Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Jul 15;373(Pt 2):409–421. doi: 10.1042/BJ20030343

EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells.

Stéphanie Charrin 1, François Le Naour 1, Valérie Labas 1, Martine Billard 1, Jean-Pierre Le Caer 1, Jean-François Emile 1, Marie-Anne Petit 1, Claude Boucheix 1, Eric Rubinstein 1
PMCID: PMC1223506  PMID: 12708969

Abstract

Several tetraspanins bind directly to a few molecular partners to form primary complexes, which might assemble through tetraspanin-tetraspanin interactions to form a network of molecular interactions, the tetraspanin web. We have produced a monoclonal antibody directed to a 63 kDa molecule (determined under non-reducing conditions) associated with CD9. This molecule was first identified by MS as a molecule with four Ig domains, EWI-2. Like the related molecule CD9P-1, EWI-2 was found to be a partner not only for CD9, but also for CD81, a tetraspanin required for hepatic infection by the parasite responsible for malaria, and also a putative hepatitis C virus receptor. Using chimaeric CD9/CD82 molecules, two separate regions of CD9 of 40 and 47 amino acids were demonstrated to confer the ability to interact with EWI-2. Both EWI-2 and CD9P-1 were detected in the human liver at the surface of hepatocytes and were found to associate with CD81 on freshly isolated hepatocytes. EWI-2 also co-localized with CD81 in the liver. CD9P-1 was not detected on most peripheral blood cells, whereas EWI-2 was expressed on the majority of B-, T- and natural killer cells and was not detected on monocytes, polynuclear cells or platelets. This distribution is identical to that of CD81. Finally, EWI-2 associated with all tetraspanins studied after lysis under conditions preserving tetraspanin-tetraspanin interactions, showing that EWI-2 is a new component of the tetraspanin web.

Full Text

The Full Text of this article is available as a PDF (447.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ali S. A., Steinkasserer A. PCR-ligation-PCR mutagenesis: a protocol for creating gene fusions and mutations. Biotechniques. 1995 May;18(5):746–750. [PubMed] [Google Scholar]
  2. Angelisová P., Hilgert I., Horejsí V. Association of four antigens of the tetraspans family (CD37, CD53, TAPA-1, and R2/C33) with MHC class II glycoproteins. Immunogenetics. 1994;39(4):249–256. doi: 10.1007/BF00188787. [DOI] [PubMed] [Google Scholar]
  3. Azorsa D. O., Moog S., Cazenave J. P., Lanza F. A general approach to the generation of monoclonal antibodies against members of the tetraspanin superfamily using recombinant GST fusion proteins. J Immunol Methods. 1999 Oct 29;229(1-2):35–48. doi: 10.1016/s0022-1759(99)00102-7. [DOI] [PubMed] [Google Scholar]
  4. Berditchevski F. Complexes of tetraspanins with integrins: more than meets the eye. J Cell Sci. 2001 Dec;114(Pt 23):4143–4151. doi: 10.1242/jcs.114.23.4143. [DOI] [PubMed] [Google Scholar]
  5. Berditchevski F., Gilbert E., Griffiths M. R., Fitter S., Ashman L., Jenner S. J. Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis. J Biol Chem. 2001 Jul 30;276(44):41165–41174. doi: 10.1074/jbc.M104041200. [DOI] [PubMed] [Google Scholar]
  6. Berditchevski F., Zutter M. M., Hemler M. E. Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins). Mol Biol Cell. 1996 Feb;7(2):193–207. doi: 10.1091/mbc.7.2.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Berditchevski Fedor, Odintsova Elena, Sawada Shigeaki, Gilbert Elizabeth. Expression of the palmitoylation-deficient CD151 weakens the association of alpha 3 beta 1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signaling. J Biol Chem. 2002 Jul 10;277(40):36991–37000. doi: 10.1074/jbc.M205265200. [DOI] [PubMed] [Google Scholar]
  8. Boucheix C., Perrot J. Y., Mirshahi M., Giannoni F., Billard M., Bernadou A., Rosenfeld C. A new set of monoclonal antibodies against acute lymphoblastic leukemia. Leuk Res. 1985;9(5):597–604. doi: 10.1016/0145-2126(85)90139-0. [DOI] [PubMed] [Google Scholar]
  9. Boucheix C., Rubinstein E. Tetraspanins. Cell Mol Life Sci. 2001 Aug;58(9):1189–1205. doi: 10.1007/PL00000933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bouloc A., Bagot M., Delaire S., Bensussan A., Boumsell L. Triggering CD101 molecule on human cutaneous dendritic cells inhibits T cell proliferation via IL-10 production. Eur J Immunol. 2000 Nov;30(11):3132–3139. doi: 10.1002/1521-4141(200011)30:11<3132::AID-IMMU3132>3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  11. Charrin S., Le Naour F., Oualid M., Billard M., Faure G., Hanash S. M., Boucheix C., Rubinstein E. The major CD9 and CD81 molecular partner. Identification and characterization of the complexes. J Biol Chem. 2001 Jan 18;276(17):14329–14337. doi: 10.1074/jbc.M011297200. [DOI] [PubMed] [Google Scholar]
  12. Charrin Stéphanie, Manié Serge, Oualid Michael, Billard Martine, Boucheix Claude, Rubinstein Eric. Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation. FEBS Lett. 2002 Apr 10;516(1-3):139–144. doi: 10.1016/s0014-5793(02)02522-x. [DOI] [PubMed] [Google Scholar]
  13. Chen B., Przybyla A. E. An efficient site-directed mutagenesis method based on PCR. Biotechniques. 1994 Oct;17(4):657–659. [PubMed] [Google Scholar]
  14. Clark K. L., Zeng Z., Langford A. L., Bowen S. M., Todd S. C. PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins. J Immunol. 2001 Nov 1;167(9):5115–5121. doi: 10.4049/jimmunol.167.9.5115. [DOI] [PubMed] [Google Scholar]
  15. Crotta Stefania, Stilla Annalisa, Wack Andreas, D'Andrea Annalisa, Nuti Sandra, D'Oro Ugo, Mosca Marta, Filliponi Franco, Brunetto R. Maurizia, Bonino Ferruccio. Inhibition of natural killer cells through engagement of CD81 by the major hepatitis C virus envelope protein. J Exp Med. 2002 Jan 7;195(1):35–41. doi: 10.1084/jem.20011124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fearon D. T., Carter R. H. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu Rev Immunol. 1995;13:127–149. doi: 10.1146/annurev.iy.13.040195.001015. [DOI] [PubMed] [Google Scholar]
  17. Flint M., Maidens C., Loomis-Price L. D., Shotton C., Dubuisson J., Monk P., Higginbottom A., Levy S., McKeating J. A. Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81. J Virol. 1999 Aug;73(8):6235–6244. doi: 10.1128/jvi.73.8.6235-6244.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gharahdaghi F., Weinberg C. R., Meagher D. A., Imai B. S., Mische S. M. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis. 1999 Mar;20(3):601–605. doi: 10.1002/(SICI)1522-2683(19990301)20:3<601::AID-ELPS601>3.0.CO;2-6. [DOI] [PubMed] [Google Scholar]
  19. Hasuwa H., Shishido Y., Yamazaki A., Kobayashi T., Yu X., Mekada E. CD9 amino acids critical for upregulation of diphtheria toxin binding. Biochem Biophys Res Commun. 2001 Dec 14;289(4):782–790. doi: 10.1006/bbrc.2001.6053. [DOI] [PubMed] [Google Scholar]
  20. Hemler M. E. Specific tetraspanin functions. J Cell Biol. 2001 Dec 24;155(7):1103–1107. doi: 10.1083/jcb.200108061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horváth G., Serru V., Clay D., Billard M., Boucheix C., Rubinstein E. CD19 is linked to the integrin-associated tetraspans CD9, CD81, and CD82. J Biol Chem. 1998 Nov 13;273(46):30537–30543. doi: 10.1074/jbc.273.46.30537. [DOI] [PubMed] [Google Scholar]
  22. Kitadokoro K., Bordo D., Galli G., Petracca R., Falugi F., Abrignani S., Grandi G., Bolognesi M. CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs. EMBO J. 2001 Jan 15;20(1-2):12–18. doi: 10.1093/emboj/20.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Le Naour F., Rubinstein E., Jasmin C., Prenant M., Boucheix C. Severely reduced female fertility in CD9-deficient mice. Science. 2000 Jan 14;287(5451):319–321. doi: 10.1126/science.287.5451.319. [DOI] [PubMed] [Google Scholar]
  24. Loke S. L., Leung C. Y., Chiu K. Y., Yau W. L., Cheung K. N., Ma L. Localisation of CD10 to biliary canaliculi by immunoelectron microscopical examination. J Clin Pathol. 1990 Aug;43(8):654–656. doi: 10.1136/jcp.43.8.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mabit H., Vons C., Dubanchet S., Capel F., Franco D., Petit M. A. Primary cultured normal human hepatocytes for hepatitis B virus receptor studies. J Hepatol. 1996 Apr;24(4):403–412. doi: 10.1016/s0168-8278(96)80160-7. [DOI] [PubMed] [Google Scholar]
  26. Maecker H. T., Do M. S., Levy S. CD81 on B cells promotes interleukin 4 secretion and antibody production during T helper type 2 immune responses. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2458–2462. doi: 10.1073/pnas.95.5.2458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mannion B. A., Berditchevski F., Kraeft S. K., Chen L. B., Hemler M. E. Transmembrane-4 superfamily proteins CD81 (TAPA-1), CD82, CD63, and CD53 specifically associated with integrin alpha 4 beta 1 (CD49d/CD29). J Immunol. 1996 Sep 1;157(5):2039–2047. [PubMed] [Google Scholar]
  28. Miyado K., Yamada G., Yamada S., Hasuwa H., Nakamura Y., Ryu F., Suzuki K., Kosai K., Inoue K., Ogura A. Requirement of CD9 on the egg plasma membrane for fertilization. Science. 2000 Jan 14;287(5451):321–324. doi: 10.1126/science.287.5451.321. [DOI] [PubMed] [Google Scholar]
  29. Nakamura K., Mitamura T., Takahashi T., Kobayashi T., Mekada E. Importance of the major extracellular domain of CD9 and the epidermal growth factor (EGF)-like domain of heparin-binding EGF-like growth factor for up-regulation of binding and activity. J Biol Chem. 2000 Jun 16;275(24):18284–18290. doi: 10.1074/jbc.M907971199. [DOI] [PubMed] [Google Scholar]
  30. Orlicky D. J., Miller G. J., Evans R. M. Identification and purification of a bovine corpora luteal membrane glycoprotein with [3H]prostaglandin F2-alpha binding properties. Prostaglandins Leukot Essent Fatty Acids. 1990 Sep;41(1):51–61. doi: 10.1016/0952-3278(90)90131-4. [DOI] [PubMed] [Google Scholar]
  31. Orlicky D. J. Negative regulatory activity of a prostaglandin F2 alpha receptor associated protein (FPRP). Prostaglandins Leukot Essent Fatty Acids. 1996 Apr;54(4):247–259. doi: 10.1016/s0952-3278(96)90055-1. [DOI] [PubMed] [Google Scholar]
  32. Pileri P., Uematsu Y., Campagnoli S., Galli G., Falugi F., Petracca R., Weiner A. J., Houghton M., Rosa D., Grandi G. Binding of hepatitis C virus to CD81. Science. 1998 Oct 30;282(5390):938–941. doi: 10.1126/science.282.5390.938. [DOI] [PubMed] [Google Scholar]
  33. Rice C. M. Is CD81 the key to hepatitis C virus entry? Hepatology. 1999 Mar;29(3):990–992. doi: 10.1002/hep.510290356. [DOI] [PubMed] [Google Scholar]
  34. Rubinstein E., Le Naour F., Billard M., Prenant M., Boucheix C. CD9 antigen is an accessory subunit of the VLA integrin complexes. Eur J Immunol. 1994 Dec;24(12):3005–3013. doi: 10.1002/eji.1830241213. [DOI] [PubMed] [Google Scholar]
  35. Rubinstein E., Le Naour F., Lagaudrière-Gesbert C., Billard M., Conjeaud H., Boucheix C. CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. Eur J Immunol. 1996 Nov;26(11):2657–2665. doi: 10.1002/eji.1830261117. [DOI] [PubMed] [Google Scholar]
  36. Seigneuret M., Delaguillaumie A., Lagaudrière-Gesbert C., Conjeaud H. Structure of the tetraspanin main extracellular domain. A partially conserved fold with a structurally variable domain insertion. J Biol Chem. 2001 Aug 1;276(43):40055–40064. doi: 10.1074/jbc.M105557200. [DOI] [PubMed] [Google Scholar]
  37. Serru V., Le Naour F., Billard M., Azorsa D. O., Lanza F., Boucheix C., Rubinstein E. Selective tetraspan-integrin complexes (CD81/alpha4beta1, CD151/alpha3beta1, CD151/alpha6beta1) under conditions disrupting tetraspan interactions. Biochem J. 1999 May 15;340(Pt 1):103–111. [PMC free article] [PubMed] [Google Scholar]
  38. Shevchenko A., Wilm M., Vorm O., Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996 Mar 1;68(5):850–858. doi: 10.1021/ac950914h. [DOI] [PubMed] [Google Scholar]
  39. Silvie Olivier, Rubinstein Eric, Franetich Jean-François, Prenant Michel, Belnoue Elodie, Rénia Laurent, Hannoun Laurent, Eling Wijnand, Levy Shoshana, Boucheix Claude. Hepatocyte CD81 is required for Plasmodium falciparum and Plasmodium yoelii sporozoite infectivity. Nat Med. 2002 Dec 16;9(1):93–96. doi: 10.1038/nm808. [DOI] [PubMed] [Google Scholar]
  40. Sincock P. M., Fitter S., Parton R. G., Berndt M. C., Gamble J. R., Ashman L. K. PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function. J Cell Sci. 1999 Mar;112(Pt 6):833–844. doi: 10.1242/jcs.112.6.833. [DOI] [PubMed] [Google Scholar]
  41. Soares L. R., Tsavaler L., Rivas A., Engleman E. G. V7 (CD101) ligation inhibits TCR/CD3-induced IL-2 production by blocking Ca2+ flux and nuclear factor of activated T cell nuclear translocation. J Immunol. 1998 Jul 1;161(1):209–217. [PubMed] [Google Scholar]
  42. Stipp C. S., Kolesnikova T. V., Hemler M. E. EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily. J Biol Chem. 2001 Aug 14;276(44):40545–40554. doi: 10.1074/jbc.M107338200. [DOI] [PubMed] [Google Scholar]
  43. Stipp C. S., Orlicky D., Hemler M. E. FPRP, a major, highly stoichiometric, highly specific CD81- and CD9-associated protein. J Biol Chem. 2000 Nov 21;276(7):4853–4862. doi: 10.1074/jbc.M009859200. [DOI] [PubMed] [Google Scholar]
  44. Tseng Chien-Te K., Klimpel Gary R. Binding of the hepatitis C virus envelope protein E2 to CD81 inhibits natural killer cell functions. J Exp Med. 2002 Jan 7;195(1):43–49. doi: 10.1084/jem.20011145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wack A., Soldaini E., Tseng C., Nuti S., Klimpel G., Abrignani S. Binding of the hepatitis C virus envelope protein E2 to CD81 provides a co-stimulatory signal for human T cells. Eur J Immunol. 2001 Jan;31(1):166–175. doi: 10.1002/1521-4141(200101)31:1<166::aid-immu166>3.0.co;2-l. [DOI] [PubMed] [Google Scholar]
  46. Waterhouse Roseann, Ha Cam, Dveksler Gabriela S. Murine CD9 is the receptor for pregnancy-specific glycoprotein 17. J Exp Med. 2002 Jan 21;195(2):277–282. doi: 10.1084/jem.20011741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wistow Graeme, Bernstein Steven L., Ray Sugata, Wyatt M. Keith, Behal Amita, Touchman Jeffrey W., Bouffard Gerald, Smith Don, Peterson Katherine. Expressed sequence tag analysis of adult human iris for the NEIBank Project: steroid-response factors and similarities with retinal pigment epithelium. Mol Vis. 2002 Jun 15;8:185–195. [PubMed] [Google Scholar]
  48. Yang Xiuwei, Claas Christoph, Kraeft Stine-Kathrein, Chen Lan Bo, Wang Zemin, Kreidberg Jordan A., Hemler Martin E. Palmitoylation of tetraspanin proteins: modulation of CD151 lateral interactions, subcellular distribution, and integrin-dependent cell morphology. Mol Biol Cell. 2002 Mar;13(3):767–781. doi: 10.1091/mbc.01-05-0275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yauch R. L., Berditchevski F., Harler M. B., Reichner J., Hemler M. E. Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration. Mol Biol Cell. 1998 Oct;9(10):2751–2765. doi: 10.1091/mbc.9.10.2751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yauch R. L., Kazarov A. R., Desai B., Lee R. T., Hemler M. E. Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151. J Biol Chem. 2000 Mar 31;275(13):9230–9238. doi: 10.1074/jbc.275.13.9230. [DOI] [PubMed] [Google Scholar]
  51. Yáez-Mó M., Tejedor R., Rousselle P., Sánchez -Madrid F. Tetraspanins in intercellular adhesion of polarized epithelial cells: spatial and functional relationship to integrins and cadherins. J Cell Sci. 2001 Feb;114(Pt 3):577–587. doi: 10.1242/jcs.114.3.577. [DOI] [PubMed] [Google Scholar]
  52. Zemni R., Bienvenu T., Vinet M. C., Sefiani A., Carrié A., Billuart P., McDonell N., Couvert P., Francis F., Chafey P. A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation. Nat Genet. 2000 Feb;24(2):167–170. doi: 10.1038/72829. [DOI] [PubMed] [Google Scholar]
  53. Zhang Xin A., Kazarov Alexander R., Yang Xiuwei, Bontrager Alexa L., Stipp Christopher S., Hemler Martin E. Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis. Mol Biol Cell. 2002 Jan;13(1):1–11. doi: 10.1091/mbc.01-10-0481. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES