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J. Biol. Chem., Vol. 281, Issue 28, 19665-19675, July 14, 2006
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1
From the
Servicio de Inmunología, Hospital Universitario de La Princesa, UAM, Madrid 28006, Spain, INSERM U602, Hôpital Paul Brousse, 94807 Villejuif Cedex, France, and ¶Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115
EWI-2 and EWI-F, two members of a novel subfamily of Ig proteins, are direct partners of tetraspanins CD9 (Tspan29) and CD81 (Tspan28). These EWI proteins contain a stretch of basic charged amino acids in their cytoplasmic domains that may act as binding sites for actin-linking ezrin-radixin-moesin (ERM) proteins. Confocal microscopy analysis revealed that EWI-2 and EWI-F colocalized with ERM proteins at microspikes and microvilli of adherent cells and at the cellular uropod in polarized migrating leukocytes. Immunoprecipitation studies showed the association of EWI-2 and EWI-F with ERM proteins in vivo. Moreover, pulldown experiments and protein-protein binding assays with glutathione S-transferase fusion proteins containing the cytoplasmic domains of EWI proteins corroborated the strong and direct interaction between ERMs and these proteins. The active role of ERMs was further confirmed by double transfections with the N-terminal domain of moesin, which acts as a dominant negative form of ERMs, and was able to delocalize EWIs from the uropod of polarized leukocytes. In addition, direct association of EWI partner CD81 C-terminal domain with ERMs was also demonstrated. Functionally, silencing of endogenous EWI-2 expression by short interfering RNA in lymphoid CEM cells augmented cell migration, cellular polarity, and increased phosphorylation of ERMs. Hence, EWI proteins, through their direct interaction with ERM proteins, act as linkers to connect tetraspanin-associated microdomains to actin cytoskeleton regulating cell motility and polarity.
Received for publication, March 6, 2006 , and in revised form, April 20, 2006.
* This work was supported in part by Biología Fundamental 2005-08435/Biología Molecular y Celular from the Spanish Ministry of Education and Science and the Ayuda a la Investigación Básica 2002 from Juan March Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported by Contrato-Investigador FIS 0019 from Instituto de Salud Carlos III. To whom correspondence should be addressed: Servicio de Inmunología, Hospital Universitario de la Princesa, Diego de León 62, 28006 Madrid, Spain. Tel.: 34-91-5202307; Fax: 34-91-5202374; E-mail: myanez.hlpr{at}salud.madrid.org.
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