R., Matsushima, K., Miller, L. H., Oppenheim, J. J. and Power, C. A. (2000) International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev 52:145-76.

Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S.-i., Kitamura, Y., Yoshida, N., Kikutani, H. and Kishimoto, T. (1996) Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382:635-8.

Najakshin, A.M., Mechetina, L.V., Alabyev, B.Y. and Taranin, A. V. (1999) Identification of an IL-8 homolog in lamprey (Lampetra fluviatilis): early evolutionary divergence of chemokines. Eur J Immunol 29:375-82.

Olson, T. S. and Ley, K. (2002) Chemokines and chemokine receptors in leukocyte trafficking. Am J Physiol Regul Integr Comp Physiol 283:R7-28.

Orimo, A., Gupta, P. B., Sgroi, D. C., Arenzana-Seisdedos, F., Delaunay, T., Naeem, R., Carey, V. J., Richardson, A. L. and Weinberg, R. A. (2005) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121:335-48.

Palevitch, O., Abraham, E., Borodovsky, N., Levkowitz, G., Zohar, Y. and Gothilf, Y. (2010) Cxcl12a-Cxcr4b signaling is important for proper development of the forebrain GnRH system in zebrafish. Gen Comp Endocrinol 165:262-8.

Pfeiffer, M., Hartmann, T. N., Leick, M., Catusse, J., Schmitt-Graeff, A. and Burger, M. (2009) Alternative implication of CXCR4 in JAK2/STAT3 activation in small cell lung cancer. Br J Cancer 100:1949-56.

Phillips, R. J., Burdick, M. D., Lutz, M., Belperio, J. A., Keane, M. P. and Strieter, R. M. (2003) The stromal derived factor-1/CXCL12-CXC chemokine receptor 4 biological axis in non-small cell lung cancer metastases. Am J Respir Crit Care Med 167:1676-86.

Qin, Q. W., Shi, C., Gin, K. Y. H. and Lam, T. J. (2002) Antigenic characterization of a marine fish iridovirus from grouper, Epinephelus spp. J Virol Methods 106:89-96.

Rimland, J., Xin, W., Sweetnam, P., Saijoh, K., Nestler, E. J. and Duman, R. S. (1991) Sequence and expression of a neuropeptide Y receptor cDNA. Mol Pharmacol 40:869-75.

Rosenkilde, M. M., Smit, M. J. and Waldhoer, M. (2008) Structure, function and physiological consequences of virally encoded chemokine seven transmembrane receptors. Br J Pharmacol 153:S154-S66.

Rostene, W., Kitabgi, P. and Parsadaniantz, S. M. (2007) Chemokines: a new class of neuromodulator? Nat Rev Neurosci 8:895-903.

Schuitemaker, H., Kootstra, N. A., de Goede, R. E., de Wolf, F., Miedema, F. and Tersmette, M. (1991) Monocytotropic human immunodeficiency virus type 1 (HIV-1) variants detectable in all stages of HIV-1 infection lack T-cell line tropism and syncytium-inducing ability in primary T-cell culture. J Virol 65:356-63.

Scotton, C. J., Wilson, J. L., Scott, K., Stamp, G., Wilbanks, G. D., Fricker, S., Bridger, G. and Balkwill, F. R. (2002) Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res 62:5930-8.

Shen, X., Artinyan, A., Jackson, D., Thomas, R. M., Lowy, A. M. and Kim, J. (2010) Chemokine receptor CXCR4 enhances proliferation in pancreatic cancer cells through AKT and ERK dependent pathways. Pancreas 39:81-7.

Spano, J. P., Andre, F., Morat, L., Sabatier, L., Besse, B., Combadiere, C., Deterre, P., Martin, A., Azorin, J., Valeyre, D., Khayat, D., Le Chevalier, T. and Soria, J. C. (2004) Chemokine receptor CXCR4 and early-stage non-small cell lung cancer: pattern of expression and correlation with outcome. Ann Oncol 15:613-7.

Strieter, R. M., Polverini, P. J., Kunkel, S. L., Arenberg, D. A., Burdick, M. D., Kasper, J., Dzuiba, J., Van Damme, J., Walz, A., Marriott, D., Chan, S. Y., Roczniak, S. and Shanafelt, A. B. (1995) The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J Biol Chem 270:27348–57.

Tachibana, K., Hirota, S., Iizasa, H., Yoshida, H., Kawabata, K., Kataoka, Y., Tachibana, K., Hirota, S., Iizasa, H., Yoshida, H., Kawabata, K., Kataoka, Y., Kitamura, Y., Matsushima, K., Yoshida, N., Nishikawa, S., Kishimoto, T. and Nagasawa, T. (1998) The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 393:591-4.

Tanaka, S., Takagi, M. and Miyazaki, T. (2004) Histopathological studies on viral nervous necrosis of sevenband grouper, Epinephelus septemfasciatus Thunberg, at the grow-out stage. J Fish Dis 27:385-99.

Tsuchida, S., Kagi, A. and Takahashi, T. (2007) Characterization of cDNA and genomic sequences encoding a canine chemokine receptor, CXCR4 and its ligand CXCL12. Vet Immunol Immunopathol 116:219-25.

Ueno, M., Yamada, S., Zako, M., Bernfield, M. and Sugahara, K. (2001) Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. J Biol Chem 276:29134-40.

Vasyutina, E., Stebler, J., Brand-Saberi, B., Schulz, S., Raz, E. and Birchmeier, C. (2005) CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells. Genes Dev 19:2187-98.

Wang, L., Wang, L., Yang, B., Yang, Q., Qiao, S., Wang, Y. and Sun, Y. (2009) Strong expression of chemokine receptor CXCR4 by renal cell carcinoma cells correlates with metastasis. Clin Exp Metastasis 26:1049-54.

Wu, Y. and Yoder, A. (2009) Chemokine coreceptor signaling in HIV-1 infection and pathogenesis. PLoS Pathog 5:e1000520.

Zhou, Y., Larsen, P. H., Hao, C. and Yong, V. W. (2002) CXCR4 is a major chemokine receptor on glioma cells and mediates their survival. J Biol Chem 277:49481-7.

Zlotnik, A. (2008) New insights on the role of CXCR4 in cancer metastasis. J Pathol 215:211-3.

Zou, Y. R., Kottmann, A. H., Kuroda, M., Taniuchi, I. and Littman, D. R. (1998) Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595-9.