HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 42, 28038-28047, October 17, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/42/28038    most recent M804991200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Gouveia, R. M. Articles by Costa, J. Search for Related Content PubMed PubMed Citation Articles by Gouveia, R. M. Articles by Costa, J. Social Bookmarking                      What's this?

Kinetic Analysis of L1 Homophilic Interaction

ROLE OF THE FIRST FOUR IMMUNOGLOBULIN DOMAINS AND IMPLICATIONS ON BINDING MECHANISM^*

Ricardo M. Gouveia¹, Cláudio M. Gomes, Marcos Sousa, Paula M. Alves, and Júlia Costa²

From the Instituto de Tecnologia Química e Biológica, Avenida da República, Apartado 127, 2780-157 Oeiras, and the Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal

L1 is a cell adhesion molecule of the immunoglobulin (Ig) superfamily, critical for central nervous system development, and involved in several neuronal biological events. It is a type I membrane glycoprotein. The L1 ectodomain, composed of six Ig-like and five fibronectin (Fn) type-III domains, is involved in homophilic binding. Here, co-immunoprecipitation studies between recombinant truncated forms of human L1 expressed and purified from insect Spodoptera frugiperda Sf9 cells, and endogenous full-length L1 from human NT2N neurons, showed that the L1 ectodomain (L1/ECD) and L1/Ig1–4 interacted homophilically in trans, contrary to mutants L1/Ig1–3 and L1/Ig2-Fn5. All mutants were correctly folded as evaluated by combination of far-UV CD and fluorescence spectroscopy. Surface plasmon resonance analysis showed comparable dissociation constants of 116 ± 2 and 130 ± 6 nM for L1/ECD-L1/ECD and L1/ECD-L1/Ig1–4, respectively, whereas deletion mutants for Ig1 or Ig4 did not interact. Accordingly, in vivo, Sf9 cells stably expressing L1 were found to adhere only to L1/ECD- and L1/Ig1–4-coated surfaces. Furthermore, only these mutants bound to HEK293 cells overexpressing L1 at the cell surface. Enhancement of neurite outgrowth, which is the consequence of signaling events caused by L1 homophilic binding, was comparable between L1/ECD and L1/Ig1–4. Altogether, these results showed that domains Ig1 to Ig4 are necessary and sufficient for L1 homophilic binding in trans, and that the rest of the molecule does not contribute to the affinity under the conditions of the current study. Furthermore, they are compatible with a cooperative interaction between modules Ig1–Ig4 in a horseshoe conformation.

Received for publication, July 1, 2008 , and in revised form, August 6, 2008.

^* This work was supported in part by CellPROM number 500039-2 and Signalling & Traffic number LSHG-CT-2004-503228 from the European Commission. 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 Ph.D. fellowship SFRH/BD/24013/2005 from Fundação para a Ciência e a Tecnologia, Portugal.

² To whom correspondence should be addressed: Instituto de Tecnologia Química e Biológica, Avenida da República, Apartado 127, 2780-157 Oeiras, Portugal. Tel.: 351214469437; Fax: 351214411277; E-mail: jcosta{at}itqb.unl.pt.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?