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Interaction of the cytoskeletal protein talin with the integrin β3 subunit cytoplasmic tail: Characterization of the talin rod IBS2 integrin binding site.

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UNIVERSITÉ LIBRE DE BRUXELLES Faculté des Sciences – Biologie Moléculaire

Centre de Biologie Structurale et Bioinformatique

Interaction of the cytoskeletal protein talin with the integrin β3 subunit cytoplasmic tail:

Characterization of the talin rod IBS2 integrin binding site.

Thèse présentée en vue de l’obtention du grade de Docteur en Sciences

par

Moes Michèle

Promoteur : M. Eric Goormaghtigh, Professeur, Université Libre de Bruxelles Co-promoteur : Mme Nelly Kieffer, Professeur, Université du Luxembourg

Octobre 2007

(2)

ABSTRACT

Talin is a multifunctional cytoskeletal protein that plays a critical role in linking the actin cytoskeleton to the integrin family of transmembrane cell adhesion receptors. Two distinct integrin binding sites have been identified in talin, one present in the globular head domain (IBS1) and involved in integrin activation, and a second (IBS2), that has been delineated to a 130 residue fragment of the talin rod domain, but whose functional role is still elusive (Tremuth et al., 2004). The objective of the present study was to define the minimal structure of talin IBS2 and to investigate its functional role in the integrin-cytoskeleton connection.

In the first part of this study, we used a combination of three different experimental approaches to define the minimal structure of talin IBS2: 1) an in silico bioinformatics approach to analyse sequence conservation of talin IBS2, 2) an in vivo cell biology approach to study the subcellular localization of recombinant talin fragments covering IBS2 in CHOαIIbβ3 cells, and 3) an in vitro biochemical approach consisting in protein overlay, pull down and Surface Plasmon Resonance (SPR) assays, to study the direct interaction between talin IBS2 and the integrin β3 subunit. We delineated IBS2 to a single amphipathic α-helical repeat of 23 residues within the talin rod domain. We further provided evidence that a two amino acid mutation (L

2094

I

2095

/AA) was sufficient to inactivate the IBS2 site, due to a disruption of the α helix structure, as demonstrated by infrared spectroscopy. In addition, we identified 2 lysine residues (K

2085

, K

2089

) exposed on the solvent face of α helix 50, which are directly involved in the talin IBS2-integrin interaction.

In the second part of this study, we investigated the functional role of talin IBS2 in spreading-

defective talin (-/-) cells and showed that in contrast to full-length wild type talin, an IBS2 LI/AA

mutant talin was unable to fully rescue the spread phenotype of these cells. These results provide

the first direct evidence that IBS2 in the talin rod is essential to link integrins to the actin

cytoskeleton.

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